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ITC Data Analysis in Origin®

1. cccceeees 7 Lesson 1 Routine ITC Data Analysis and Fitting cccccccsssssssssssccccccsccessssssecs 9 Roubiie WT CD ata Aid y S05 sei sreceecehcetsie a EE E eins ala tatee ere ated 9 CUNE FINNO eosa aaa Mere eee Seen ree Mee E ter een ee Mee Ser nee Mace ene eet ree tener 13 Fitime Parameter OMS UALS vac cuutapoieidedstieacuutenitudesalaitie E E E TEA 14 Fimme Parameters Lexin Eee Rene ne ee mre een oe eee eee ene Mee nnn ener ae een eer cree nee neers 14 Creatine a Final Figure for PUD MCA Ot i i5 ce scat aise sientneteasaddi ed cuetoumiantatasnnd ad E 17 Lesson 2 Setting Baseline and Integration Range ecccccssessecccoocssssesceeccosssssseeeeoso 21 Lesson 3 Deleting Bad Data es cciesdccsstesdeheciscievenctele sexta sactees se detucdsessescwscualiesasesteeee Uetaerss 27 Lesson 4 Analyzing Multiple Runs and Subtracting Reference sceees 29 Openine Multiple ata TICS eneee TT T T 29 Ps ite the Molar Rati serei a E A E E E AE E 33 Subtracting Relerem e Dila anir a a E EE oat E AEAEE 34 Subtracting Reference Data Additional Topics c ccc cceessssssssesesssesssssssssssssssssssssssssssssssseeeas 36 Lesson S ITC Data Handing 2ssisecsccceecedeccotscenswespeievetesctiosercidcsettscenssutesiecetesctiosescivens 43 Reading Worksheet Values from Plotted Data ccccccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 43 Copy and Paste Worksheet Data ig vcsi ca tacseveseveseivsvseseness
2. e O 2 O O Molar Ratio Using the Sequential Binding model with non identical sites The model of Sequential Binding can also be useful for systems with non identical sites The Two Sets of Sites model discussed earlier considers the saturation of individual sites on the same molecule assumes they saturate independently of one another and uses three fitting parameters for each site N K and H The model of Sequential Binding Sites assumes a fixed sequence of binding 1 e the first ligand which binds to an individual molecule always binds to site 1 the second ligand which bids to an individual molecule always binds to site 2 etc The number of sequential sites must be exactly integral 1 2 3 so there is no fitting parameter equivalent to N and best fit is determined by only two parameters K and H at each site once the total number of sites has been selected by the operator For a molecule which has 2 sites of quite different affinity e g K values different by a factor of five or more the two models will tend to give equivalent values of K and H since thermodynamics will dictate binding to the site of highest affinity first However when K values at two independent sites are more nearly equivalent then sequential binding will not be strictly followed MAU130010 Rev D 65 ITC Tutorial Guide 66 One inherent advantage of the sequential model is the smaller number of fitting parameters
3. P K X F l P 20 2 _ KK X 2 dP F K K K X P where P 1 K X K K X 4K K K X 21 X X M iF t i Once n and values of fitting parameters K through K are assigned then equations 20 21 may be solved for X by numerical methods the Bisection method is used After X is known all F may be calculated from equation 20 and the heat content after the i injection is determined from Q M V F AH F AH AH 4 F AH AH AH 4H 22 And as before 22 Q i 1 OG 1 23 5a cae AQ B O Which then leads into the Marquardt minimization routine MAU130010 Rev D 109 ITC Tutorial Guide V Enzyme substrate inhibitor Assay Assaying enzymes inhibitors or substrates by calorimetric activity has the major advantage that it works well for any enzyme substrate inhibitor system with no prior chemical modification of any participants in the reaction The rate R of the substrate decomposition reaction is directly proportional to the power output in the calorimeter cell 1 e DNE ARV 24 where P is the power generated by the reaction AH is the heat of decomposition of the substrate and V is the cell volume The units of R will be moles l sec if P is expressed in ucal sec AH in ucal per mole of substrate and V in liters for example If Michaelis Menten kinetics are assumed then the experimental values for the rate R ca
4. 1 17 oon av AQ B O which may be used in the Marquardt algorithm to obtain best values for the six fitting parameters IV Sequential Binding Sites For sequential binding the binding constants K K3 K must be defined relative to the progress of saturation so that K MX MX ma H e In the sequential model there is no distinction as to which sites are saturated but only as to the total number of sites that are saturated If the sites are identical then there is a statistical degeneracy associated with the sequential saturation since the first ligand to bind has more empty sites of the same kind to choose from than does the second ligand etc For identical interacting sites then we can distinguish between the phenomenological binding constants K defined by eq 18 and the intrinsic binding constants K i where the effect of degeneracies has been removed The relationship between the two binding constants is given by ke hy 19 All calculations given below as well as parameters reported from curve fitting are in terms of K values but the operator may convert to K values if desired using eq 19 Since concentrations of all liganded species ML can be easily expressed in terms of the concentration of the non liganded species M then the fraction of total macromolecule having 1 bound ligands F is simply 108 MAU130010 Rev D Appendix Equations Used for Fitting ITC Data l F
5. ITC Tutorial Guide Enzyme Assay Method 2 Substrate Only Begin this lesson by opening the ITC data file M2NoInhibitor itc as follows e Select File New Project A new Origin project opens to display the RawITC plot window e Click on the Read Data button Click on the scroll down arrow of the Files of type text box and select Enzyme Assay IT file type e Navigate to the C Origin70 Samples folder and select M2NoInhibitor from the File Name list and click OK The Enzyme Assay dialog box will open allowing you to select one of the four models e Select Method 2 Substrate only and click OK The Method 2 Substrate only dialog box will open up as shown below e Enter 10500 cal mole for AH Note In Method 2 the AH must be independently determined in a separate single injection expermiement Method 1 and that value is entered here F M2NoInhibiRAW_cp Average Time P i i er Calculate Rate B 10500 cal mole cancel _ Ss mi in wo cL S00 1000 1500 2000 2500 S000 Timefsec e Click the Concentration button The concentration dialog box will open allowing you to verify or edit the concentrations Average Time P I2 MAU130010 Rev D Lesson 7 Advanced Curve Fitting The rate of substrate decomposition reactions are determined by measuring the change in the power output in the calorimeter cell which results after each addition of the substrate The new power l
6. Keaen l K 11 1 0 1 0 X X X M n 0 7 0 12 Solving equation 11 for and and then substituting into equation 12 gives XK nM X K mM X K nM X K 13 A T 1 X K mm Clearing equation 13 of fractions and collecting like terms leads to a cubic equation of the form XT p X 4 X r 0 14 where l p n n M SA K 2 p 15 1 The first infinitesimal volume element in the i injection contributes no heat effect since it has already equilibrated at existing concentrations after the 1 1 injection The last volume element of an injection contributes heat effects equal to the liquid remaining in V since its concentrations are equivalent to those in V after the 1 injection Assuming linearity over the small AV volume increment then the liquid in the displaced volume is only half as effective in producing heat relative to the liquid in V MAU130010 Rev D LO ITC Tutorial Guide Equations 14 and 15 can be solved for X either in closed form or as done in Origin numerically by using Newton s Method if parameters n n2 K and K are assigned Both and may then be obtained from equation 11 above As discussed earlier in section II the heat content after any injection 1 is equal to O MV n 0 AH n AH 16 After a similar correction for displaced volume the pertinent calculated heat effect for the 1 injection is 20 Q i 1 7 Ol
7. E OnelnjBase sim Programming OneInj001 sim E OneInj002 sim File name Onelnj001 sim Files of type ITC Data sim e Click Open The file is then read in and the following operations take place on the data set 1 The data is read into a worksheet that is created with the corresponding name and RAW appended 1 e the worksheet is named OneInjOO1RAW 2 The time before the injection starts 60 seconds 1s subtracted from the x data so that the injection starts at t 0 and all data points are shifted to the left Note The x data before t 0 is removed from the worksheet but the data is still plotted in the graph for use in baseline subtraction MAU130010 Rev D So ITC Tutorial Guide 3 The data is corrected for the time constant of the instrument 4 The noise introduced by the time constant correction is filtered using the standard Fourier transform filter of Origin and a bandwidth of 15 data points 5 The corrected and filtered data is then plotted in the ARawITCsi window To zero the baseline Subtract Options The next operation will be to zero the baseline the E operation is similar to the Subtract Options see page 92 Control Baseline Subt gii DE x button used in removing dilution effects from the area data in 7 the AdeltaH window for standard ITC data fe Input final numerical Y position f Subtract a constant e Click the Subtract Options button from the Single C Subtract reference data
8. ITC Tutorial Guide To Normalize Data points Click the Normalize Data button from the Single Injection group After clicking the Normalize Data button the concentration continuum will be calculated and the normalized heat will be plotted in a new window named DeltaH The data are now in the form of conventional ITC normalized data and may be fit with the methods described in pages 93 and 94 5 ili a 5 Ore Ci LH E H Ore MOLHO H Ore COSL LH OH Ore ODAH O H 3 Mokar Fa be 100 MAU130010 Rev D Lesson 9 Other Useful Details Lesson 9 Other Useful Details Chi square chi 2 Minimization The aim of the fitting procedure is to find those values of the parameters which best describe the data The standard way of defining the best fit is to choose the parameters so that the sum of the squares of the deviations of the theoretical curve s from the experimental points for a range of independent variables is at a minimum For the ITC models where there is no weighting the theoretical models can be represented by S Pie Pos Pas where Pi the fitting parameters the expression for x simplifies to l x e sA peP where n the total number of experimental points used in the fitting p total number of adjustable parameters y experimental data points SP LPzp fitting function Note the difference d n p is usually referred to as the number of degrees of freedom The abov
9. but will default back to 01 the next time Origin for ITC is opened Contact MicroCal if you need to permanently change the Delta value The Parameters Significant Digits Group Select values for the display of significant digits for each parameter from the associated drop down list Select Free from the drop down list to use the current Origin setting This will only effect the text box display in the Fitting Sessions dialog box MicroCal has preset the significant digits to be 4 for all parameters The Weighting Method Drop Down List The bottom part of the Control Parameters dialog box enables you to select how different dataset points are to be weighted when computing chi square during the iterative procedure The choices are No weighting Instrumental Statistical Arbitrary dataset and Direct Weighting We recommend that the default option of No weighting be used for all ITC data unless the user has strong reason to feel another choice 1s more appropriate for a particular data set No weighting assumes that each data point has the same absolute error probability Return to the Fitting Session dialog box by clicking on the button or by selecting Action Fit Deconvolution with Ligand in the Cell and Macromolecule in the Syringe Whenever the ligand and macromolecule each have only one site for interaction with the other then the system is symmetrical and it does not matter which of the two 1s loaded into the cell and which into the
10. concentration in the syringe this concentration OK must always be entered as equivalent monomer ey concentration Click on the Concentration button In this case the concentration has been C in Syringe mM ABA correctly entered and stored in the data file Click Inject Volfull 4 OK or Cancel Note Unlike typical ITC files there is no entry for eae macromolecule concentration in the cell as the concentration is zero Select the Dissociation button The NonLinear Curve Fitting dialog box for the Dissociation model will open Click the 100 Iterations button one or two times to ensure that Ch1 2 is no longer decreasing then click OK The fitting parameters should be similar to below MAU130010 Rev D Lesson 7 Advanced Curve Fitting Data Dissociatio DH Model Dissociation Chi 2 0 1881 DH cal mole 9994 16 7 K mM 0 623 0 0037 Competitive Ligand Binding Competitive binding experiments are carried out by injecting a strong binding ligand A into a solution which contains both the macromolecule and the competing ligand B The ligand A appears to bind more weakly to the macromolecule in the presence of the competing ligand In order to do curve fitting on results from a competitive binding experiment a second non competitive experiment must first be carried out in the conventional way to determine the binding parameters for ligand B Ng Kg and AHp itself These three parameters are used as input which then
11. or choose RawITC from the Window menu to make it the active window Select RnahhhBASE from the Data menu RnahhhBASE becomes checkmarked to show it is selected Math ITC Tools Format Ww Set Weoley Range Beset to Full Range Data Markers Move Data Points Remove Bad Data Points 1 AnahhhRaM tine cpi HARBASE Select Plot from the Format menu The Plot Details dialog box opens Click on the Worksheet button The RnahhhBASE worksheet opens MAU130010 Rev D Lesson 5 ITC Data Handling me Origin 7 UNTITLED RnahhhBASE Si File Edit View Plot Column Math Sb 14 04571 C3 20 15694 28 09143 20 17892 42 13714 5 20 20089 56 18286 20 22986 70 22857 7 20 24483 84 27429 70 26681 98 32 70 28878 112 36571 To export the worksheet data as an ASCII file Shortcut Right click on the upper left containing white space then select Export ASCH Select Export ASCII from the File menu The Export ASCII dialog box opens with RnahhhBASE DAT selected as the file name Export ASCII Save ir E Onin E AddOn Setup F Templates B Buttons J Origin Tutorial FitFune Palettes Updates _ Help pClarnp J Localization Samples _ Modified Files Tables File name BREYER Save as type EDAT Cancel Click Save After you click Save in the Export ASCII dialog box the ASCHI Export Into dialog box opens MAU130010 Rev D 4 I
12. 5 Molar Ratio To print the page in the ITCFINAL window select Print from the File menu Before you print make sure ITCFINAL is the active window When a window is active its title bar changes from gray to blue this can vary depending on your Windows setup to view or change your setup select Start Settings Control Panel then double click on Display and click on the Appearance tab Click on a window to make it active or select the window from the Window List in the Window menu e Choose Save Project As from the File menu The file Save As dialog box opens e Enter a name for the project for example Lesson 1 in the File Name text box The name for the project may contain up to 255 characters and include spaces MAU130010 Rev D 19 ITC Tutorial Guide e Click on the Save button The entire contents of this project including all data sets and plot windows are saved into a file called Lesson 1 OPJ Choose Exit from the File menu Origin closes 20 MAU130010 Rev D Lesson 2 Setting Baseline and integration Range Lesson 2 Setting Baseline and Integration Range In Lesson you learned how to use Origin to perform routine data analysis of ITC files In routine data analysis integration details baselines and integration ranges are determined automatically Sometimes however the automatically determined values are not sufficiently accurate and you will want to set integration details manually This is especially t
13. E m t 2 5 a 8 Deconvolution with the Sequential Binding Sites Model All models discussed until now are concerned only with independent sites It often occurs in biological systems that the binding of a ligand to one site will be influenced by whether or not ligands are bound to any of the other sites If the sites happen to be non identical in the first place then binding studies alone cannot determine whether the sites are independent or interacting On the other hand if the sites within a molecule are known to be identical then it is possible sometimes to determine if they are interacting Consider the simplest case that of a macromolecule with two identical sites this might be a homodimeric protein for example Ifthe sites are identical then we can no longer distinguish between binding at the first site and binding at the second site so the bookkeeping must be done in terms of the first ligand bound K1 H1 and the second ligand bound K2 H2 as described in the Appendix A system with positive cooperativity means K2 gt K1 while negative cooperativity means K1 gt K2 Positive cooperativity is generally more difficult to distinguish from binding studies alone since the tendency is for both sites on any single molecule to saturate together with heat change H1 H2 so that only one phase is seen in the titration curve To determine if cooperativity were present one could use another technique which was able
14. MAU130010 Rev D Appendix Equations Used for Fitting ITC Data where S o is the starting substrate concentration Knowing AH the substrate concentration can be determined as a function of time from the equation Pdt S S EI 27 After obtaining the time dependent rate from equation 24 then these data can be equated to the Michaelis expression in equation 25 to provide the final equation to be fit by non linear least squares In the absence of inhibitor kea and Ky are used as variable parameters during iterative fitting In the presence of inhibitor I it is best to enter previously determined values of kcat and Ky and use K as the only variable fitting parameter Method 2 Multiple injections In this method multiple injections of substrate solution from the syringe are made into the reaction cell containing enzyme solution with or without inhibitor After each injection a sufficient time is allowed for the instrument to equilibrate at the new power level resulting from the increased substrate concentration Measurements are carried out quickly enough however so that little hydrolysis of substrate takes place relative to the total substrate contained in the cell That is S is calculated directly from the total added substrate assuming no significant hydrolysis Equations 24 25 are still valid for Method 2 except that R and S now correspond to discrete values of the rate and substrate concentration after each in
15. Rate millimolesjfsec O 00000 S Cm Mi e Click on the Fit to Model button The Fitting Sessions dialog box will open e Click on the 100 Iter button one or two times then click Data M2Nolnhibi R Done to end the fitting session Model M2SubstrateOnly Chi 2 DoF 2 129E 12 Keat 67 8 0 423 MAU130010 Rev D Km 0 084 0 00219 AH 1 050E4 ITC Tutorial Guide Kcat and Km are used as the variable parameters during the iterative fitting and along with the entered AH reported in the output parameter box Enzyme Assay Method 2 Substrate plus inhibitor Begin this lesson by opening the ITC data file M2Inhibitor01 itc as follows e Select File New Project A new Origin project opens to display the RawITC plot window e Click on the Read Data button Click on the scroll down arrow of the Files of type text box and select Enzyme Assay IT file type e Navigate to the C Origin70 Samples folder and select M2NoInhibitor from the File Name list and click OK The Enzyme Assay dialog box will open allowing you to select one of the four models e Select Method 2 Substrate plus inhibitor and click OK The Method 2 Substrate plus inhibitor dialog box will open up as shown below Enter 0 01 mM for I inhibitor concentration 10500 cal mole for AH as determined in the previous example enter 67 8 sec for K a and 0 084 mM for Ky c M2InhibitoR AA cep cal mole sec KM mh Cancel oF
16. a system with two identical sites the first ligand has two empty sites at which to bind while the second ligand has only one The binding constants reported in the parameter box are phenomenological binding constants which include effects from degeneracy To remove these effects and compare intrinisic binding constants K at each site refer to eq 19 in the Appendix Binding of multiple ligands to transition metal ions The binding of multiple ligands to transition metal ions is another example where the sequential binding model is appropriate and where all sites are identical in the apo metal ion The sample file Persson7 ite contains data on the binding of four Br to Cd to 64 MAU130010 Rev D Lesson 7 Advanced Curve Fitting form CdBry For practice call up the file and do curve fitting to obtain binding parameters for each of the four bromide ions using the Sequential Binding Sites model The concentrations of both Br and Cd are correct as contained in the file Convergence occurs you must click on 100 Iter button several times without operator selection of initial parameters but you may want to try to improve the initialization for practice m Persson7_NDH Persson7_B Data Persson7_ NDH Model Sequential Binding Sites Chi 2 DoF 50 98 Kl 2 34E3 37 AH1 318 7 6 33 ASI 14 4 K2 114 5 0 AH2 4565 125 AS2 24 6 K3 2 22E3 99 AH3 1119 141 AS3 19 0 K4 25 0 0 86 AH4 6690 75 1 AS4 15 9 w Cc a9 w O
17. button you will be prompted to enter a final Y position in kCal mole The end point of each plotted data set will be placed at that position and the rest dataset will be offset proportionately You would typically use 0 an the final Y position When you click this button you will be prompted to enter a constant kCal mole that will be used to subtract from all datasets plotted in the ADeltaH graph When you click this button the heats from a control experiment can be subtracted from all data sets that are plotted in the ADeltaH graph When you click this button all data sets are moved out of the graph layer the cursor will change to the data reader tool then the first data set will be moved back into the graph You may click once to see the y axis position of the data reader tool When you click twice or press enter the end point of the data set will be moved to that y position the data set will be removed from the graph and the next file of the series will be plotted in the graph and you may repeated the process for all data sets that were originally plotted in the graph When you click this button all data sets are moved out of the graph layer the cursor will change to the data reader tool then the first data set will be moved back into the graph Double click at the point on the graph where you want the line to begin then double click at the point where you want the line to end A straight line will be created between the two p
18. e Click on the Read Data button Click on the scroll down arrow of the Files of type text box and select Dissociation IT file type Loak in E samples a A ce Analysis Ay MT rhibitorO 5 ite C Data a H1 Nolnhibitor itc _ Graphing a Mal nhibitorO ite _ Programming a MeN olnhibitor itc a Dissociation TC a Persson ite Febur 0 ite a Rnahhh ite Files of type Dissociation it Cancel ITC Data 1t Omega Data 1 e Navigate to the C Urigin U Samples folder and select Vissociation itc trom the rile Name list and click OK MAU130010 Rev D T3 ITC Tutorial Guide 76 Similar to the normal ITC files the Dissociation file is read and plotted as a line graph in the RawlTC window in units of ucal second Vs minutes Origin then automatically performs the following operations 1 2 3 Selects Auto Baseline routine Each injection peak is analyzed and a baseline is created Selects Integrate All Peaks routine The peaks are integrated and the area ucal under each peak is obtained Opens the DeltaH window The difference for this model is that Origin then plots the area ucal injection Vs equivalent monomer concentration mM pAg _ O x 1 Disecado Dn One Set of Sites kalm ob ot hle cta t G G G E Gd 7 6 3 3 1 0 1 z 14 Egulua koe Monomer Concentra don im W ss LLU At this point you may wish to check the For Data Dissociation
19. for each site Using a model for independent sites 1t would be extremely difficult to obtain a unique fit for more than two sets of sites which is why no fitting model for three sets of independent sites has been included in this software As shown above for the Persson7 itc file the sequential model is capable of providing a unique fit even for systems with four binding sites if the K and or H values are sufficiently different for each site Thus for some multi site systems the model of sequential binding may be the only choice available for providing a unique phenomenological characterization of binding parameters Enzyme substrate inhibitor Assay There are two different methods described below for carrying out an enzyme assay These methods are discussed in the Appendix where the appropriate equations are included Both methods assume that no significant product inhibition occurs In Method 1 an enzyme solution is in the sample cell and the experiment consists of a single injection of substrate solution into the sample cell Immediately after injection the calorimeter baseline shifts prominently to reflect heat effects which occur from the decomposition of substrate as it comes into contact with the enzyme because of the finite response time of the instrument it takes a few minutes before the calorimetric signal becomes equilibrated with the actual heat from substrate turnover Eventually after all substrate has been reacted the basel
20. how to open the worksheet associated with a particular data plot copy paste the data export the data to an ASCII file and import ASCII data Reading Worksheet Values from Plotted Data k Shortcut Select the New Project button Begin this lesson by opening the ITC Rnahhh ITC data file series as follows Select File New Project A new Origin project opens to display the RawITC plot window Click on the Read Data button The File Open dialog box opens with the ITC Data IT file name extension selected If you have not previously Set Default Folder to the samples folder then navigate to the C Origin70 samples folder Select Rnahhh from the file name list and click OK As you saw in Lesson 1 Origin plots the Rnahhh data as a line graph in the RawITC plot window automatically creates a baseline integrates the peaks normalizes the integration data and plots the normalized data in the DeltaH plot window As a result the following eight data sets are created rnahhh_ dh Experimental heat change resulting from injection i in ucal injection not displayed rnahhh_ mt Concentration of macromolecule in the cell before each injection i after correction for volume displacement not displayed rnahhh_ xt Concentration of injected solute in the cell before each injection not displayed rnahhh_injv Volume of injectant added for the injection i rnahhh_ndh Normalized heat change for injection 7 in calories pe
21. note that the original K1 value of ca 3 2 x 10 has reappeared and that the new Chi Sqr value is only slightly smaller at ca 31000 Thus the two fits having very different fit parameter values have approximately the same Chi Sqr This is because these data show rather large scatter from the smooth theoretical fit curve and are therefore not capable of defining precise values for the fitting parameters You may run across other instances in your own experiments when fitting parameters are even more poorly defined than for OTFFe3 DH The most likely situation for this to occur 1s with two sets of sites where K1 and K2 values are less than 10 fold different It is even possible that the set of best fit parameters may be quite different depending on the initialization parameters which are used to start the fit 1 e the curve fitting routine can become trapped in a local minimum for ch1 2 and be unable to find the global minimum You can usually detect this by starting with several different sets of initialization parameters to see if you arrive at the same final minimum with nearly the same fitting parameters When you are satisfied with the fit click on the Done button in the dialog box to paste the fitting parameters to the Results Window the plot window and end the fitting session Go to the ITC menu and reselect the default option Ligand is in Syringe MAU130010 Rev D Lesson 7 Advanced Curve Fitting Ls ovea NDH Ovted_Fi
22. page 49 for details To view the Results Log When you save an Origin project the contents of the Results Log is saved with the project 16 Origin automatically routes most analysis and fitting results to the Results Log a sub window of Origin s Project Explorer In most cases when results are output to the Results Log it opens automatically although it may be positioned out of view docked to the lower edge of the workspace However to manually open and Close the Results Log click the Results Log button on the standard toolbar Opening and closing the Results Log only controls its view state You do not lose results by closing the log When the Results Log first opens it displays docked to the lower edge of the workspace You can dock it to any other edge or display it as a window in the workspace To prevent the Results Log from docking when positioning it it as a window press CTRL while dragging Each entry in the Results Log includes a date time stamp the window name a numeric stamp which is the Julian day the type of analysis performed and the results Please refer to the Origin User s Manual or on line help for more information about the Results Log and Project Explorer MAU130010 Rev D Lesson 1 Routine ITC Data Analysis and Fitting Creating a Final Figure for Publication To create a final figure for publication select Final Figure from the ITC menu The ITCFINAL plot window opens This window contains two
23. related graphs The top graph shows raw data in terms of ucal second plotted against time in minutes after the integration baseline has been subtracted The bottom graph shows normalized integration data in terms of kcal mole of injectant plotted against molar ratio The two X axes are linked so that the integrated area for each peak appears directly below the corresponding peak in the raw data Time min 20 ucal sec C S O eck 2 O lt lt 1 0 Molar Ratio Note The user should understand that the raw data in the upper frame of the ITCFinal template is the original raw data after the integration baseline has been subtracted from it Once this subtraction has been made by creating the ITCFinal figure there is no way to recover the original raw data except by starting a new project and calling in the raw data file again since the subtracted data has been stored under the original filename and the original integration baseline replaced by the Y 0 baseline If you modify the integration data or the fit curve in the DeltaH window or the raw data in the RawITC window simply select Final Figure again to update the ITCFINAL window with your changes Note that the top graph in the ITCFINAL window still includes the integration baseline at Y 0 You may wish to remove this baseline before printing the graph MAU130010 Rev D L7 ITC Tutorial Guide To remove the baseline from the raw data Origin has draw
24. shown below Enter the following values obtained from the first experiment Ng 0 993 Kg 216000 AHg 11700and Cg 0 887 then click OK The NonLinear Curve Fitting dialog box for the Competitive Binding model will open MaE E Ne J 393 sites mole Ke 216000 Wi T 11700 cal mole Ca es ahi Cancel g e Click the 100 Iterations button one or two times to ensure that Ch1i 2 is no longer decreasing then click OK Competitiv_NDH Com petitiw_EB 78 Lesson 7 Advanced Curve Fitting Simulating Curves To simulate You may want to simulate titration experiments without actually going through the fitting routine The simulated curve may or may not be related to actual data which you have obtained To simulate data there must be some ITC results in computer memory either raw data called up or an Origin project that contains data but these results need not be related to the simulations you carry out The data in memory needs to contain at least as many data points or number of injections as the curve you wish to simulate Note for proper simulation you must use a data file that had all injections of the same volume so do not use a file which used a preliminary 1 injection of a different size a fit curve Exit the fitting session and start a new project by selecting File New Project or click on the New Project button from the menu Click on the Read Data button in the RawITC wi
25. the standard ITC data as described in the previous lessons of this tutorial These buttons are fully functional on data collected from the AutoITC but only operate on a single data set Auto ITC Buttons Apart from the buttons associated with the standard ITC there is a group of buttons labeled AutoITC that perform data manipulations on multiple ITC files Examples of the use of each button are presented in the following pages Button Name Function Subtract Mode When you click this button the Control Baseline Subtraction dialog box will pop up providing 4 different means to adjust the experimental datasets to minimize the heats of dilution One Set of Sites When you select this button the One Set of Sites fitting model will be applied to all data sets plotted in the AdeltaH graph The calculated fitting parameters will then be printed in a summary table Two Sets of Sites This button will apply the Two Sets of Sites fitting model to all data sets plotted in the active layer then prints the fitting parameters in a summary table Raw Data Window This button provides a quick method to return to the ARawITC window MAU130010 Rev D g1 ITC Tutorial Guide 92 To check and edit concentration values The concentration values for the cell and syringe are usually entered into the data files when setting up the experiments in VPViewer You should always check that concentration values are correct for each experiment Incorrect va
26. to Lesson 5 which describes how to open worksheets from plotted data copy and paste data and export data to an ASCII file Now would be a good time to save the area data the RNAHHH integration results as a separate data file We will use this data again in Lesson 4 when we subtract reference data To save area data to a separate file e Select Window DeltaH to make DeltaH the active window Alternatively you may press and hold the Ctrl key and press the tab key to scroll through Origin s open windows e Click on the Save Area Data button Origin opens the File Save As dialog box with Rnahhh DH selected in the File name text box e Select a folder for the file and click OK Before fitting a curve to the data it is a good idea to check the current concentration values for this experiment To edit concentration values e Click on the Concentration button in the DeltaH window e A dialog box opens showing the concentration values for the RNAHHH data For Data Rnahhh Cancel C in Syringe mM 21 16 C in Cell mb 0 651 Inject ol ul 4 Cell ol ml 1 345 Click OK or Cancel to close the dialog box You should always check that the concentration values are correct for each experiment Incorrect values will negate the fitting results If you need to edit the concentration values simply enter a new value in the appropriate text box The concentration and injection volume values which appear initially are those
27. which the operator enters before the experiment starts The cell volume is a constant which is stored in the data collection software This value is read by Origin whenever you call up an ITC data file 12 MAU130010 Rev D Lesson 1 Routine ITC Data Analysis and Fitting Before you proceed to fit the data you may want to manually adjust baselines or integration details These procedures are discussed in Lesson 2 but here we will simply accept the computer generated results Curve Fitting Origin provides six built in curve fitting models One Set of Sites Two Sets of Sites Sequential Binding Sites Competitive Binding Dissociation and Enzyme Assays To invoke one of these models click on the appropriate button in the DeltaH window The following describes the basic procedure for fitting a theoretical curve to your data See Lesson 7 for advanced curve fitting lessons and the Appendix for a discussion of fitting equations To fit the area data to the One Set of Sites model e Click anywhere on the DeltaH plot window to make it the active window Or select DeltaH from the Window menu e Click on the One Set of Sites button A new command menu display bar appears The Fitting Function Parameters dialog box opens there are two modes for the Fitting Sessions dialog box basic and advanced see page 58 for more information showing initial values for the three fitting parameters for this model N K and H Origin initializes the
28. will be assumed throughout that the macromolecule M is in the cell at an initial bulk concentration M H moles liter before the first injection and the ligand X to be injected is initially at zero concentration in the cell The working volume cross hatched area below of the lollipop shaped cell is V the size of the ith injection is AV and the total liquid which has been injected at any point during the experiment AV is simply the sum of the individual AV for all injections AV pesca wis At the beginning of an experiment both the cell and the long communication tube are filled with macromolecule solution but it is only that contained within V that is sensed calorimetricallly Because of the total fill nature of the cell each injection acts to drive liquid out of the working volume and up into the inactive tube as shown by the darkened portion representing AV Thus the concentration of macromolecule in V changes a small amount with each injection since the total number of moles of macromolecule initially in V i e M times V at the beginning of the experiment is later distributed in a larger volume V AV Since the average bulk concentration of macromolecule in AV is the mean of the beginning concentration M and the present concentration M in the active volume then conservation of mass requires that l M Y MV M M AV 1 so that M M 2 2V Using similar reasoning it is easily shown tha
29. window A second way to recover the bad point without reintegrating is to click on the Concentration button and then click OK Even if you did not change the concentration in the dialog box Origin goes back to the worksheet and normalizes on the concentration again which then restores the deleted point MAU130010 Rev D Zil ITC Tutorial Guide This page was intentionally left blank 28 MAU130010 Rev D Lesson 4 Analyzing Multiple runs and subtracting Reference Lesson 4 Analyzing Multiple Runs and Subtracting Reference Origin allows you to open multiple runs of ITC data into the same project and subtract one run from another The heat of ligand dilution into buffer can thus be subtracted from the reaction heat by performing the control experiment of injecting into a buffer solution and subtracting this reference data from the reaction heat data In order to subtract the reference injections you must have both the sample and reference area data in memory This lesson shows you how to read two area data files into Origin and subtract one from the other In the first example below you will read two area DH data files from disk In the second example you will work directly with two raw ITC ITC data files This second example also illustrates some helpful procedures for dealing with difficult data Two reference data files BUFFER DH and FEBUF10 ITC have been included in the samples subfolder for your use with this lesson B
30. you may click OK or Cancel Note Once you apply the Time Constant correction the original data is replaced in the active window by the corrected data OK Cancel Response lime sec 18 5 The response time of your instrument is dependant on the FeedBack gain mode used during the experiment Typical values for the relaxation time are ca 18 5 seconds for high gain 51 seconds for low gain and 72 seconds for no active feedback none gain mode The actual values are measured for your instrument and stored in the VPViewer ini file Note Older instruments may not have these values stored please contact MicroCal if you wish to save the values for your instrument Zero axis e Select the Zero Y Axis button The cursor will turn to a cross hair allowing you to double click a point as indicated in the graph on the previous page to place that point at y 0 Choose a point on the flat part of the baseline before the injection is made You may also pick a point to Zero X axis The point where the injection was made should be used to zero the X axis where you see the first small deflection in the baseline Note you may single click a point then use the arrow keys to move the point then press enter to select that point Calculate Rate e Click the Calculate Rate button The Rate will be calculated and plotted in a new window in a graph of the Rate millimoles sec Vs S mM i e the concentration of the unreacted substrate in the cell Tw
31. you may select either the High Point or Low Point check box then when you click OK Origin will search each data set and delete all data before the corresponding High or Low point in the data The data will then be plotted on the graph with the beginning data removed Alternatively you may select the Remove selected range option When you select this option button and click OK each data set will be sequentially plotted on the graph with two data markers displayed on the trace You may click and drag on a marker to move it to your desired point on the trace then double click or press enter to set the point All data between the two markers will be removed from the graph and eliminated from future analysis e Click on Remove Bad Data button from Single Injection group e Select the Remove data before option then put a check into the Low Point box and click OK The graph should look as shown below IG 67 Time imin Hormalize Data To Normalize Data points MAU130010 Rev D Hint When moving a data marker you may press the space bar to increase the size of the cross hair Lesson 7 Advanced Curve Fitting Click the Normalize Data button from the Single Injection group After clicking the Normalize Data button the concentration continuum will be calculated and the normalized heat will be plotted in a new window named DeltaH The data is now in the form of conventional ITC normalized data and may be fit with the methods des
32. 10_ndh area data replaces Rnahhh_ndh in the DeltaH window You need to plot both area data sets into layer 1 To show both area data sets in layer 1 e Double click on the layer 1 icon a in the DeltaH plot window The Layer Control dialog box opens e Select Rnahhh_ndh in the Available Data list then click on the gt button Rnahhh_ndh joins Febuf10_ndh in the Layer Contents list e Click OK Rnahhh_ndh joins Febuf10_ndh in the DeltaH plot window The axes automatically rescale to show all the data Your plot window should now look like the illustration below Loe Fenutio Nox E FAnahnhhh_HOH E m t 1 p 2 u E E As we discussed earlier in this lesson page 33 you should now check that the ligand concentrations for both data sets are identical Make each data set active in turn then MAU130010 Rev D 317 ITC Tutorial Guide click on the Concentration button in the DeltaH window and check that value in the C in Cell mM field for Febuf10_ndh is identical to that value for Rnahhh ndh In this case you will find that the two values are the same Notice that Febuf10_ndh shows only twelve injections while Rnahhh_ndh shows twenty How do you subtract one data set from another when the number of injections doesn t match The quick and dirty way is to subtract a constant A more precise method would be to fit a straight line to the reference data then subtract the line Let s look briefly at each method To s
33. ASCII file from the Files list Enter the appropriate Initial Graph button from X Value 0 for RnahhhBASE dat and Increment in X 28 25287 and click OK the Standard toolbar 48 MAU130010 Rev D Lesson 6 Modifying Templates Lesson 6 Modifying Templates The RawITC DeltaH and ITCFinal plot windows and all other plot windows in Origin are created from template files OTP file extension A template file is a file that contains all of the attributes of a plot window or a worksheet except the data The important thing about template files is that you can change a plot window and then save the changes into the template file for that window The next time you open the window it will include your changes Thus template files let you customize plot windows to meet your specifications You can change any of Origin s template files In this lesson we will edit both the DeltaH and ITCFinal plot windows then save the changes into the corresponding template file Though the changes we make will be minor you can actually change any property of a template For more information about customizing templates refer to the Origin User s Manual or press the F1 key for Online Help Caution In this lesson you will be modifying plot window templates that are basic to Origin s operation In the unlikely event that you make a mistake you are unable to correct simply copy the original template file from the Custom folder of the installation CD ROM
34. Biochemistry 277 260 266 MicroCal would like to thank Dr Sigurskjold for providing this fitting routine VIII Single Injection Method Creating New Worksheet The raw data after time constant correction Fourier filtering baseline subtraction and eliminating inappropriate data are used to form a new worksheet which is modeled after the existing worksheet used with multi injection binding data 1 Input Parameters In addition to the raw data parameters AP ucal sec from the Y axis and time t sec from the X axis of the corrected raw data the known parameters are the injection rate R ul sec stored in header total delivery volume V ul stored in header active cell volume Veen ml the initial macromole concentration in the cell M mM before any dilution the dilution factor dy for the macromolecule solution resulting from autosampler loading the initial ligand concentration in the syringe X before any dilution the dilution factor dx for the ligand concentration resulting from loading The approximate values are 0 95 for dy and 0 91 for dy and the values will be independent of the instrument which is used in the experiment 2 Point numbering In the existing Origin worksheet for multiple injections the rows are numbered 1 2 3 according to the injection number In the worksheet for single injection experiments the numbering corresponds to the data point number The data points will be spaced at one for each filter
35. H 1 049F4 Rate millimoles I sec S mM 70 MAU130010 Rev D Lesson 7 Advanced Curve Fitting Enzyme Assay Method 1 Substrate Plus Inhibitor In the presence of inhibitor I it is necessary to enter previously determined values of Kyat Km and H as determined in the previous example and use K as the only fitting variable Begin this example by opening the ITC data file M1Inhibitor0175 itc as follows E A ie ES e Select File New Project a A new Origin project opens to display the RawITC plot Method 1 Substrate only window M e Click on the Read Data button Method 2 Substrate only Method 2 Substrate plus inhibitor Click on the scroll down arrow of the Files of type text box and select Enzyme Assay IT file type Cancel e Navigate to the C Origin70 Samples folder and select M1Inhibitor0175 ITC from the File Name list and click OK The Enzyme Assay dialog box will open allowing you to select one of the four models e Select Method 1 Substrate plus inhibitor and click OK i Ix The Method 1 Substrate plus inhibitor dialog box will 1 o5 ant open up Enter 0175 mM for I inhibitor concentration 10500 cal mole for AH as determined in the previous AH ioo cael example enter 81 9 sec for K a and 076 mM for K mg a K wu Click OK Note Your values for K y and K may be slightly different depending on where the data was truncated EMm ory r
36. H and lower ITCFinal templates 1 e data with ndh extension are normalized on moles of injectant If you ever wish to view the experimental integrated heats in ucal per injection then double click on the Layer dialog box and move the _ndh file out of the Active data and move the _ dh file into the Active data To complete the process you must double click on the Y axis tick labels and remove the factor of 1000 54 MAU130010 Rev D Lesson 7 Advanced Curve Fitting Lesson 7 Advanced Curve Fitting The model for one set of sites discussed in Lesson 1 will work for any number of sites n if all sites have the same K and AH If a macromolecule has sites with two different values of K and or AH then the model with two sets of sites must be used Whenever there are two sets of sites the automatic initialization procedure 1s rarely effective If the initialization parameters are extremely far away from best values then convergence to the best values cannot take place as iterations proceed In fact often the fit gets worse rather than better with successive iterations Therefore the user must get involved in arriving at initialization parameters before the iterations can be started An indication of poor initialization occurs when values for the K parameter become negative during the fitting procedure Fitting with the Two Sets of Sites Model The protein ovotransferrin has two very tight non identical sites for binding ferric 1ons one loc
37. Injection group of buttons BS esi The Control Baseline Subtraction dialog box will pop ae up Straight line Cancel Button Name Function Input final numerical Y position When you click this button you will be prompted to enter a final Y position in kCal mole The end point of plotted data set will be placed at that position and the rest of the data set will be offset proportionately You would typically use 0 an the final Y position Subtract a constant When you click this button you will be prompted to enter a constant kCal mole that will be used to subtract from all datasets plotted in the ADeltaH graph Subtract reference data When you click this button the heats from a control experiment can be subtracted from data set that is plotted in the ADeltaH graph Y axis shift When you click this button the cursor will change to the data reader tool You may click once to see the y axis position of the data reader tool When you click twice or press enter the end point of the data set will be moved to that y position Straight Line When you click this button the cursor will change to the data reader tool Double click at the point on the graph where you want the line to begin then double click at the point where you want the line to end A straight line will be created between the two points and extrapolated to subtract from all data points For this tutorial practice two different options to zero the baseline e Click on
38. MicroCal LLC 22 Industrial Drive East Northampton MA 01060 USA Phone 413 586 7720 Fax 413 586 0149 ITC Data Analysis in Origin Tutorial Guide Version 7 0 January 2004 ae A a Using Origin scientific plotting software to analyze calorimetric data from all MicroCal Isothermal Titration Calorimeters Time min 6 10 20 30 40 50 60 70 80 0 100 110 Apa _ ce 5 A hjen Qo 2 o at b 1 Ultrasensitive Calorimetry RUMLA NCNIA MicroCal MAU130010 Rev D Table of Contents Table of Contents Introduction to ITC Data AnalysiS eeccoossssssccccocssssssececcosossssccccoocssssecccessssssseseeeesso 1 Getine Stared ccia E S 3 Systemi FR CUI CNC INS asur E ln re ee T E O 3 Installine Origin Single User License iiss isaciteecacvaas ihe areal ee 3 Installing Origin for ITC Custom Disk oi isc64 iss ence ct veian Gieartea are e G 3 Recisterime wathy On cits ac aces caer eect ora eas on eee see oe 4 SaN OO cof ancy occ A atau ea seehouestdetea eas cedduce send ea esiaaeaueeeans 5 Monu IS VClSio5225c ii cos he ducse bcetameen a taped tscndsadaesuted easabeeaeehandaddeandal dou teanueaeeanteaseimcnas 5 Simultaneously Running DSC ITC and PPC Configurations cccccceeessseesssesssesssenssseeees 6 MEW MOG casia N A das ouuad sdvunsehmawaddduded aidouenand auenaangeadeuacats 6 A NOE About Data TIMPO a aa a a E 7 Opening and Analyzing Previous Versions of Origin ORG Documents
39. Note About Units Raw data in ITC files are stored in terms of ucal second versus seconds as you will see if you open a worksheet containing raw data The integrated area under the peaks data are stored in the worksheet column DH in units of ucal per injection This is apparent if you open a worksheet containing integrated data However for curve fitting and for better publication presentation both the DeltaH and ITCFinal plot windows present the integrated heat data as H kcal per mole of ligand injected which is more closely related to the fitting parameter H calories per mole of ligand bound That is H will be nearly equal to H except for the factor of 1000 in early injections when nearly all of the ligand added is bound The factor of 1000 is achieved by entering that factor to the Y axis tick labels as discussed earlier in this lesson Also both the RawITC plot window and the upper graph in the ITCFinal plot window display X axis values in minutes while the stored values are in seconds In this case the X axis labels are factored by 60 as we discussed for the RawITC window earlier in this lesson If you double click on the top X axis labels in the ITCFinal window you will notice there is a factor of 60 in the Divide by Factor text box just as there was with the RawITC window Again this factor setting is saved as part of the ITCFinal template MAU130010 Rev D 93 ITC Tutorial Guide The Y axis data plotted in the Delta
40. O AE EA E A EEA 113 DaKa o E E E E E A E E E A E E E E se 115 MAU130010 Rev D Getting Started Introduction to ITC Data Analysis Origin is a general purpose scientific and technical data analysis and plotting tool In addition Origin can carry add on routines to solve specific problems Analyzing isothermal titration calorimetric 1 e ITC data from the OMEGA MCS or VP ITC instruments is one such specific application This version of Origin includes routines designed to analyze ITC data Most of the ITC routines are implemented as buttons in plot window templates designed specifically for the ITC data analysis software Some routines are located in the ITC menu in the Origin menu display bar This tutorial will show you how to use all of the ITC routines Lesson provides an overview of the ITC data analysis and fitting process and should be read first The subsequent lessons each look in more detail at particular aspects of ITC data analysis and may be read in whatever order you see fit If you are unfamiliar with the basic operation of Origin you may find it helpful to read through OriginLab s Origin Getting Started Manual particularly the introductory chapter Note that this ITC tutorial contains information about Origin only in so far as it applies to ITC data analysis If you have questions or comments we would like to hear from you Technical support and customer service can be reached at the following numbers Toll Free
41. Subtract Options button and select Straight line option The cursor changes to the data reader tool 84 MAU130010 Rev D Lesson 7 Advanced Curve Fitting e Double click the data reader tool on the near the end of the curve about 30 minutes and then double click the data reader tool on the curve at 0 minutes e Origin quickly creates a straight line which is extrapolated and subtracted from all data points Your plot should look like this i Oneingo1RAW_ooll 0 5 0 0 0 5 2 0 ucal sec 2 5 3 0 3 5 4 0 0 00 8 33 16 67 25 00 33 33 Time min Now Click on the Read Data button and re open the OnelInj001 sim data file Click on Subtract Options button and select Input final numerical Y position option Enter 0 in Value window Click OK Your plot should look like this Use this data set for subsequent curve fitting 16 67 Time min Hemove Bad Data To Remove Bad Data MAU130010 Rev D 85 ITC Tutorial Guide 86 Data at the beginning of the experiment will be distorted by the time constant correction and there may be Remove Bad Data ra Beh extraneous data points after the injection was completed The Remove Bad Data button will simplify the task of excluding these data points from subsequent analysis At the start of the injection typical experiments will have a high point exothermic reaction or a low point endothermic reaction When you select the Remove data before option
42. TC Tutorial Guide ASCII Export into RnahhhBaseE DAT Cancel ip Include Column Names M Include Column Labels Export Selection e You may format the output of this ASCII file Please refer to the Origin User s Manual for more information about Exporting worksheet data This file may then be opened into any application that recognizes ASCII text files Importing Worksheet Data ASCII files can be imported directly into an Origin worksheet or plot window The basic worksheet Origin menu supports a number of additional file formats for importing data Lotus Excel dBASE LabTech etc while the menus for ITC or DSC Data Analysis support routine ASCII import To import an ASCII file into a new worksheet e Choose Worksheet from the New sub menu under the File menu A new Origin worksheet Data1 opens e Select the File Import ASCII command If you like you can select File ASCII Options This will allow you to set ASCII file import options The Import ASCII dialog box opens set to open a data file with a DAT extension e Double click on a file in the File Name list for example the RnahhhBASE DAT file you just exported The RnahhhBASE data imports into the worksheet To import an ASCII data file into a plot window m e Choose Graph from the New sub menu under the File menu bo Shortcut e Choose Import ASCII Single File from the File menu To create a graph click on the New e Select the rnahhhBASE dat
43. This will correct any problem that may arise Modifying the DeltaH Template The DeltaH template shows units of kilocalories mole of injectant along the left Y axis The scale for this axis 1s actually defined in terms of calories mole of injectant but the axis is factored by 1000 to yield units of kilocalories mole The right Y axis labels for the DeltaH template are hidden from view In the following example we will modify the template so that the right Y axis labels are visible We will then factor the labels by 1000 so they will be identical to the left Y axis labels and then save these changes into the DeltaH template file To open the DeltaH plot window e If you are continuing from a previous lesson click on the New Project button from the Standard toolbar or select Project from the New sub menu under the File menu to create a new project e Click on the Read Data button in the RawITC window The File Open dialog box opens with the ITC Data ITC file extension selected e Navigate to the C Origin70 Samples folder and open any ITC data file for example FEBUFI10 ITC The DeltaH template opens to show the normalized area data MAU130010 Rev D 49 ITC Tutorial Guide m Febuf10_NDH kcal mole of injectant Molar Ratio To show the right Y axis units e Double click on the right Y axis in the DeltaH window Altrnatively select Format Axis Y Axis The Y Axes dialog box opens Y Asis Layer 1 Tick Labels Min
44. W appended i e OneInjo001 sim is read into a worksheet named OneInjOOIRAW 2 The time before the injection starts 60 seconds is subtracted from the x data so that the injection starts at t 0 and all data points are shifted to the left Note The x data before t 0 is removed from the worksheet but the data is still plotted in the graph for use in baseline subtraction 3 The data is corrected for the time constant of the instrument 4 The noise introduced by the time constant correction is filtered using the standard Fourier transform filter of Origin and a bandwidth of 15 data points 5 The corrected and filtered data is then plotted in the ARawITCsi window 96 MAU130010 Rev D Lesson 9 Other Useful Details To zero the baseline Subtract Options The next operation will be to zero the baseline the operation is similar to the Subtract Options see page 92 button used in removing dilution effects from the area data in the AdeltaH window for standard ITC data e Click the Subtract Options button from Single Control Baseline Thad t B x Injection the group of buttons The Control Baseline Subtraction dialog box f Input final numerical position will pop up Button Name Input final numerical Y position Subtract a constant Subtract reference data Y axis shift Straight Line C Subtract a constant CO Subtract reference data Co 4 axis shift C Straight line zm Function When you click this
45. a are plotted in the DeltaH window as a scatter plot called Rnahhh_ndh Rnahhh_ndh shows area data as kilo calories per mole of injectant plotted against molar ratio m Rnahhh_NDH kcal mole of injectant 0 0 0 5 1 0 1 5 2 0 Molar Ratio The Rnahhh_NDH normalized area data 30 MAU130010 Rev D Lesson 4 Analyzing Multiple runs and subtracting Reference e Return to the RawITC template and repeat the above steps to open the reference data file Buffer dh Buffer dh is also located in the samples subfolder A new plot Buffer_ndh replaces Rnahhh_ndh in the DeltaH window m Buffer_NDH kcal mole of injectant 0 0 0 5 1 0 1 5 Molar Ratio The Buffer_ndh normalized buffer data When you open the second ITC data file Buffer_ndh the Rnahhh_ndh data are cleared from the DeltaH plot window The Rnahhh_ndh data have not been deleted from the project but are simply removed from the window display Since the data are not deleted they can be retrieved from memory and replotted To show both the sample and the reference area data Shortcut to Layer 7 Control Right e Double click on the layer 1 icon at the top left corner of the DeltaH window click on any white The Layer Control dialog box opens space between the axis and select e Click on Rnahhh_ndh in the Available Data list then click on the gt button Layer Contents Rnahhh_ndh copies to the Layer Contents list MAU130010 Rev D 31 ITC Tutorial Guid
46. ach time you open an ITC raw data file series Origin creates eight data sets These eight data sets will always follow the naming convention shown below that is the name of the ITC source file followed by an identifying suffix injection number is indicated by the row number i Double click on the layer icon to view the available data sets rnahhh_ dh rnahhh mt rnahhh xt rnahhh_injv rnahhh_ndh rnahhh xmt rnahhhbase rnahhhraw_ cp Experimental heat change resulting from injection i in ucal injection not displayed Concentration of macromolecule in the cell before each injection i after correction for volume displacement not displayed Concentration of injected solute in the cell before each injection not displayed Volume of injectant added for the injection i Normalized heat change for injection 7 in calories per mole of injectant added displayed in DeltaH window Molar ratio of ligand to macromolecule after injection i Baseline for the injection data displayed in red in the RawITC window All of the original injection data displayed in black in the RawITC window l Two temporary data sets are also created rnahhhbegin contains the indices row numbers of the beginning of an injection rnahhhrange contains the indices of the integration range for the injection MAU130010 Rev D 11 ITC Tutorial Guide Origin creates three worksheets to hold these data sets To open these worksheets refer
47. ak If you wish to keep the same expanded Y axis limits for integrating other peaks then double click on the Y axis to bring up the Y Axes Dialog Box see below Click on the Scale tab then in the lower left corner of the dialog box change the Rescale option from Normal to Manual and click OK Now the Y axis will maintain these limits and will not rescale when you proceed on to adjust integration for other peaks Contact MicroCal if you wish to permanently change the default values for the y axis scaling Y Axis Tick Labels Minor Tick Labels Custom Tick Labels Scale Title amp Format Grid Lines Break Selection C Increment 0 5 HH Horizontal To 20 5 t Major Ticks 3 Type Linear Minor Ticks 1 Bescale Jia Tore First Tick L Cancel e Once the baseline is where you want it you may press the escape key or the Enter key to set the baseline The data points will disappear and the cursor will change from the cross hair to the pointer tool so you may adjust the integration range If the MAU130010 Rev D 23 ITC Tutorial Guide integration range is already set you may click on the Integrate button and click on an arrow key to show an adjacent peak To adjust the integration range e Ifthe baseline data points are still visible double click on any data point or press the Enter key or press the Esc key The data points will disappear and the cursor will change from the cross hai
48. alculate AH using the formula on page 110 in the appendix Method 1 Substrate only E Ea cal mole Cancel Click Cancel The data file will be read in and plotted in a new window as shown MAU130010 Rev D 67 ITC Tutorial Guide fay Origin 7 UNTITLED EnzymeAssay z File Edit View Graph Data Math ITC Tools Format Window Help Snes 2S 2 oe ses ass H a sss Saul M1 NolnhibiRAW_cp ee Calculate Rate Piucalec 0 500 1000 1500 2000 2500 Time sec B nn wal AO A S s ESS key to change size Concentration The concentration and injection volume values which appear initially are those which the operator enters before the experiment starts The cell volume is a constant which is stored in the data collection software This value is read by Origin whenever you call up an ITC data file You should always check that the concentration values are correct for each experiment incorrect values will negate the fitting results If you need to edit the concentration values simply enter a new value in the appropriate text box e Click the Concentration button The Concentration dialog box will open showing the concentration values and the cell volume The values are correct for this example so you may click OK or Cancel For Data Mi Nolnhibi OK Cancel C in Syringe mM in C in Cell mM 7 5E 6 Cell Vol ml 1 26 68 MAU130010 Rev D Lesson 7 Adv
49. allows Na Ka and AH to be determined from results of the competitive experiment When designing a competitive experiment the total concentration of the competing ligand B iot should be selected so that K KIB ho 10 10 M where Ka is the estimated value of K4 This insures that the apparent binding constant in the competitive experiment will be in the best window 10 to 10 M to be easily measured by ITC In the following example results from a conventional non competitive experiment have already been analyzed to obtain the parameters Ng 0 993 Kp 21600 M and AHp 11700 cal mole The data from the competitive experiment have been saved in an area data file named Competitive dH which will be analyzed below Begin this lesson by opening the ITC data file Competitive dH as follows e Select File New Project A new Origin project opens to display the RawITC plot window e Click on the Read Data button Click on the scroll down arrow of the Files of type text box and select Area Data DH file type e Navigate to the C Origin70 Samples folder and select Competitive dH from the File Name list and click OK The Competitive dh file opens the data are normalized on concentration and plotted in the DeltaH window MAU130010 Rev D Ta ITC Tutorial Guide Competitiv_NDH kcal mole of injectant 1 5 Molar Ratio e Click the Competitive Binding button and the dialog box will open as
50. anced Curve Fitting Apply Time Constant When substrate is injected into the enzyme solution substrate decomposition starts to occur immediately However as can be seen in the figure above it takes approximately one minute after the injection before the baseline has reached the position where it reflects the full amount of heat which is being released in the cell This lag is caused by the finite response time of the instrument There are two software procedures designed to reduce the effect which instrument response time exerts on final parameters obtained from the data The first procedure is activated from the Apply Time Constant button Knowing the actual time constant for the instrument determined by MicroCal before shipment and stored in Origin the experimental data are mathematically corrected to remove the effect which this time constant exerted on the experimental data When this operation is carried out the old data is transferred out of the active window and the corrected data appears in the active window The second procedure is activated from the Truncate Data button Here the operator is able to remove that portion of the data immediately after the injection where distortion remains even after the time constant correction e Click the Apply Time Constant button The time constant dialog box will open allowing you to check and edit the time constant for your instrument The value of 18 5 is correct for high gain feedback mode so
51. and calculating mean value for 93 and injection time spacing 36 41 42 and opening multiple runs 29 33 subtracting a constant 38 39 subtracting a straight line 39 40 with a different number of injections than reaction data 36 41 System requirements for Origin 3 T Templates defined 49 DeltaH 18 21 52 ITCFinal and creating a final figure for publication 17 19 modifying and saving 49 52 units used to display data in 53 54 Text annotations formatting 15 16 MAU130010 Rev D repositioning 15 Time Constant 69 Tolerance Text Box 59 Two Sets of Sites fitting equations 97 98 fitting procedure 55 58 U Units used in default templates 53 54 Index Vv View Mode and available types 6 W Weighting Method 60 Window view mode 6 Windows Restore button 6 9 set as active 46 Worksheet copy and paste worksheet data 45 46 MAU130010 Rev D LA
52. and holding the Ctrl key then pressing the Tab key or selecting RawITC from the Window menu Double click on the bottom X axis tick labels or select Format Axis X Axis The X Axis dialog box opens Click on the Tick Labels Tab Select Bottom from the Selection List Box Enter 3600 in the Divide by Factor text box Since the worksheet X values for raw ITC data are in terms of seconds a factor of 3600 gives us axis tick label values in units of hours for this axis Set Decimal Places to 2 MAU130010 Rev D Lesson 6 Modifying Templates Scale Title amp Format Grid Lines Break Tick Labels Minor Tick Labels Custom Tick Labels Selection M Show Major Labels er T A Ippe Numeric Format Decimal 1000 Bolton Divide by Factor 3600 Font Default Arial M Set Decimal Places E Color Black Pretix Hl T Bold Point 18 Suffix This Layer zj Paint This Layer zj Color This Layer zj Bold This Layer zj e Click OK to close the dialog box e Finally double click on the X axis title it reads Time min to open the Text Control dialog box and edit the text to read Time hrs To save the changes into the RawITC template file e Just as we did for the DeltaH template select File Save Template As e Click Cancel in the Attention dialog box if you do not want to change the original RawlITC template If you click OK Origin will save the modified RawITC window as RawITC OTP A
53. ated in the N domain and one in the C domain The Origin area data FeOTF54 NDH shown below were obtained by titrating ovotransferrin with ferric ion Injections 1 5 titrate primarily the stronger N site injections 7 11 primarily the C site while injections 13 15 result in no binding since both sites are already saturated e Select File New Project or click on the New Project button to create a new project Click on the Read Data button in the RawITC window and select Area Data DH from the File of type drop down list go to the C Origin70 Samples folder and open FeOTF54 DH m Feotf54_ NDH m Z o s 2 amp e 2 je E O x 1 5 2 0 Molar Ratio MAU130010 Rev D oD ITC Tutorial Guide e Nowclick on the Two sets of Sites button in the DeltaH window Origin opens the NonLinear Curve Fitting Fitting Session dialog box but produces an attention dialog box and a very poor initial fit to the curve Click OK in the warning dialog to proceed Attention Ez al oa 5 Auto initialization failed ou must initialize the parameters yourself NonLinear Curve Fitting Fitting Session Feot4_NDH ae Category Function Action Options Scripts amom Farameter Value Warm Eror 5 7434 z 0 600 a I I I I Press Esc key to stop fitting iterations J C fei 5 g E o uu a E a Chi S qr Titer T00 Tieri 100 Simplex Iter 10 15 l 30 Enter fitti
54. ation P plus 2 times the aggregate concentration P gt Using the expression for the dimer dissociation constant to obtain P in terms of P2 leads to the equation Cli K P B 28 A similar expression applies to the solution in the syringe of fixed concentration C C K P 2 B 29 Syr Since C is known and C can be determined from C knowing injection volumes then P2 syr and P2 can be determined from equations 28 29 if K is assigned The heat released q when the i injection of volume dV is made into a fixed volume V cell will be dV q AH iise P Jaw aV AH iise P P Ja v a 30 The first term in equation 30 is the heat content of the aggregate contained in the injection volume prior to injection while the second term is the net heat content due to the difference in aggregate present in the cell dV before and after the injection The i a a factor in the final term is an effective volume which takes into account the displacement which occurs in a total fill cell see Appendix section I Assuming experimental parameters Vo dV and C y are known equations 28 30 are simultaneous equations which can be solved for q whenever values are assigned to K and AHaise Only the latter two parameters are varied during iterative fitting VII Competitive Binding Model Using conventional ITC methods binding constants from 10 M to 10 M can be measured most acc
55. ch in Co D CL S00 1000 1500 2000 2500 S000 Timetsec 74 MAU130010 Rev D Lesson 7 Advanced Curve Fitting e Click the Calculate Rate button As illustrated in the previous example the Rate will be calculated and plotted in a new window Vs the concentration of the injectant in the cell Two new button will be made available Truncate Data will allow you to eliminate the abberation at the start of the experiment Fit to Model will open Origin s Non linear Least Squares curve fitter to perform the fitting iterations In Method 2 you do not need to use the Truncate Data button e Click on the Fit to Model button The Fitting Sessions dialog box will open e Click on the 100 Iter button one or two times then click Done to end the fitting session K is used as the variable parameter during the iterative fitting and reported in the output parameter box You should get a value near 0076 mM Dimer Dissociation Model This model is intended for the analysis of heats of dilution data where the sample compound in the syringe has a tendency to form dimers 1 e AH 2 P 2P K I LP Multiple injections are made from the syringe and the resulting heats analyzed to give best values for the dissociation constant K and the heat of dissociation AH Begin this lesson by opening the ITC data file Dissociation itc as follows e Select File New Project A new Origin project opens to display the RawITC plot window
56. ch requires multiple injections In this single injection procedure only one slow continuous injection of ligand solution is made from the injection syringe into the macromolecule solution contained in the sample cell It should be pointed out that the binding parameters obtained from a well designed multiple injection experiment usually have higher degree of accuracy than the single injection experiment If the sample turnover rate 1s not a prime concern users should perform the multiple injection experiment for more precise binding parameters Correction of Raw Data for time constant of the instrument etc There are several operations which are automatically performed by Origin when the data file is opened a data corrected for the time constant of the instrument and b corrected data set is filtered using the standard Fourier transform filter in Origin 7 0 and a bandwidth of 15 data points The user then a sets the zero baseline from which the experimental data will be subtracted page 97 98 and b exclude distorded or extraneous data points prior to subsequent analysis page 99 Creating New Worksheet The raw data after time constant correction Fourier filtering baseline subtraction and eliminating inappropriate data can then be used to form a new worksheet which is modeled after the existing worksheet used with multi injection binding data To create SIM Auto ITC icon on desktop e Right click any Origin 7 icon on the des
57. click anywhere in the text box and select copy After you update to Origin 7 0 you may right click in any of the 7 0 templates and select paste 8 MAU130010 Rev D Lesson 1 Routine ITC Data Analysis and Fitting Lesson 1 Routine ITC Data Analysis and Fitting In this lesson you will learn to perform routine analysis of ITC data For reasonably good data Origin makes a very good guess on the baseline the range to integrate the injection peaks and the initialization of the fitting parameters These factors can be adjusted manually as described in the following chapters but it is not always necessary to do so Routine ITC Data Analysis A series of sample ITC files have been included for your use with this tutorial A typical file is designated RNAHHH ITC This file contains data from a single experiment which included 20 injections It is located in the samples subfolder of the origin70 folder Note The 1 file extension indicates an ITC file generated with the non Windows MicroCal data acquisition software The ITC extension indicates an OMEGA MCS ITC or VP ITC file generated with the Windows version of the MicroCal data acquisition software The two file types are identical except that the procedure for opening them differs slightly as described below To open the RNAHHH LITC ITC file e Start Origin for ITC as described in the previous section The program opens and automatically displays the RawITC plot window Along the le
58. copy then right click wher e you 4 Click on the ITCFINAL window or select ITCFINAL from the Window menu want to position the ITCFINAL becomes the active window Click once on a position in the graph where text label and select you want the parameter box to appear paste Click on the fitting parameters text label we had placed in the upper left corner of the window A colored selection square surrounds the text Select the Edit Copy command 5 Select the EDIT PASTE command The fitting parameters paste to the ITCFINAL window 18 MAU130010 Rev D To print the final figure To save the project and exit Click the Save Project button on the Standard toolbar Shortcut Lesson 1 Routine ITC Data Analysis and Fitting 6 We want to position the text label next to the integration data but first we need to reduce the size of the label Right click inside the text box then select Properties from the drop down menu to open the Text Control dialog box Select 10 or type 10 in the Size drop down list to reduce the point size to 8 Click OK to close the dialog box 7 Click and drag the label to position it next to the integration data as shown below ucal sec Cc 4 _ O SA I ae O O O x Time min 30 Data Rnahhh_NDH Model OneSites Chi 2 DoF 2856 N 1 02 0 0016 K 5 54E4 1 1E3 AH 1 361E4 29 8 AS 22 0 1
59. cribed in previous Lessons e g Lesson 1 One Set of Sites on page 13 If necessary you can modify macromolecule and ligand concentrations by clicking Concentration button and entering new values Page 12 E Ore LHH _ T p Ti La ba Moar Raia MAU130010 Rev D 87 Lesson 9 Other Useful Details Lesson 8 Autosampler Data Optional Accessory The Autosampler is an optional accessory for use with the ITC to allow multiple unattended experiments The AutoITC Origin module contains curve fitting routines that operate on multiple data files from the autosampler If you are not familiar with curve fitting of standard ITC data you should review the previous Lessons of this tutorial especially Lessons 1 2 3 amp 4 before starting this lesson Launching the ITC Autosampler Data Session ig The routines for Auto ITC files are located on a separate window named ARawITC from the regular ITC routines Mi LLC l E e To launch the Auto DSC double click on the MicroCal LLC Auto ITC icon on the Desk Top Or alternatively select Start Programs OriginLab MicroCal LLC Auto ITC When you start Origin the program automatically opens the ARawITC graph window To input the data Import Multiple ASCII Fa a a Raw data files from the autosampler are typically a series of data files of the form Lookin Sms A FamilyName001 ITC FamilyName002 ITC sees oe FamilyName003 ITC etc Th
60. ct LinearFit_Febufl0ND from the Available Data list then click on the lowermost gt button LinearFit_Febufl0ND copies to the Y2 text box Click in the operator box and type Available Data Y Rnahhh_NDH Y1 Rnahhh NDH Febufl ORAY cp AIEEE LinearFit FebuflOND i n YI tz Rnahhh DH operator F W Y1idata set Y 2 data Rnahhh_INJ or number Rnahhh Mt _ gt _ 2 LinearFit_Febuft0 Rnahhh NDH hi cael cane Help Febufl ORANGE Click OK Every point in LinearFit_Febufl0ND is subtracted from the corresponding point in Rnahhh_ndh The resulting data set is plotted as Rnahhh_ndh in the DeltaH plot window Note that the Febuf10_ndh reference data plot the original twelve injection points is not affected MAU130010 Rev D Lesson 4 Analyzing Multiple runs and subtracting Reference Fenutio NDH E Arahhh_WOH Linga ti FeouliOWo E m t a E 2 E Ta 10 Habr Aalia We will end this lesson with a note about injection timing You may have noticed the difference in injection time spacing between Rnahhhraw cp and Febufl0raw cp To make this difference more apparent you need to plot both raw data sets in the same plot window To plot both Rnahhhraw_cp and Febutl0Oraw_cp in the RawITC plot window e Click on the RawITC window to make it active or select RawITC from the Window menu e Double click on the layer 1 icon a in the RawITC window The Layer Control dialog box o
61. ct values Now change the concentration by multiplying the correct concentration in the cell by 2 and enter that incorrect value into the Concentration dialog box Do curve fitting again and you will see that the new incorrect value of n is exactly 50 of the correct value obtained using the correct concentration The values for binding constant and heat of binding will be the same in the two cases Single Injection Method The VP ITC is also capable of carrying out a complete binding experiment using only a single continuous injection as opposed to the normal procedure which requires multiple injections In this single injection procedure only one slow continuous injection of ligand solution is made from the injection syringe into the macromolecule solution contained in the sample cell It should be pointed out that the binding parameters obtained from a well designed multiple injection experiment usually have higher degree of accuracy than the single injection experiment If the sample turnover rate is not a prime concern users should perform the multiple injection experiment for more precise binding parameters Correction of Raw Data for time constant of the instrument etc There are several operations which are automatically performed by Origin when the data file is opened a data corrected for the time constant of the instrument and b corrected data set is filtered using the standard Fourier transform filter in Origin 7 0 and a bandwid
62. ct your print outs Only on screen display is affected A Note About Data Import MicroCal has produced three models of the ITC instrument the OMEGA the MCS ITC and the VP ITC All together there are four different data collection software packages available for use with these instruments a DOS based and a Windows based package for the OMEGA a Windows based package for the MCS and a Windows based package for the VP ITC This version of Origin will accept data files from any of the four versions of the data ITC Data t ee nae T a eee De generated by the Windows based data Omega Data 1 collection software from the OMEGA the Area Data DA MCS or the VP ITC instruments you click on r ae a the Read Data button in the RawITC plot Diseociation EDHI window and select ITC Data IT from the filename list To import a data file generated by the OMEGA DOS based data collection software click on the down arrow of the Files of type drop down list box and select Omega Data 1 PLEASE NOTE Data file names should not begin with a number nor should they contain any hyphens periods or spaces Once a data file is called into Origin all further operations on the data are identical regardless of the original source of the data Note that in this tutorial the data were generated by the VP ITC or the OMEGA Windows based data collection software and so we will be using only the Read Data ITC Data IT file opening proced
63. cted from the corresponding point in Rnahhh_ndh The result is plotted as Rnahhh_ndh in the active layer in this case layer 1 in the DeltaH plot window m Rnahhh_NDH Cc O 2 O 2 4 O 2 E O oO x Molar Ratio Note that Buffer_ndh is not affected by this operation It is cleared from the DeltaH window but is still listed as available data in the Layer Control dialog box The Original Rnahhh ndh data could be recovered by selecting Math Simple Math and adding the Buffer_ndh data set to the new Rnahhh_ndh data set To save the project and all related data files e Select the File Save Project As command from the Origin menu bar The Save As dialog box opens with untitled selected as the file name e Enter anew name Origin7 0 accepts long filenames for the project navigate to the folder in which you want to save the file and click OK It is not necessary to enter the opj file extension This will be added automatically Now that you have named the file the next time you save it you can simply use the File Save Project command In order to save Some memory space you may find it useful to delete the original injection data This may be useful when you are reading a large number of data sets into the same Origin project MAU130010 Rev D ei ITC Tutorial Guide 36 To delete a data set from a project either Double click on any layer icon in any plot window Select a data set from the Available Da
64. d by selecting Edit Update Client the changes will be transferred to the Word document Calculating a Mean Value for Reference Data You may quickly calculate a mean value for the area data of any reference or blank injections by the following method For this example we will calculate the mean of the area data of the reference file Febuf10 itc see Subtracting Reference Data starting on page 34 e From the RawITC window click on Read Data button and open the file Febuf10 itc located in the Origin70 Samples folder e Open the area data worksheet by right clicking on any data point of the Febuf10 1itc data set in the DeltaH window and selecting Plot Details Worksheet from the menu e Select the NDH column by clicking on the column heading All the cells of the column should be highlighted e Click on the Statistics on Column s button Note If you do not see the button while the worksheet is open Select View Toolbars then select the Worksheet check box A new worksheet will appear with the mean standard deviation standard error of the mean the sum of the data and the number of data points of the NDH dataset column 42 7506 12 34335 aff A1502 10 428 41935 i 151 00433 421797962 MAU130010 Rev D 103 ITC Tutorial Guide This page was intentionally left blank 104 MAU130010 Rev D Appendix Equations Used for Fitting ITC Data Appendix Equations Used for Fitting ITC Data I General Considerations It
65. e Layer 1 Syvailable Data Uelete Layer Contents Ti l mahhh_dh E Cancel mahhh_ndh mahhh inj mahhh_st Layer Properties mahhh_mt 42 mahhh_ndh Plot Associations buffer dh buther iry buffer st Ul giejpeit buffer_mt buffer _ndh Edit kange M Show Range Sort Rescale on OF T Show current folder only e Click OK Rnahhh ndh plots into the DeltaH window The axes automatically rescale to show all of the data m Buffer_NDH m Rnahhh_NDH k Qunceeeennnnnee 2 o e E 84 T aot 0 0 0 5 1 0 1 5 2 0 Molar Ratio Rnahhh_ndh and Buffer_ndh plotted together The Available Data list in the Layer Control dialog box shows all data sets currently available for plotting in this project The Layer Contents list shows all data sets currently plotted in the active layer See the Origin User s Manual or Origin s Online Help menu item or press F1 for more on handling Origin data 32 MAU130010 Rev D Lesson 4 Analyzing Multiple runs and subtracting Reference Note that you can read any number of data files into the same DeltaH window When multiple data plots appear in the same window you can set the active data plot by clicking on the plot type line symbol icons next to the i er data set name in the legend a oe oe E FAnahhh WOH A black border around the line symbol icon indicates the currently active data plot Editing fitting and other operati
66. e completion of the 1 1 injection to completion of the 1 injection The expression for Q in eq 9 only applies to the liquid contained in volume V Therefore after completing an injection it is obvious that a correction must be made for displaced volume i e AV injection volume since some of the liquid in V after the i 1 injection will no longer be in V after the i injection even though it will contribute to the heat effect assuming the kinetics of reaction and mixing are fast before it passes out of the working volume V The liquid in the displaced volume contributes about 50 106 MAU130010 Rev D Appendix Equations Used for Fitting ITC Data as much heat effect as an equivalent volume remaining in Ve The correct expression then for heat released AQ i from the i injection is Ol OG 1 agi 00 E aal 06 1 10 The process of fitting experimental data then involves 1 initial guesses which most often can be made accurately enough by Origin ofn K and AH 2 calculation of AQ i for each injection and comparison of these values with the measured heat for the corresponding experimental injection 3 improvement in the initial values of n K and AH by standard Marquardt methods and 4 iteration of the above procedure until no further significant improvement in fit occurs with continued iteration III Two Sets of Independent Sites Using the same definition of symbols as above for set 1 and set 2 we have
67. e equation states that the Chi squared value of the fit is equal to the sum of the squares of the deviations of the theoretical curve s from the experimental points divided by the number of degrees of freedom Since there is no weighting it can be seen that the calculated values are dependent on the magnitude of the scale and the number of data points After fitting this value is reported as Chi 2 DoF Line Types for Fit Curves You may select a line type to plot your data or fit curves from the Plot Details dialog box The Plot Details dialog box is available by double clicking on the data plot right clicking on the data plot and selecting Plot Details from the shortcut menu or selecting the desired data plot from the Data menu data list and selecting Format Plot MAU130010 Rev D 101 ITC Tutorial Guide 102 The Line Group Select the desired line connection from the associated Connect drop down list The line connection type affects interpolation results The default line type for fit curves is Straight line The most common method of connecting the fit curve data points are straight spline or B spline Straight A straight line is displayed between data points This type of line connection will not give a smooth representation of the fit curve if there are few data points Spline This option generates a cubic spline connection To use the connection the X values must be discrete and increasing Furthermore the number of data poi
68. e family Graphing name may have up 11 alphanumerical mn characters VPViewer adds the sequential 3 a indat See digit number therefore there may be a maximum of 14 characters for the filename To open the file series click on File name Jite0523c001 ite Add File s Gk the Read Data button and the Import EREA ITC Daa kte E f f iles of type uta ata itc Remove File s Cancel Multiple ASCII dialog box will open m a Select the Auto ITC Data ITC as the Files of type the upper File Name list box will list only the first file of the series File Name 001 ITC but when selected all files of the same file name will be read in until the end of the series is reached Alternatively you may open individual files or a selection of files similar to the normal ITC method for multiple file input When you select the Files of type to be ITC Data ITC then all files with the ITC extension will be listed in the upper list box You may then manually select the files to add to the lower list box and read into Origin MAU130010 Rev D 89 ITC Tutorial Guide To open serial Auto ITC 001 ITC files e Click on the Read Data button The Import Multiple ASCII dialog box opens Select Auto ITC Data itc as the Files of type and only ITC files that have 001 before the extension dot will be listed e Navigate to the C Origin70 Samples folder and then select itc0523c001 ITC from the Files list Please Note Raw data fi
69. e icon from Select File SaveTemplate As Origin opens a dialog box asking if you want to save the file as DELTAH OTP the DeltaH template file Click Cancel at this point if you do not want to change the original DeltaH template If you click OK Origin will save the modified DeltaH window as DELTAH OTP If you saved the modified template and now select the File Read Data buttonthe modified DeltaH window will appear Note that plotted data cannot be saved to a template file so there 1s no need to delete the plotted area data before saving the DeltaH window To revert to the original DeltaH template If you do decide to modify the DeltaH template it is easy to recreate the original Simply reverse the steps you used to create the modified template That is open the DeltaH window open the Y Axis dialog box click on the Tick Labels tab remove the check mark from the Show Major Labels check box then select File Save Template AS Modifying the RawITC template The RawITC plot window shows bottom X axis tick labels in units of minutes Let s use what we have just learned about factoring tick labels to change this axis scale so that the tick labels are in units of hours rather than minutes To factor the RawITC X axis tick labels by 3600 Shortcut Right click on the bottom X axis tick labels and select Tick Labels from the drop down menu list 52 Set the RawITC window as the active window by either pressing
70. efault a Symbol Color EE Automatic Overlapped Points Offset Plotting T Show Construction 4 Plot Type Scatter gt gt Worksheet Cancel Aol e Click on the Worksheet button The Rnahhh worksheet opens Please note If a worksheet cell is not wide enough to display the entire number Origin will fill the cell with HH signs The full number is used by Origin If you wish to view the full number you may increase the column width by placing the cursor on the left or right border of the column name waiting till the cursor changes to a double headed arrow then moving the column edge to the left or right to increase the column width Alternatively you may right click the column name select properties from the drop down list and increase the value in the Column Width text box 44 MAU130010 Rev D Lesson 5 ITC Data Handling me Origin 7 UNTITLED Rnahhh p File Edt View Plot Column Math Statistics Tools Format Window Help PaaS ael S S aa al Copy and Paste Worksheet Data Data can be copied from a worksheet to the clipboard then pasted from the clipboard into another Origin worksheet a plot window or another Windows application To copy and paste worksheet data Select a range of worksheet values e Select the initial cell row or column in the range To select a cell click on the cell To select an entire row click on the row number To select an entire column click on the c
71. efore you begin this lesson open a new project This will clear any old data that may be in memory ik To open a new project Shortcut Click the New e Choose Project from the New sub menu under the File menu Project button on the Standard toolbar Opening Multiple Data Files In the following example you will open two area DH data files and subtract one from the other Both area files were previously saved to disk This example assumes that you have previously opened the ITC file Rnahhh ite into Origin and saved the resulting area data as Rnahhh DH as described in Lesson 1 If you have not yet done so you should refer to Lesson 1 now To read the sample and reference data into memory e Click on the Read Data button in the RawITC plot window The File Open dialog box opens click on the down arrow in the Files of type drop down list box and select Area Data DH e Navigate to the C Origin70 samples subfolder Several DH files will be listed in the File Name list box MAU130010 Rev D Zig ITC Tutorial Guide Open x Look jr E Samples J amp l c Analysis 2 Otfte3 dh C Data Protb dh C Graphing 3 Anahhh DH L Programming 3 Buffer dh FeatfS4 dh File name Finahhh ite Files of type Area Data DH Cancel P Open as read only e Double click on Rnahhh dh Alternatively you can single click and click Open The Rnahhh dh file opens the data are normalized on concentration then the dat
72. es a Samples iC Tables inf Updates H Origin70BC H OriginPPC H OriginPPC2 5 _Setup dll _inst32i ex_ E SDelere Double click on Setup exe file _s ys1 cab _sys1 hdr T usert cab _userl hdr Data tag i datal cab ini setup ins setup lid EX_ File Application Application E WinZip File HDR File WinZip File HDF File TAG File WinZip File HDF File DAT File BIN File DAT File BMP File Application Configuration Internet Com LID File 02 18 2002 3 00 Pk 02 18 2002 3 00 Pk 02 18 2002 3 00 Pl 02 18 2002 3 00 Pl 02 18 2002 3 00 Pk 02 18 2002 3 00 Pk 02 18 2002 3 00 Pk 02 18 2002 3 00 Pk 02 18 2002 3 00 Pk 02 18 2002 3 00 Pk 02 18 2002 3 00 Pl 02 18 2002 3 00 Pl 02 18 2002 3 00 Pk 02 18 2002 3 00 Pk 02 18 2002 3 00 Ph 02 18 2002 3 00 Pk 02 18 2002 3 00 Ph 02 18 2002 3 00 Pk Note you may also launch the setup up program by selecting Run from the Start Menus then entering c Origin70 AddOn Setup Setup exe into the text box and clicking OK e Follow the instructions on screen Choose Folder e Wcushtomdisk customdisk origine samples fe e Origini Registering with OriginL 4 MAU130010 Rev D Getting Started OriginLab Corporation a separate company from MicroCal LLC produces and supports the Origin software package MicroCal LLC produces and supports the calorimetric fitting routines imbedded in the Origin for DSC and Origin for ITC
73. evel is determined by averaging the power level for a specified time prior to the next injection After each injection you need to allow enough time for the instrument to equilibrate at the new power level but not so much time to allow significant hydrolysis of substrate to occur With this in mind a default value of 15 seconds is entered for the time period to average the power signal before each injection You may change this default value with the Average Time P button eS LULU e Click the Average Time P Average P Value The dialog box will open allowing you the opportunity to change or accept the default Cancel value of 30 seconds Click OK to accept 30 seconds for average the power level Enter time i e Click the Zero Y Axis button The cursor will turn to a cross hair allowing you to double click a point to place at y 0 e Click the Calculate Rate button As illustrated in the previous examples the Rate will be calculated and plotted in a new window Vs the concentration of the injectant in the cell Two new button will be made available Truncate Data will allow you to eliminate the abberation at the start of the experiment Fit to Model will open Origin s Non linear Least Squares curve fitter to perform the fitting iterations For this examle you will not need to use the Truncate Data nor the Apply Time Constant buttons Eix m Method 2 a Substrate only Pre ae Truncate Data Fit to Model 0 00005
74. f injections data points as any of the plotted data sets Subtract a constant When you click this button you will be prompted to enter a constant kCal mole that will be used to subtract from all datasets plotted in the ADeltaH graph Input final numerical Y position When you click this button you will be prompted to enter a final Y position in kCal mole The end point of each plotted data set will be placed at that position and the rest dataset will be offset proportionately Y axis shift When you click this button all data sets are moved out of the graph layer the cursor will change to the data reader tool then the first data set will be moved back into the graph You may click once to see the y axis position of the data reader tool When you click twice or press enter the end point of the data set will be moved to that y position the data set will be removed from the graph and the next file of the series will be plotted in the graph and you may repeated the process for all data sets that were originally plotted in the graph One Set of Sites Pong eee OEEO A 2S SS e Click the One Set of Sites button Each data set that is plotted in the graph will be fitted to the One Set of Sites model and the parameters n number of sites K binding constant AH enthalpy change and AS entropy change are calculated These parameters will then be printed in the summary table e Select the menu item Window MicroCal VP ITC Autosa
75. fitting parameters and plots an initial fit curve as a straight line in red please see page 101 for a discussion of line types in the DeltaH window Rnahhh_NDH F Category Function Action Options Scripts EA Pies Fi ES Bra Parameter Value Var Eror Dependency EZE A a 1 545E4 Iv E Press Esc key to stop fitting iterations a D D o 2 o 8 lt xl ChiSar 1 Iter _100 Iter 100 Simplex Iter Done 1 0 i i Enter fitting session and perform nonlinear curve fitting Basic Mode Molar R atio e Click 1 Iter or 100 Iter button in the Fitting Session dialog box to control the iteration of the fitting cycles Click 1 Iter to perform a single iteration 100 Iter to perform up to 100 iterations It may be necessary that the 100 Iter command be used more than once before a good fit is achieved Repeat this step until you are satisfied with the fit and Ch1 2 is no longer MAU130010 Rev D L3 ITC Tutorial Guide 14 decreasing Note that the fitting parameters in the dialog box update to reflect the current fit Fitting Parameter Constraints Each fitting model has a unique set of fitting parameters For the One Set of Sites model these are N number of sites K binding constant in M and AH heat change in cal mole A fourth parameter AS entropy change in cal mole deg is calculated from AH and K and displayed after fitting You can use
76. ft side of the window you will notice several buttons Clicking on these buttons gives you access to many of the ITC routines EF Cian i UNTITLED Rawi TE B Ee ES View Grech Das Meth TC Iost Foma Window Heb Cf i 5 25 2 ajajaja Sle a CHS A aa l g a cal sec T T T T T T f T 0 00 8 33 16 67 25 00 33 33 41 67 Time min If you are not seeing the entire window as shown above click on the Restore button F in the upper right corner of the RawITC template MAU130010 Rev D 9 ITC Tutorial Guide e Click on the Read Data button The Open dialog box opens with the ITC Data it selected as the Files of type e Navigate to the C Origin70 Samples folder then select Rnahhh ite from the Files list PLEASE NOTE Data file names should not begin with a number nor should they contain any hyphens periods or spaces Also Origin truncates the filenames to the first 15 characters therefore when reading in multiple files the first 15 characters of the filename must be a unique combination to prevent overwriting the data You may select a default folder for Origin to Look in for a data file by selecting File Set Default Folder and entering the default path e g for this tutorial you may wish to choose the path to be C Origin70 Samples Your dialog box may not show the DOS filename extension ITC you may view the extension by opening Windows Explorer and selecting View Options and removing the check ma
77. g options e Use the controls to format the text Select Black Line from the Background drop down list box to change the background style from shadow to border to remove the background style altogether select None from the Background drop down list If the background line is not removed it may be necessary to select Window Refresh Click OK when done The DeltaH window redraws to show the changes in the text box To move the text in the plot window e Click once on the text in the plot window e A colored outline appears indicating that the text is selected and the cursor will change into an arrow headed cross MAU130010 Rev D LD ITC Tutorial Guide Slowly Click again within the colored outline to move the text Click outside the colored outline to deselect the text Rnahhh_NDH Rnahhh_Fit Data Rnahhh NDH Sies Madel OneSie Chi2 2856 29 H 1 023 10 00 1628 K SaJEa 41066 aH 1 a 429 89 kcal mole of injectant 0 0 0 5 1 0 15 2 0 Molar Ratio Hint If you click anywhere along the edge of the text background border a colored size box appears around the text with various size boxes positioned around the perimeter Click and drag on one of the small perimeter boxes to change the size of the text background frame Origin will automatically change the font size to fit within the size of the box Note that any formatting changes can be saved as part of the DeltaH plot window template file See
78. hese numbers are located inside your registration card in the Origin product package Please refer to the Origin Getting Started Booklet for further information When choosing a Destination directory or folder name to place Origin make sure this name or any other name in the path does not include a space otherwise Origin will not operate properly After installation is complete you will see the Origin70 program folder with the program icons If you wish to create a shortcut desktop icon you may do the following Right click the MicroCal LLC ITC icon and select copy from the drop down menu then right click anywhere on the desktop and select paste to install a desktop icon for Origin ITC Installing Origin for ITC Custom Disk After installing Origin you will need to install the Custom disk for the ITC routines if you have purchased other options the relevant Autosampler PPC and DSC routines will be included on this disk e Insert the Custom Disk CD into your CD ROM MAU130010 Rev D ITC Tutorial Guide e Using Windows Explorer navigate to the AddOn Setup sub folder of the Origin 7 0 folder and open the Setup exe application file by double clicking on the filename Select the AddOn Setup Folder 24 Exploring AddOn Setup Reese CI C Origin70 AddOn Setup H Origin H Origin Setup Disks H Origin50 H Origin50T SA ODrigin70 3 AddOn Setup 2 FitFune Modified Files i NAG PDFs H Origin of Palett
79. hich includes the central peak and each neighboring peak In most cases you may want to adjust only the central five points for the central peak of interest The outermost points are usually more closely associated with the neighboring peaks Click on a point then drag the mouse or use the and keys to move the point note that baseline points can only move vertically Use the lt and keys or the mouse to select the next point to the right or left Repeat for each point you want to move Note When any point on the baseline is moved the position of the moved point automatically becomes part of the baseline and any future integration will be calculated from this new baseline MAU130010 Rev D Lesson 2 Setting Baseline and integration Range 2150 2200 2250 2300 2350 2400 Note For a closer look you may use the Magnifying Glass from the Toolbox to expand the flat baseline portion of your data for more accurate adjustment of the integration baseline To do this click on the appropriate icon in the Toolbox Then place the icon pointer near the indicated position I in the above figure click and drag it to position 2 shown above When you release the mouse button that part of the graph contained within the solid rectangle will expand to fill the plot window for better viewing of the baseline If you wish to return to the original non expanded display double click on the Magnifying Glass icon or proceed on to integrate the next pe
80. igand is in Syringe has the check mark y next to it indicating it is the active mode If the check mark is next to the Ligand is in Cell select the menu item Ligand is in Syringe This causes the mode to switch to having the ligand in the syringe To fit the data to the interacting sites model click on the Sequential Binding Sites button in the DeltaH window then enter 2 for the number of sites Enter guesses of 1e8 8000 1e6 3000 for the parameters K1 H1 K2 and H2 respectively Click on the Chi Sqr button to enter the above guesses then click on the 100 Iter button several times until you are satisfied with the convergence These data can be deconvoluted with the default initialization parameters The binding constant for the second ligand is about 70 times weaker than for the first ligand and the heat of binding is also less exothermic Note that stoichiometric parameters n1 and n2 are not included as floating parameters with the model of interacting sites since this would allow a non integral number of ligand molecules to bind in each step which is a physical impossibility This means that accurate concentrations of ligand and macromolecule are more important here since concentration errors cannot be covered up by non integral values of nl and n2 as is the case with the model of two independent sites Note Systems with identical binding sites have statistical degeneracy that influences the saturation profile For example in
81. in North America 800 633 3115 Telephone 413 586 7720 Fax 413 586 0149 Email info microcalorimetry com Web Page www microcalorimetry com MAU130010 Rev D di ITC Tutorial Guide This page was intentionally left blank 2 MAU130010 Rev D Getting Started Getting Started In this chapter we describe how to install Origin on your hard drive how to configure Origin to include the ITC add on routines and how to start Origin System Requirements Origin version 7 requires the following minimum system configuration Microsoft Windows 95 or later or Windows NT version 4 0 or later 133 MHz or higher Pentium compatible CPU 64 megabytes MB of RAM CD ROM drive 50 MB of free hard disk space Internet Explorer version 4 0 or later we recommend version 5 0 or later Internet Explorer need not be your default browser but it must be installed for viewing Origin s compiled HTML Help Installing Origin Single User License Note We recommend that when installing Origin you do not accept the default directory but choose the folder C Origin70 To install a new copy of Origin or to upgrade an existing copy insert the Origin 7 CD into your CD ROM A window opens with a number of options including installing Origin Click the link to install Origin If the CD does not start automatically browse the CD and run ORIGINCD EXE directly The Setup program prompts you to type in your Origin serial number and license key T
82. ine will return to its original position prior to injection of substrate From analysis of the decay curve resulting from substrate decomposition the Michaelis parameters Ky mM and Kat sec may be determined as well as the heat of substrate decomposition AH Ifa second similar experiment is carried out but with an inhibitor in the sample cell along with the enzyme then the resulting decay curve may be analyzed in a similar way to determine the Michaelis inhibitor constant K mM In the analysis of this second decay curve the parameters determined in the first experiment Km Keat and AH are used as input parameters and only K is used as a fitting parameter In Method 2 the enzyme solution is again placed into the reaction cell but the enzyme concentration will generally be lower than in method 1 A number of injections of substrate solution are carried out allowing baseline equilibration to occur subsequent to each injection The equilibrated baseline value after each injection is then used to construct a plot of reaction rate versus total substrate concentration assuming no appreciable substrate degradation takes place during measurement From curve fitting these data parameters Kum Kea and AH may be determined As in Method 1 if the experiment is repeated again but this time with added inhibitor in the enzyme solution then the inhibitor constant K may also be determined Before starting this example you should have Origin up and
83. injection syringe The operator must only be careful to record the proper concentration of the species in the syringe and the species in the cell In cases where the ligand is sparingly soluble and the macromolecule is not then it is sometimes advantageous to load the ligand into the cell since the starting concentration then need not be so high The situation 1s a little more complicated if the macromolecule has more than one site even if there is only one set of sites Let s assume that it has two fairly strong sites with differing affinity for ligand Jf we put the macromolecule in the cell and the ligand in the MAU130010 Rev D Lesson 7 Advanced Curve Fitting syringe then the tightest of the two sites with heat change H1 will titrate in the early injections and the weakest of the two with heat change H2 will titrate in subsequent injections until both sites are saturated whereupon the heat change goes to zero However if the ligand is loaded into the cell and the macromolecule in the syringe then the situation is different since the ligand will be in excess in the early injections so that both sites will titrate with heat change H1 H2 Once sufficient macromolecule 1 e molar ratio of macromolecule ligand of 0 5 has been added to tie up all of the ligand as the 2 to 1 complex then further injections of the apo macromolecule will result in some of the ligand being removed from the weaker site in the 2 to 1 complex so that it can bi
84. is often the most useful since it combines reasonably good WYSIWYG accuracy with fast operation also see Origin User s Manual or Origin s Help menu item for more information on view mode To enter the Adjust Integration session e Click on the Adjust Integrations button in the RawITC window The cursor changes into a cross hair MAU130010 Rev D Zee ITC Tutorial Guide 22 e Move the cursor into the RawITC plot window and click on or near the injection peak you want to adjust For this example click on peak 19 second peak from the right The window zooms to shows the baseline region of peaks 18 19 and 20 Note Origin will show the injection peak before and the injection peak after the injection chosen but any manipulations will only affect the integrated area between the center injection A new set of buttons appears along the top edge of the window Two blue lines appear the section of the plot between the lines is the integration range The basic procedure for adjusting integration details is to select a peak adjust the baseline and the integration range integrate the peak and then move on to the next peak and repeat the process The expanded screen is shown below Baseine _intewrate To adjust the baseline e Click on the Baseline button in the RawITC window which has been temporarily titled Peak 19 The automatically generated points for this baseline appear For the baseline Origin displays 15 points w
85. jection rather than time dependent values To determine AH from equation 26 it is necessary to carry out another single injection experiment where hydrolysis is allowed to go to completion Having done this then discrete values of R at different S are calculated so that equation 25 can then be fit to obtain best values of kea and Ku in the absence of inhibitor In the presence of a competitive inhibitor data are also fit to equation 25 but using keat and Km as fixed results obtained from previous experiment with no inhibitor present and treating K as the only fitting parameter VI Dimer Dissociation Model A protein molecule P may associate at high concentrations to form a dimer The dilution of this concentrated protein solution by injection into the calorimeter cell containing buffer can then result in some heat effects from dissociation AH disc 2 Pre 2P K Py P where P and P2 are the concentrations of monomer and dimer and where AH gj is the heat of dissociation of the dimer It is assumed in this model that the stoichiometry is well defined 1 e no aggregates with stoichiometry higher than 2 are present By measuring heats for a series of injections it is then possible using curve fitting to determine the dissociation constant K and heat of dissociation MAU130010 Rev D LEL ITC Tutorial Guide The equivalent monomer concentration after the ha injection C is the sum of the actual monomer concentr
86. ktop Choose Copy then right click mouse on desktop and choose Paste to create copy of Origin 7 icon Rename icon Right click the copy of the icon choose Rename and enter SIM AutoITC Change target of desktop icon to SIM Right click SIM AutoITC icon choose Properties In Target window change final number of target to 9 enter OK To input SIM data Double click SIM AutoITC icon on desktop Read Data e Click on the Read Data button in the Single Injection group The Import Multiple ASCII dialog box opens Select Auto ITC Data sim as the Files of type and only ITC files that have 001 before the extension dot will be listed e Navigate to the C Origin70 Samples folder and then select OneInj001 sim from the Files list MAU130010 Rev D 95 ITC Tutorial Guide Please Note Raw data file names should not begin with a number nor should they contain any hyphens periods or spaces Import Multiple ASCII HA Look jr Samples Tl c Analysis Data Graphing Programming a itc0523c001 ite File name Jitc0523c001 itc Files of type Auto ITC Data itc A Remove Cancel File Hame teO523c001 ite e Select OneInj001sim file and click Add File s e Click OK TheOnelInj001 family of files is then read in sequentially As the files are read in the following operations take place on each data set 1 The data is read into a worksheet that is created with the corresponding name and RA
87. lacing the checkmark for N1 and N2 and iterate until you return to the earlier fit with a ch1 2 of about 33 000 Note that sometimes you may have to click on the 10 Iter command many times before you achieve the smallest chi 2 value as the fitting can become trapped in a local minimum for several iterations Click on the Done button to end the fitting session NonLinear Least Squares Curve Fitting Session For more information on the Fitting Session press the F1 after opening the dialog box 58 Advanced Mode NonLinear Curve Fitting Fitting Session Aborting the NLSF Session Two NLSF Modes Basic and Advanced Origin offers two modes of its nonlinear least squares fitter Basic and Advanced While both modes allow you to fit your data they differ substantilallly in the options they provide as well as in the degree of complexity By default when you enter the NLSF Curve Fitting Session by selecting one of the three ITC curve fitting models the Origin s nonlinear least squares fitter starts in the mode most NonLinear Curve Fitting Fitting Session M m Ea recently used Parameter Value Varn Error Dependency Basic Mode hae we oo Allows for performing iterative curve fitting to Can Wal the built in functions and plotting the results to the graph Click on the More button to enter the _ educed 6370546 Ch gt ea Sar 1 Iter 30 Tter 3 Advanced mode Select Function Select Datase
88. le Data list in Layer Control dialog box 31 32 37 41 in Math dialog box 38 40 in Subtract Reference Data dialog box 34 B Baseline and Auto ITC Baseline command 10 creating automatically 10 modify in adjust integration session 22 24 Binding equations Competitive 102 3 Dimer Dissociation 101 2 Enzyme Assay 100 101 general considerations 95 96 Sequential Binding Sites 98 99 SingleSet of Identical Sites 96 97 Two Sets of Sites 97 98 Blank injections See Subtracting reference data C Chi Sqr button 57 64 80 81 91 Competitive binding 79 Control Parameters dialog box 59 60 Copy and paste worksheet data 45 46 Copy command 45 Curve fitting Competitive binding 79 Dimer Dissociation 75 77 invoking one of the three fitting models 13 line types 91 92 Nonlinear Least Squares Fitting Session 14 One Set of Sites 13 14 Sequential Binding Sites fitting procedure 63 66 Two Sets of Sites 55 58 with ligand in cell and macromolecule in syringe 63 Custom disk installing 3 4 Data deleting 36 Importing 7 Data markers and setting integration range 24 Data Reader tool 38 Data set see also ITC data active 39 deleting 36 mean value 93 naming 44 naming conventions 11 12 43 44 set as active 33 Data deleting points 27 Deconvolution see Curve fitting Deleting bad data 27 DeltaH plot window see templates Derivative Delta Group 60 Dimer Dissociation 77 equati
89. le names should not begin with a number nor should they contain any hyphens periods or spaces Note You may select a default folder for Origin to Look in for a data file by selecting File Set Default Folder and entering the default path e g for this tutorial you may wish to choose the path to be C Origin70 Samples Import Multiple ASCII E x Look ir E Samples E Til cl Analysis Data Graphing Programmirig a itcO523c001 ite File name te0523c001 itc Files of type Auto ITC Data itc T Remove Cancel File Hame ite 0523c001 ite e Select itc0523c001 ITC file and click Add File s e Click OK The itc0523c001 family of files is then read in sequentially 1 e 1tc0523c001 itcO523c002 itc0523c006 As the files are read in the following operations take place on each data set 1 The data is plotted as a line graph in the ARawITC window 2 Each injection peak is analyzed and a baseline is created 3 The peaks are integrated and the area ucal between the peaks and the baseline is obtained 4 The normalized area data is then plotted in the ADeltaH window 90 MAU130010 Rev D Lesson 9 Other Useful Details E ADeltaH Oo One Set of Sites Two Sets of Sites healmole of inectant to0S290002_NDH Load HD ROS2I0004 HOH AO0S200005_ MDH AS29008 HOH Wabr Aalto Note The Data Control and Model Fitting group of buttons as shown above are normally used with
90. lues will produce erroneous fitting results e Select the menu item Window MicroCal VP ITC Autosampler Summary Table The summary table will open and display the concentration values mM for the data If you need to edit the concentration values enter the value in the appropriate cell then exit the cell to make the change effective Filename Welle EN itcO523c001 1TC 1 itc0523c002 1TC Oe ee itcO523c003 1C O 1 CPS OPP ssid itcO523c004 1TC O 10 01825 Oe sis itcO523c005 1C Ss 12 01825 OO O O OZO O o itcO523c006 1C O 1 i 01825 OP O OZO OoOO O O e Select the menu item Window ADeltaH to return to the ADeltaH window You may use the Subtract Options button to adjust each integral heat data set thereby minimizing the effects of the heats of dilution Control Baseline Subtraction Ra ie ka e Click the Subtract Options button 0 cccccseesussestsseseseseereeeee The Control Baseline Subtraction dialog Q Subtract reference data box will pop up To subtract the heats of dilution effect from area data C Subtract a constant C Input final numerical t position C Y axis shift Cancel MAU130010 Rev D Lesson 9 Other Useful Details Button Name Function Subtract reference data When you click this button the heats from a control experiment can be subtracted from all data sets that are plotted in the ADeltaH graph The control experiment must have same or greater number o
91. mpler Summary Table The summary table will open display the parameter values Well Cellc N K H Comments iteO523c001 1TC O1 al 0 1825 0 0122 1 04 1 1466 16090 25 4 itc0523c002 1T Bl 0 1825 0 0122 1 04 1 266 16030 251 a C B 01825 0 0122 0 984 1 01E6 16860 28 1 10 0 1825 0 0122 1 03 1 1E6 16090 25 4 s_ 12 0 1825 0 0122 1 03 1 1E6 16080 _ 25 4 itc0523c006 IT 14 01825 0 0122 1 02 1 06E6 16420 26 6 s MAU130010 Rev D 93 ITC Tutorial Guide Two Sets of Sites To fit your data to the Two Sets of Sites model perform the same preliminary operations as described in the previous pages then Using the Two Sets of Sites model with multiple data sets e Click the Two Sets of Sites button Each data set that is plotted in the graph will be fitted to the Two Sets of Sites model and the eight parameters nl n2 number of sites K1 K2 binding constants AH1 AH2 enthalpy change and ASI AS2 entropy change are calculated These parameters will then be printed in the summary table Raw Data Window To return to the ARawlITC window e Click the Raw Data Window button to return to the ARawlITC window 94 MAU130010 Rev D Lesson 9 Other Useful Details Single Injection Method with AutoITC Auto ITC is also capable of carrying out a complete binding experiment using only a single continuous injection as opposed to the normal procedure whi
92. n a baseline rnahhhbase for the raw data at Y 0 If you like you can remove this baseline as follows Shortcut Right click anywhere 1 Double click on the baseline The Plot Details dialog box opens inside the axis of the graph and select Plot Details Piot potai lt Ey RawiTC Line a Layer fee RinahhhRaw time te 2 7 Connect Straight r Delete Style Solid Width 0 5 r Color Red ka Remove Fill Area Under Curve Hormeal r Plot Type Line gt gt Worksheet Cancel Apply 2 Inthe Plot Details dialog box right click on the RnahhhBASE data name then select Remove from the drop down menu The baseline data are removed from the project Note You may also remove the baseline from the plotted data by double clicking on the Layer Control button in the upper left corner of the TCFinal window and then move the rmahhhbase data from the Layer Contents list to the Available Data list by first highlighting it and then selecting the left pointing arrow To paste the fitting parameters to the ITCFinal window Earlier in this lesson the fitting parameters were pasted to the DeltaH window Before printing let s copy these parameters and paste them to the ITCFINAL window 1 Click on the DeltaH window or select DeltaH from the Window menu DeltaH becomes the active window Alternatively to copy gt and paste you may right click anywhere inside the text box 3 select
93. n about Creating a Graphic Presentation Importing your graph into Word e Create you graph in Origin and when you are satisfied with its appearance select Edit Copy page e Open your Word document and click at the location where you want the graph to be located e Select Edit Paste Special e Select Origin Graph Object from the As list box e Select the Paste radio button e Click OK MAU130010 Rev D Lesson 9 Other Useful Details Linking your graph into Word e You must first create your graph in Origin and then save it as part of an Origin project OPJ e Open the saved Origin project if it is not already opened that includes the desired graph window e Make the desired graph window active select Edit Copy Page e Open your Word document and click at the location where you want to insert the graph e Select Edit Paste Special e Select Origin Graph Object from the As list box e Select the Paste Link radio button e Click OK After your Origin graph is linked to Word you may return to the original Origin graph and make changes to the graph These changes can be reflected in the Word document by e Select Edit Update Client from the Origin menu to make immediate changes to the Word document graph Shortcut You may quickly start Origin and load the linked graph by simply double clicking on the graph while in Word Origin will be started with the original document loaded the changes can be made an
94. n be expressed as R aa 25 S K os I where keat 1s the catalytic rate constant for substrate decomposition Ky 1s the Michaelis constant E tot is the total enzyme concentration and S is the instantaneous concentration of substrate The equation as written is valid both in the absence or presence of a competitive inhibitor at concentration I and with inhibition constant Kj The use of equation 25 assumes no effects from product inhibition This assumption has been discussed by Todd and Gomez Todd M J amp Gomez J 2001 Analytical Biochemistry 296 179 187 and found to be quantitative in many cases In those cases where product inhibition is significant then equation 25 can only be used to express initial rates of reaction prior to accumulation of product Todd and Gomez discussed in some detail the two methods by which assays can be carried out in a titration calorimeter and these are summarized below Method 1 Single injection Using this approach the reaction 1s initiated by injecting enzyme solution from the syringe into the sample cell containing substrate solution or vice versa If desired a competitive inhibitor may also be included in one solution or the other The reaction is allowed to go to completion in the calorimeter cell and the power P is recorded as a function of time t Integration of the excess power P associated with the reaction enables AH to be determined 1 e 110 Pat t 0
95. nd to the stronger site on the newly injected macromolecule The heat change for this second phase of the titration will then be H1 H2 assuming that site 1 is sufficiently stronger than site 2 This being the case then all of the ligand will be in the 1 to 1 complex when the molar ratio reaches 1 0 and further injections of macromolecule will give zero heats For example take a look at the ITC integration data file OTFFe3 DH To open this file first click Done in the fitting session menu bar to exit from the fitting session then select File New Project or click on the New Project button to create a new project Now click on the Read Data button in the RawITC window select Files of type Area Data DH then navigate to the C Origin70 Samples folder then double click on OTFFe3 in the File Name list The normalized NDH data plot into the plot window outed NDH m p gi Lm TA oA kair Aalia The data in file OTFFe3 DH were obtained with the macromolecule ovotransferrin in the syringe and the ligand a chelated form of ferric ion in the cell Injections 1 5 correspond to formation of the diferric form of ovotransferrin with heat change H1 H2 Injections 8 14 involve conversion of the diferric form into the mono ferric form with heat change H1 H2 Before fitting to this data select Ligand is in Cell from the ITC menu MAU130010 Rev D 61 62 ITC Tutorial Guide Click OK in the At
96. nd holding down the Alt key then pressing the Tab key till the application s icon is selected Paste the copied values from the clipboard to the destination Select Paste from the Edit menu alternatively click the apy BY right mouse button and select Paste i 0 09681 REHHHHR The selected values are pasted from the clipboard 2 0 19391 PHHHHHH ee Note It may happen that your worksheet does not show the 4 0 38896 RRHH 5 0 48692 gevenaens data but only displays pound signs as shown on the right The data is available for manipulations but is not displayed 6 0 58517 IHHHHHH a 0 78252 IHHHHHHHHI C9 0 88163 9748 91693 10 0 98103 7922 79781 faa 1 0807 i2 1 1806 because the column is not wide enough You may increase the column width by moving the cursor to the right edge of the column header the cursor will change into a double headed arrow then clicking and dragging the cursor to the right or you may right click the column heading select properties then increase the number for the column width Exporting Worksheet Data The contents of any worksheet can be saved into an ASCII file In this section you will open the worksheet for the RnahhhBASE baseline data plotted in the RawITC window and export the X and Y data to an ASCII file To open the Rnahhhbase worksheet Shortcut to worksheet Right click on the data trace and select Open Worksheet 46 Click on the RawITC window
97. ndow and select ITC Data ITC from the List Files As type box and open the Rnahhh ITC data file located in the Origin70 samples folder alternatively you may read in the Area Data dh data file Rnahhh dh The DeltaH window becomes the active window Click on the Concentration button in the DeltaH window When the concentrations dialog box opens change the concentrations and injection volume values to those desired for the simulation For this example set concentration in syringe to 14 concentration in cell to 0 3 and injection volume to 3 0 Note If you do not see the Inject Vol ul text box the file contained injections which were not all the same size you will need to read in a different data file MAU130010 Rev D T9 ITC Tutorial Guide For Data Rnahhh Cancel C in syringe mbd 14 C in Cell mbd 0 3 Inject Vol ul 3 0 Cell ol mlj 1 345 e Click OK e For this example we will simulate to the Two Sets of Sites model Click on the Two sets of Sites button from the model fitting box The Fitting Session dialog box will open with the Two Sets of Sites model selected e Enter the following parameters in the parameters text boxes nl 1 KI 1e7 H1 10000 n2 1 K2 1e5 H2 10000 NonLinear Curve Fitting Fitting Session jie E Parameter Value a Error Dependency Chi A 1 Iter l 10 Iter Select Function Select Dataset More Done e Click on the Chi Sqr butt
98. ng session and perform nonlinear curve fitting Basic kode hdolar R atio You will see that the auto initialization produces a curve which represents the data very poorly If iterations are started from this the fit will not converge A little intuition however will allow the operator to obtain a satisfactory initialization which will lead to convergence Examination of the experimental points shows that the first few injections at a molar ratio below 1 produce ca 1 kcal per mole of injectant changing to ca 12 kcal for molar ratio 1 to 2 and finally changing to zero at molar ratios larger than 2 Begin manual initialization then by entering 1 into both the N1 and N2 parameter boxes in the Fitting Session dialog box Because of the behavior noted above H1 must be near 1000 and H2 close to 12 000 so enter these guesses into the appropriate parameter boxes Since the experimental heats fall off quickly from the H1 value to the H2 value it is clear that K1 must be much larger than K2 and because the heat changes abruptly from the H2 value to zero 1 e beginning with the eleventh injection it is also clear that K2 itself must be large 1 e even though it is smaller than K1 Enter 1e8 into the K1 parameter box and 1e6 into the K2 parameter box Be careful not to insert a space before or after the e when using exponential notation or Origin will not accept the value 56 MAU130010 Rev D Lesson 7 Advanced Curve Fitting NonLinea
99. nts cannot exceed 900 if the data set exceeds this number the operation will fail Since curvature information is held in memory the spline resolution remains the same regardless of page magnification The SplineStep variable in the ORIGIN INI file controls the spline calculation increment It is expressed in units of 1 point This is usually the most satisfactory representation of the fit curve but may exhibit an excursion from the actual fit curve if there is a sharp corner in the data B Spline The B spline curve can be described by parametric equations Unlike spline curves which pass through the original data points the B spline curve winds around the original data points without passing through them Thus this curve may not produce a satisfactory representation of the fit curve For a complete discussion of the B spline connection see the Origin User s Manual Inserting an Origin graph into Microsoft Word There are two ways to include your Origin graph into Word or other applications you may import your graph into Word or you may link share your graph with Word When you import your graph Word will display the graph as an object and it cannot be edited by Origin tools although it may be resized or reposition in the Word document When you link share your graph Word displays the graph as an object which can be edited by Origin and updated when the Origin graph changes Please refer to the Origin manual for more informatio
100. o new buttons will be made available Truncate Data will allow you to eliminate the abberation at the start of the experiment Fit to Model will open Origin s Non linear Least Squares curve fitter to perform the fitting iterations MAU130010 Rev D 69 ITC Tutorial Guide O 00050 0 0004 5 0 00005 0 00020 0 00025 Truncate Data Fit to Model 0 00020 0 00045 0 00070 O 00 005 a Lh w wo a co E a t N a 0 00000 0 00005 0 00010 0 00013 0 00020 5 Cm hi Truncate Data e Select the Truncate Data button Move the data markers to the positions shown and double click on one of the markers or press enter This will eliminate the data obtained immediately after the injection of the substrate before the calorimeter equilibrated with the on going reaction Fit to Model e Click on the Fit to Model button The Fitting Sessions dialog box will open e Click on the 100 Iter button two or three times to ensure that the chi 2 value is no longer decreasing then click Done to end the fitting session Kcat and Km are used as the variable parameters during the iterative fitting and the best values along with AH are reported in the output parameter box Note AH is determined from the total area contained in the negative peak of the raw data but is not used as a fitting parameter Data M1NoInhibiRAW_Rt Model M1SubstrateOnly Chi 2 DoF 3 148E 11 Keat 81 9 0 223 Km 0 076 5 06E 4 A
101. ob mea e Click the Concentration button The concentration dialog box will open allowing you to verify or edit the concentrations e Click the Zero Y Axis button The cursor will turn to a cross hair allowing you to double click a point to place at y 0 e Click the Apply Time Constant button The time contant dialog box will open allowing you verify or edit the time constant for your data e Click the Calculate Rate button As illustrated in the previous example the Rate will be calculated and plotted in a new window Vs the concentration of the injectant in the cell Two new button will be made available Truncate Data will allow you to eliminate the abberation at the start of the experiment Fit to Model will open Origin s Non linear Least Squares curve fitter to perform the fitting iterations e Select the Truncate Data button Move the data marker to remove the abberation on the right side of the data display then double click on one of the markers or press enter to set the point to truncate the curve e Click on the Fit to Model button The Fitting Sessions dialog box will open e Click on the 100 Iter button two or three times then click Done to end the fitting session The inhibition constant K is used as the variable parameter during the iterative fitting and reported in the output parameter box The values for the three entered paramters Koa K yy and AH are also displayed in the parameter box MAU130010 Rev D Val
102. oeane uses eeste ean tense ade es 45 Exporine Worksheet Dalasi ia uaadanes E ic lottita ate outa 46 Dapon ne Worksheet Dataord a R E TAE EEE IA 48 Lesson 6 Modifying Templates sssseeeecoocsssssscccccccssssseccecsosssssscecsosssssssceecssssssssseeesso 49 Modine me Deta H Template isa Pan ar AE E A E R E AAE 49 Modityine tme RaW TEC tempa en a E E oe eee 52 e eo e E E E A E E E A A E A E E A E 53 Lesson 7 Advanced Curve Fitting sssssssssccccccsssssssssssccccsscccsssssssccccssssssesssssecs 55 Fitting with the Two Sets of Sites Model soncraersinorian ranae ba ee a 55 NonLinear Least Squares Curve Fitting SeSSIOM ou cccccccccecceeeceeeecccecececeeeeeeeececceseeeeseeeeeeess 58 Controlling the Fitting Procedure assoc tartan Gee ee iS 59 Deconvolution with Ligand in the Cell and Macromolecule in the Syringe ccccceeeeeeees 60 Deconvolution with the Sequential Binding Sites Model ccececcccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 63 Fnzyine Substrate iii bitOt AS Sayona a a E E A 66 Dimer Dissocianon Mode liinicaseaen a a a doeee a ceuacats 75 Competitive Tele and Bindina S E 77 SANO CUVE esses 2 ace de cotaws T A a eran ceed 79 Using Macromolecule Concentration rather than n as a Fitting Parameter 000000000000000000000 82 Sele Injection Methodo eea E A E A E ALE tne ae Sern en ee 82 MAU130010 Rev D Lesson 8 Autosampler Data Optional ACCeSSOLY sccccccsssssssssssssccccessceses
103. oints and extrapolated to subtract from all data points The first data set will be removed from the graph and the next file of the series will be plotted in the graph and you may repeated the process for all data sets that were originally plotted in the graph MAU130010 Rev D a ITC Tutorial Guide For this tutorial practice two different options to zero the baseline e Click on Subtract Options button and select Straight line option Data set OneInj001 is the only data set in the active window and the cursor changes to the data reader tool e Double click the data reader tool on the near the end of the curve about 30 minutes and double click again on the curve at about 0 minutes Origin quickly creates a straight line which is extrapolated and subtracted from all data points e Data set OneInj002 is now the only data set in the active window Repeat above steps for this data set e Repeat for remaining data sets Your plot should look like this ucal sec 0 00 8 33 16 67 25 00 33 33 Time min Now Click on the Read Data button and re open the OneInj001 data file Click on Subtract Options button and select Input final numerical Y position option Enter 0 in Value window Click OK Your plot should look like this Use this data set for subsequent data analysis 1G G7 Time imin 98 MAU130010 Rev D Lesson 9 Other Useful Details Remove Bad Data To Remove Bad Data Data at the beginning of the ex
104. olumn heading e To select a contiguous portion of worksheet values click on the first cell row or column keep the mouse button depressed drag to the final cell row or column that you want to include in the selection range then release the mouse button The entire selection range will now be highlighted Note If you ever wish to select a range of cells where the initial cell but not the final cell is in view then click on the first cell and scroll to the final cell press and hold the shift key then click the final cell Copy the selected values to the clipboard e Choose Copy from the Edit menu alternatively you may click the right mouse button inside the highlighted text and select Copy from the menu The selected values are copied to Windows clipboard Select a destination for the copied values e To paste into a plot window click on the plot window to make it active Shortcut To open a new e To paste into a worksheet click on the worksheet or select File New Worksheet to worksheet click on open a new worksheet then click to select a single cell This cell will be in the upper the New left corner of the destination range Worksheet button from the Standard toolbar MAU130010 Rev D 45 ITC Tutorial Guide To paste into another Windows application switch to the target application then follow the pasting procedure for that application Shortcut If an application is already open you may switch to it by pressing a
105. on The simulated curve yfit appears in the DeltaH window Be careful not to click on the 1 or 30 Iter buttons or the parameters will be changed 80 MAU130010 Rev D Lesson 7 Advanced Curve Fitting m Rnahhh_NDH YFIT Cc wy O 2 e O a2 O E O x 1 0 1 5 Molar Ratio The original data detracts from the simulated data but this data is required to be in memory for simulated data You cannot delete this data but you can hide it from view Right click directly on any of the square data points of the Rnahhbh curve and select Hide Right click on the simulated data trace and select Change to Line Symbol Notice that the simulated data has twenty data points the same as the original Rnahhh curve Also it appears that the simulated curve has not leveled due to complete binding This can be corrected by clicking on the Concentration button to increase the concentration in the syringe decrease the concentration in the cell or increasing the volume of the injection Alternatively you could start over and read in a data set with more data points or injections Select Window DeltaH then click on the Concentrations button Enter 2 mM for the concentration in the cell The graph will rescale on the X axis but the simulated curve will not be affected until you click on the Chi Sqr button again Click on the Chi Sqr button The curve will be simulated using the new concentration MAU130010 Re
106. ons 101 2 Draft view mode 7 21 E Enzyme Assay 66 75 equations 101 Method 1 Substrate Only 66 70 Method 1 Substrate Plus Inhibitor 71 Method 2 Substrate Only 72 74 Method 2 Substrate Plus Inhibitor 74 75 Exiting Origin 20 F Final figure 17 19 Fitting Function dialog box 13 see also Curve fitting Fitting parameters and Sequential Binding Sites 63 66 98 99 and the single set of sites model 13 14 96 97 and the two sets of sites model 55 58 97 98 general considerations 96 text box 14 using macromolecule concentration 82 MAU130010 Rev D LIS ITC Tutorial Guide Importing data 7 Importing Worksheet Data 48 Initialization parameters 55 58 Installing Custom Disk 3 4 Origin 3 Integrate injection peaks in routine data analysis 10 Integrate on All Peaks command 10 25 single peak 24 Integration details and modifying baseline 22 24 defined 21 To adjust the integration range 24 23 25 ITC data see also Subtracting reference buffer data Area data Routine ITC data analysis and opening multiple area data runs 37 and subtracting reference buffer data from reaction heat data 29 36 42 data sets created in routine data analysis 1 1 12 43 44 opening multiple area data runs 29 3 1 plotting in DeltaH window 37 ITCFinal plot window see Templates Iterations Max Number of Iterations 60 L Layer Contents list 31 32 37 41 Layer Control dialog b
107. ons can only be carried out on the active plot Adjusting the Molar Ratio Note in the above figure that the Buffer_ndh data plots from molar ratio 0 to ca 1 3 while the Rnahhh_ndh data plots from 0 to ca 2 0 In the case of the Buffer_ndh data the molar ratio is in fact infinity since injections of 21 16 mM ligand solution were made into a cell which contained only buffer and no macromolecule 1 e in order to determine heats of dilution of ligand into buffer Origin automatically assigns a concentration of 1 0 mM in order to obtain non infinite values for the molar ratio to allow plotting of the Buffer ndh points Before subtracting the reference data you should check that the molar ratio is identical for both data sets This will ensure that the final result is accurate and will also ensure that the two data sets plot in register that is injection 1 of the control experiment plots at the same molar ratio as injection 1 of the sample experiment etc To adjust the molar ratio 1 Click on the Data menu and check that Rnahhh is checkmarked If not select Rnahhh from the menu This sets Rnahhh as the active data set BEER Math ITC Tools Format Alternatively you may Get Denia Range right click on any open Reset to Full Range space between the axis Data Markers and click on Rnahhh Move Data Points Remove Bad Data Points 1 Buffer Mts MEOH Finahhh skiis NDODHE as a simpler alternative to the above p
108. or Tick Labels Custom Tick Labels Scale Tithe amp Format Grid Lines Break Selection Increment 0 02 To Jo aaa C Major Ticks 1U Type Linear H Minor Ticks 4 H H Horizontal mi Rescale E Ae First Tick i 50 MAU130010 Rev D Lesson 6 Modifying Templates Selection e Click on the Tick Labels Tab e Select Right from the Selection List Box e Click the Show Major Labels check box to insert a check mark e Click OK Right B The dialog box closes The DeltaH window redraws to show tick labels along the right Y axis The right axis labels are in units of calories mole of injectant while the left axis labels are in kcal mole of injectant m Febuf10_NDH 0 45 mamma 450 3 Cc o amp 2 E O E O a4 Molar Ratio To factor the axis label values by 1000 e Double click on the right Y axis tick labels or select Format Axis Y Axis The Y Axis dialog box opens Selection e Click on the Tick Labels Tab a a bo E z e Select Right from the Selection List Box a 1 e Enter 1000 in the Divide by Factor text box e Click OK to close the dialog box T l L Note Now both axes plot in kcal since both are factored by 1000 MAU130010 Rev D ol ms the Standard toolbar ITC Tutorial Guide To save the changes into the DeltaH template file Shortcut Click on the Save Templat
109. ote that this menu level is available only if you purchased the optional DSC with the PPC attachment Short Menus Select this option to run Origin with menus that are an abbreviated version of the General Full Menus configuration Note that you cannot switch to a new menu level if there is a maximized plot window or worksheet in the current project A warning prompt will appear if you try to switch levels while a window is maximized If this happens simply click on the window Restore button u You will then be able to switch levels Simultaneously Running DSC ITC and PPC Configurations If you purchased the DSC ITC Autosampler and PPC software modules the installation program will have automatically created icons in the MicroCal OriginLab program group for the relevant software This allows you to run the configurations simultaneously The most likely reason to do this would be if you have the MicroCal DSC with the PPC or Autosampler attachment and the MicroCal ITC microcalorimeters and you intend to run them on the same computer Double click on any icon to run that configuration View Mode Each Origin plot window can be viewed in any of four different view modes Print View Page View Window View and Draft View These are available under the View menu option Print View is a true WYSIWYG What You See is What You Get view mode This view mode displays a page that corresponds exactly to the page from your hard copy device E
110. ox 31 32 37 41 Layer icon double clicking on 31 37 41 Line types for fit curves 91 92 Linear Regression command 39 M Math dialog box using to subtract reference data 38 39 40 Mean value of a data set 93 Menu Levels 5 N Naming a data set 11 12 43 44 O Opening and Analyzing Previous Versions of Origin ORG Documents 7 P Page view mode 6 21 Paste command 46 116 Plot command 44 46 Plot Details dialog box 18 25 36 44 91 Plot window templates see Templates Plotting data see Area data ITC data Raw ITC data files Routine ITC data analysis Print view mode 6 Project opening 21 29 36 43 66 71 72 74 75 77 saving 20 R Raw ITC data files opening 9 10 37 43 RawlITC plot window see Templates Reaction heat subtracting reference from see ITC data Subtracting reference buffer data Read Data button 7 10 49 79 Reference data See Subtracting reference data Registering with OriginLab 5 Routine ITC data analysis see also ITC data and integrating injection peaks 10 and opening raw data files 9 10 37 43 S Save area data 12 Sequential Binding Sites 63 66 binding equations 98 99 multiple ligands to transition metal ions 65 non identical sites 65 66 Significant digits 60 Simple Math command 38 39 40 Simulating Curves 79 81 Starting Origin 5 Subtract Reference Data button 34 Subtracting reference buffer data see also ITC data
111. packages MicroCal LLC will provide technical support for all aspects of the software without registration OriginLab Corp will not provide technical support for the calorimetric fitting routines but if the copy is registered will provide standard technical support for the general purpose routines of the program Upon receipt of Origin please fill out and return the registration form included with your package to OriginLab You may also register at any time by contacting the Customer Support Department at OriginLab Starting Origin To start Origin double click on the Origin 7 0 program icon on the Desk Top Alternatively click Start then point to Programs then point to the OriginLab folder then click on the MicroCal LLC ITC program icon from the submenu Menu Levels This ITC version of Origin comes with a minimum of three distinct menu configuration options or menu levels there are other levels available if you purchased the optional DSC PPC or Autosampler DSC software Each menu level has its own distinct menu commands After Origin has opened you may change a menu level option under the Format Menu option The nine menu levels are General Full Menus Select this option to run Origin in the generic non instrument mode This menu level contains no instrument specific routines but does contain many general data analysis and graphics routines not present in the instrument specific menus that you may find useful for othe
112. pens for layer 1 e Select rnahhhraw _ cp in the Available Data list then click on the gt button rnahhhraw_cp is added to the Layer Contents list Layer 1 Svailable Data Delete Laver Contents feburl Craw cp Cancel rhahhhrav cp rmnahhhbase Layer Properties Plot Associations Urnamup Edit Hange T Show Range Rescale on OF rhahhh xt rhahhh_ mt mahhh_ndh mahhhraw_cp mahhhbegin mahhhrange mahhhbaze feburl 0_ dh feburl Ory febuflO_ xt MAU130010 Rev D Al ITC Tutorial Guide e Click OK Both rnahhhraw cp and febuf10raw_cp are now plotted in the RawITC window Note the difference in the time spacing of the injections woo O 0b 72 Z 49 O 16 67 25 00 33 33 41 67 50 00 Time min The difference in peak spacing is not a problem when subtracting reference data You can work with data files having different time spacing since you are interested primarily in the integration area data for each peak 42 MAU130010 Rev D Lesson 5 ITC Data Handling Lesson 5 ITC Data Handling Every data plot in Origin has an associated worksheet The worksheet contains the X Y and if appropriate the error bar values for the plot A worksheet can contain values for more than one data plot It is always possible to view the worksheet from which data were plotted This lesson shows you
113. periment will be distorted by the time constant correction and there may be extraneous data points Remove Bad Data EE after the injection was completed The Remove Bad Data button will simplify the task of excluding these data points from subsequent analysis At the start of the injection typical experiments will have a high point exothermic reaction or a low point endothermic reaction When you select the Remove data before option you may select either the High Point or Low Point check box then when you click OK Origin will search each data set and delete all data before the corresponding High or Low point in the data The data will then be plotted on the graph with the beginning data removed Alternatively you may select the Remove selected range option When you select this option button and click OK each data set will be sequentially plotted on the graph with two data markers displayed on the trace You may click and drag on a marker to move it to your Hint When desired point on the trace then double click or press enter to set the point All data moving a data between the two markers will be removed from the graph and eliminated from future marker you may analysis press the space bar to increase the size e Click on Remove Bad Data button from Single Injection group of the cross hair e Select the Remove data before option then put a check into the Low Point box and click OK I ST Time min MAU130010 Rev D ce
114. period 2 sec MAU130010 Rev D LES ITC Tutorial Guide 114 3 4 5 6 7 DH and time t columns The DH column corresponds to the column of the same name in the existing Origin ITC worksheet while the time t column is one which doesn t exist in the existing worksheet and must be added The DH and time t columns should be filled with the data points from the above data set after TC correction FT smoothing control subtraction and data trimming DH is the Y axis value AP ucal sec and time t sec is the corresponding X axis value INJV column All entries into this column should be identical and equal to the injection rate R ul sec times the filter time 2 sec X d Rt Eee eS R L000V 2000V cell po 2000V M column M M dyu lie 2000V cell X t XM column XM t Note Indexing for X M and INJV refer to values before the ith injection while DH XM NDH refer to indexing after the ith injection the new column time t is also indexed after the ith injection MAU130010 Rev D Index A Aborting the NLSF Session 58 Active data set 33 39 Active data set icon 33 Adjust integration session see Integration details Area data see also Subtracting reference buffer data deleting 36 opening multiple runs 29 31 plotting in DeltaH window 37 saving 12 ASCII data transfer see Exporting ASCII data Importing Worksheet data Auto ITC Baseline command 10 Availab
115. r Curve Fitting Fitting Session Category Function Action Options Scripts Caress Me Parameter Value Yam Error Dependency I I I I El Chisa 1 Iter 100 Iter 100 Simples Iter Done Basic Mode Click on the Chi Sqr button in the dialog box Origin draws a new fit curve using the entered parameters which is a much better representation of the data mmn an IT Though this fit is good enough to lead to correct convergence we can still improve on it some Since the fit curve goes from H2 to zero much more gradually than the data let s increase K2 from 1e6 to 1e8 We must still keep K1 larger than K2 so increase K1 from 1e8 to 1e10 Click on Chi Sqr button and observe that we now have a very good initialization curve Select the 100 Iter button a few times and convergence occurs with a final ch1 2 of about 33 000 risa Im MAU130010 Rev D Os ITC Tutorial Guide Note that N1 and N2 are nearly the same magnitude but not quite It would be interesting to see if a fit of nearly equal quality could be obtained with N1 and N2 exactly equal to each other Although theoretically they should each be 1 0 Assign the value 1 0 into the N1 and N2 parameter value box click in the N1 and N2 checkboxes to remove the checkmark and continue the iterations The final fit is not as good as when N1 and N2 are both floated although there is no obvious explanation for this Float all variables by rep
116. r applications ITC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific ITC data analysis routines Single Injection ITC Data Analysis Select this option to run Origin in a configuration that analyzes Single Injection ITC experiments Autosampler ITC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific autosampler with ITC data analysis routines for handling multiple data files Note that this menu level is available only if you purchased the optional AutoITC software module AutoITC Single Injection Data Analysis Select this option to run Origin in a configuration that analyzes multiple data files of Single Injection ITC experiments Note that this menu option is available only if you purchased the AutoITC software module DSC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific DSC data analysis routines Autosampler DSC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific autosampler with DSC data analysis routines for MAU130010 Rev D 5 ITC Tutorial Guide handling multiple data files Note that this menu level is available only if you purchased the autosampler DSC software module PPC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific PPC data analysis routines N
117. r mole of injectant added displayed in DeltaH window rnahhh_ xmt Molar ratio of ligand to macromolecule after injection i X value of data point rnahhhbase Baseline for the injection data displayed in red in the RawlITC window rnahhhraw_cp All of the original injection data displayed in black in the RawlITC window In addition origin creates two temporary data sets MAU130010 Rev D 43 ITC Tutorial Guide rnahhhbegin Contains the indices row numbers of the start of an injection rnahhhrange Contains the indices of the integration range for the injections An Origin data set is named after its worksheet and worksheet column usually separated by an underscore Thus the first six data sets above will all be found on the same worksheet RNAHHH in columns named DH INJV Xt Mt XMt and NDH respectively The temporary two data sets above are located on separate worksheets named rnahhhbase an Origin created baseline and RnahhhRAW the experimental data The temporary data sets are indices created by Origin and do not have a worksheet created To open the Rnahhh worksheet Select the Plot command from the Format menu The Plot Details dialog box opens for the rnahhh_ndh data plot if the DeltaH window is active Shortcut to worksheet Right click on the data trace and select Open Worksheet Plot Details Foy DeltaH Symbol Drop Lines Fi Layer JE Ef Fnahhh Mitts ND E abe H Edge Thickness D
118. r to the pointer tool 2150 2200 2250 2300 2350 2400 e Set anew integration range by clicking and dragging either line with the mouse e The integration area for the central peak selected will be between the dashed blue lines To integrate the selected peak e Click on the Integrate button This integrates the peak using the current baseline and integration range The curve in the DeltaH window is updated accordingly The integration results are also updated on the worksheet containing the injection data To select another peak e Click on the and buttons to move to the next or previous peak Note that the current peak number 1s always displayed in the window title bar To end the Adjust Integration session e Click on the Quit button The RawITC window is restored to show all of the injection peaks Note that the area data in the DeltaH window will have updated to reflect any changes you made 24 MAU130010 Rev D Lesson 2 Setting Baseline and integration Range You will notice that the RawITC template includes a button to Integrate All Peaks This button integrates all injection peaks and replots the area data You will recall from the previous lesson that the area data in the DeltaH window were originally created with the Integrate All Peaks routine It is not necessary at this point to integrate on all peaks again In fact it is a good idea not to If you now integrate on all peaks you will not get the same area re
119. rk next to Hide MS DOS file extensions for file types that are registered Open Look in E Analysis 8 M1Inhibitor0175 ite _ You may view more 1 Data a M1 Nalnhibitor ite information about the files by T Graphing E M2Irihibitor01 ite clicking the Details button 2 Programming i M2Nalnhibitor ite a Dissociation TC 2 Ferss itc Febuf1 Dite EliAna File name Finahhh ite Files of type lite Data it Cancel P open as read only e Click Open Hint You may prefer the shortcut method for opening files Instead of selecting a file and clicking Open simply double click on the file name The RNAHHH file is read and plotted as a line graph in the RawITC window in units of ucal second vs minutes Origin then automatically performs the following operations 1 Selects Auto Baseline routine Each injection peak is analyzed and a baseline is created 2 Selects Integrate All Peaks routine The peaks are integrated and the area ucal under each peak is obtained 3 Opens the DeltaH window Plots the normalized area data rnahhh_ndh in kcal per mole of injectant versus the molar ratio ligand macromolecule Note that the DeltaH window contains buttons that access ITC routines 10 MAU130010 Rev D Lesson 1 Routine ITC Data Analysis and Fitting Rnahhh_NDH One Set of Sites Two Sets of Sites Sequential Binding Sites kcalimale of injectant biolar R atio E
120. rocedure you could have just clicked on the Rnahhh NDH listing in the plot type icon 2 In the DeltaH window click on the Concentration button In the dialog box that opens note the value in the C in Cell mM field it should be 0 651 3 Click Cancel to close the dialog box Now repeat step 1 but this time set the Buffer data set as active MAU130010 Rev D 33 ITC Tutorial Guide 4 In the DeltaH window click again on the Concentration button This time a dialog box opens to show the concentration values for Buffer In the C in Cell mM field enter 0 651 Click OK The two data sets will now plot in register as shown below m Butter WOH E FAnahhh_HOHd kealmole of inje dant 1 0 Ha bar Alia Subtracting Reference Data To subtract Buffer ndh from Rnahhh ndh e Click on the Subtract Reference Data button in the DeltaH window The Subtract Reference Data dialog box opens The most recent file opened in this case Buffer _NDH will appear in both the Data and Reference drop down list box Note that the data set in the Reference box will be subtracted from the data set in the Data box e Select Rnahhh_NDH from the c ubtract Relee e aE HEA Data drop down list Rnahhh NDH becomes highlighted Data Reference and will be entered as the Data Reference Buffer_NDH a 34 MAU130010 Rev D Lesson 4 Analyzing Multiple runs and subtracting Reference e Click OK Every point in Buffer_ndh is subtra
121. rue when working with very small injection peaks This lesson shows you how to manually set integration details Begin this lesson by starting Origin then opening the RNAHHH ITC data file as you did at the beginning of Lesson 1 To start Origin e Double click on the MicroCal LLC ITC icon on the DeskTop If the icon is not available on the DeskTop Start from the task bar then select Programs OriginLab MicroCal LLC ITC Origin opens and displays a new project with the RawITC template plot window To open the RNAHHH file e Click on the Read Data button The Open dialog box opens with the ITC Data it file name extension selected e If you have not previously Set Default Folder to the samples subfolder then navigate to the C Origin70 samples subfolder e Select Rnahhh from the Files list e Click OK e Raw data are plotted in the RawITC window Normalized area data are plotted in the DeltaH window e Select the RawITC window from the file list in the Window menu Note If you ever notice that the the RawITC window or another window has lost some of its formatting instructions e g text rotation this can happen from being in the Draft View mode Draft View is the fastest view mode and is very useful when precise formatting is not required The View Mode is selected from the Page menu To view the page as it will appear when printed select Page View mode which is the slowest but most accurate Page View mode
122. running The routines for Enzyme Assays become available after you read in the ITC data when the File of type was selected to be Enzyme Assay it Enzyme Assay Method 1 Substrate Only Begin this lesson by opening the ITC data file M1NoInhibitor itc as follows e Select File New Project A new Origin project opens to display the RawITC plot window e Click on the Read Data button MAU130010 Rev D Click on the scroll down arrow of the Files of type text box and select Enzyme Assay IT file type Open Look ir Samples E Lesson 7 Advanced Curve Fitting Analysis I MiInhibitor 1 75 ite 1 Data a M1 Nalnhibitar itc Graphing a Mel rihibitorQ1 itc Programming a MM olnhibitor itc a Dissociation ITC a Persson ite a Febut Otc a Rnahhb ite File name Files of type Enzyme Assay it Cancel ITC Data 1t7 Dissociation OH e Navigate to the C Origin70 Samples folder and select M1NoInhibitor from the File Name list and click OK The Enzyme Assay dialog box will open allowing you to select one of the four models E ES Enzyme Assay Method 1 Substrate plus inhibitor Method 2 Substrate only C Method 2 Substrate plus inhibitor Cancel e Select Method 1 Substrate only and click OK The Method 1 Substrate only dialog box will open up as shown below If you do not enter a value for AH the program will c
123. sson 4 Analyzing Multiple runs and subtracting Reference Available Data Y Rnahhh_NDH gt _ Y1 Rnahhh_NDH pale a Y Y1 Y2 j operator 7 Y Y1 data set Y2 data males or number Y 350 Cancel e Click OK The constant 350 is subtracted from each value in the Rnahhh_ndh data set The result is plotted as Rnahhh_ndh in the DeltaH window To subtract a straight line from RNAHHH NDH e Click on the Pointer tool k to deselect the Screen Reader tool Now check the Data menu to see that Febuf10_ndh is the active data set the active data set will be checkmarked All editing and fitting operations are carried out on the active data set Select Febuf10_ndh if it is not active Beem Math ITC Tools Format Set Weoley Rande Reset to Full Range Data Markers Move Data Ports Remove Bad Data Points t l Febu sM NOHE 2 Rnahbh sMtts MDOHIY e Select Linear Regression from the Math menu A straight line is fit to the Febuf10_ndh data Origin assigns the name LinearFit_Febufl0ND to the data set for this line MAU130010 Rev D oe ITC Tutorial Guide 40 os Fen uri HoH E Anann _ HDH LingarF i Fe oul iO m t a E E a E E n 1 0 Waker Rali Select Simple Math from the Math menu The Math dialog box opens Select Rnahhh_NDH from the Available Data list then click on the uppermost gt button Rnahhh_NDH copies to the Y1 text box Sele
124. ssssecs 89 Launching the ITC Autosampler Data Session ccccccccccccccccceeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 89 AUO EC IDB UUOUS lesa casein see celeste etd taeda ean date Mndoedrar decestaa tonanlianduGasasdua tuted ested nu deteta testis teabueddaanates 91 Single Injection Method with Autol TO cccsinseciacash cc dee sec ariedadcidataadeaaeededasnecatrmedndeandakaet 95 Ibesson 9 Other Usctul Details senro dasdodundscuaeveess 101 Chiesquate chr 2 Vin ZALIOM saeni a E a a E A 101 Line LypPES TOC F IEC UVES oann a EE eee 101 Inserting an Origin graph into Microsoft Word ccccccscscsscscesesesesesesescsvsvevsvscecscscssesesvsvevevevassens 102 Calculating a Mean Value for Reference Data nnnnnnnnnnnnennnsenerenessreereeressrerrrrrrrrrrrrrrrsrrrereen 103 Appendix Equations Used for Fitting ITC Data ssssccccocccsssssecccoccssseseeccsosssssssee 105 Is General OnsiG Craton gt 5 i4 sel tancsdel te N taseuceanl da a e a N S 105 I Sinsle Set OL identical Sites sic a hse sera atc soduhd dasaddetea cakes seesstieeieans 106 ITs TP wOsSets Of Independent SIES cect t stint esse a tah e a T a 107 Ie Seguental TS IMINO SUES uee rr E E awe iv Fa sep cass ese epee EE 108 Vo Bnzyine subst ate mmbIDitOr ASSI Varer iei E N E AE eevee la eevee a 110 VIL Dimer Dissociation Mode borsin oiri EEE ies AA eee 111 VIE Compctitive Bindine Model siririna EA AE E E AEE 112 VIE SimeleTnjecion MeMo oiriin r Ei ad auc eae
125. sult as when you integrated each peak separately To view the worksheet data Shortcut to the Worksheet Right click anywhere on the data trace and select Open Worksheet e Select the Pointer tool Lie by clicking on it in the Toolbox e Double click anywhere on the trace of the RawITC data plot in the plot window or select Format Plot The Plot Details dialog box opens for this data plot Plot Details E E RawlTC Line El Layerl E RnahhhRaw timeps RnahhhBASE Connect i Straight Style Solid Width 0 5 z Color B Black X T Fill Area Under Curve Plot Type Line gt gt Worksheet Cancel Click on the Worksheet button The Worksheet containing the injection data opens Note You may notice that the worksheet X axis values are in seconds while the plotted data is shown in minutes This is because the X axis has been factored as described in Lesson 6 You can now proceed to fit the data see Lesson 1 If necessary you can first delete any bad data points as descried in the next lesson MAU130010 Rev D 2J ITC Tutorial Guide This page was intentionally left blank 26 MAU130010 Rev D Lesson 3 Deleting Bad Data Lesson 3 Deleting Bad Data After your injection data are integrated the integration results are displayed in the DeltaH plot window You can delete bad data points from the DeltaH window before starting the fitting session To delete bad data points Shor
126. t More Done Basic Mode Category Function Action Options Scripts In addition to the basic mode features the Advanced Arn Mode allows for defining linear constraints Parameter Value Var Eror Dependency adjusting the configuration of the fitting parameters N oso i i K he4 Maipo Poo simulating data and defining your own fitting e Fee ee function Click on the Basic Mode button to return to the Basic mode al ao Press Esc key to stop fitting iterations If you wish to exit the NLSF session without printing the fitting parameters to the Results window or the graph text box select the fitting session dialog box close x button and click No to the dialog box question Do you want to end the current fitting session 30 Simplex Iter Done Chi Sqr 1 Iter i Enter fitting session and perform nonlinear curve fitting Basic Mode Advanced Mode MAU130010 Rev D Lesson 7 Advanced Curve Fitting Controlling the Fitting Procedure You may enter the NLSF Curve Fitting Session and initialize the parameters by selecting one of the three fitting models One Set of Sites Two Sets of Sites or Sequential Binding Sites After the parameters have been determined you may re enter the NLSF Curve Fitting Session and keep the same fitting parameters by selecting from the menu Math Start Fitting Session Normally the Fitter will open in the Basic mode click on the More but
127. t the actual bulk concentration of ligand in Vo X is related to the hypothetical bulk concentration X s assuming that all of the injected ligand remained in V as follows 1 X V XV sees 3 MAU130010 Rev D 105 ITC Tutorial Guide x x 1 44 4 2V O q The above expressions for M and X are used by Origin to correct for displaced volume effects which occur with each injection II Single Set of Identical Sites In the following equations K Binding constant n of sites Vo active cell volume M and M are bulk and free concentration of macromolecule in Vo X and X are bulk and free concentration of ligand and fraction of sites occupied by ligand X K _ 5 1 X X X nOM 6 Combining equations 5 and 6 above gives o o 1 p 7 nM nKM nM p The total heat content Q of the solution contained in V determined relative to zero for the unliganded species at fractional saturation is QO nOM AHV 8 where AH is the molar heat of ligand binding Solving the quadratic equation 7 for and then substituting this into eq 8 gives 2 X 1 xX 1 4X Loe Ca 1 r he 9 The value of Q above can be calculated for any designated values of n K and AH at the end of the i injection and designated Q i The parameter of interest for comparison with experiment however is the change in heat content from th
128. ta list then click on the Delete button or If the data are plotted in a plot window double click on the trace of the data plot that you want to delete The Plot Details dialog box opens The name of the data set appears in the file list box under the layer icon Right click on the file name you wish to delete then click Delete from the drop down menu In either case the data set along with any related data plots is deleted from the project If you have saved the data set to disk the saved copy will not be affected Plotting Multiple Data Sets Whenever multiple data sets are included in the same plot there may be overlap of data points from the different data sets There are two ways to eliminate this overlap by displacing one or more of the curves on the Y axis if you wish to do so First you may select Math Simple Math and add or subtract a constant from all points in one data set to displace it Remember if you are doing this on data plotted in the DeltaH template that although data is plotted in kcal the actual data is in the worksheet as cal so they must be modified by adding or subtracting cal see A Note about Units starting on page 53 Second you may make the appropriate data set active by selecting it in the list for plot type icons Then select Math Y Translate Use the resulting cross hair icon to select one data point in the active set click on it and hit enter or double click on a data point Then move the icon
129. tcut to switch ad between Origin windows Press and hold down the Ctrl key while pressing the Tab key Select Window DeltaH to make it the active window Click on the Remove Bad Data button The pointer becomes a cross hair Click on the point that you want to delete A small red cross appears on the selected data point The XY coordinates index number and data set name for the selected point are displayed immediately in the Data Display Tool floating Note If you have trouble selecting a particular data point select a point near by and use the left or right arrow keys to move to the data point you wish to select Press ENTER The selected data point is deleted Alternatively after clicking on Remove Bad Data you may double click on a data point to delete it Note The main menu bar also contains a data deletion function under Data Remove Bad Data Points and this works a little differently We recommend the user always delete data using the Remove Bad Data button located on the DeltaH plot window Though there is no Undo command available in this version of Origin with which to un delete a data point it is possible to recover if you have mistakenly deleted a point To recover simply integrate the injection peaks again by clicking on the Integrate All Peaks button in the RawITC window All of the injection peaks will re integrate and the area data including the deleted data point will replot in the DeltaH
130. tention dialog box Attention G Change to have ligands in Cell Cancel This switches the settings letting Origin know that the ligand is now in the cell You may confirm this by clicking on the ITC menu again and noting that the checkmark is next to Ligand is in Cell Note Origin defines the macromolecule as the species with n greater than 1 0 and the ligand as the species with only one site irrespective of their molecular weights DIB Tools Format windo Ligand i in Syringe W Ligand iz in Cell Final Figure Now you can click on the Two Sets of Sites button to select the appropriate fitting model The default fitting parameters will lead to a satisfactory convergence in this case but lets try to improve on them before beginning interations The first several injections indicate that H1 H2 equals about 10 000 calories per mole You might start off with values of 7000 for H1 and 3000 for H2 Set n1 and n2 equal to 1 0 and click to remove the Nland N2 checkmark Insert 1e8 for K1 and 1e6 for K2 Select Chi Sqr and use the 100 Iter command to iterate until chi 2 no longer changes Notice that the estimated errors for K1 and K2 are quite large being about 100 in the case of K1 To verify this change the K1 value from ca 3 2E7 to 5E7 remove the checkmark from K1 and carry out 30 iterations Replace the checkmark at K1 and click on Chi Sqr which then shows a value of ca 32000 Click on 100 Iter again
131. th of 15 data points The user then a sets the zero baseline from which the experimental data will be subtracted page 84 85 and b exclude distorted or extraneous data points prior to subsequent analysis page 86 Creating New Worksheet The raw data after time constant correction Fourier filtering baseline subtraction and eliminating inappropriate data can then be used to form a new worksheet which is modeled after the existing worksheet used with multi injection binding data 82 MAU130010 Rev D Lesson 7 Advanced Curve Fitting To create SIM ITC icon on desktop e Right click any Origin 7 icon on the desktop Choose Copy then right click mouse on desktop and choose Paste to create copy of Origin 7 icon Rename icon Right click the copy of the icon choose Rename and enter SIM ITC Change target of desktop icon to SIM Right click SIM ITC icon choose Properties In Target window change final number of target to 8 enter OK To input SIM data e Double click SIM ITC icon on desktop Read Data e Click on the Read Data button in the Single Injection group The Import Multiple ASCII dialog box opens The only option for Files of type is ITC Data sim Navigate to the C Origin70 Samples folder and then select OneInj001 sim from the Files list Please Note Raw data file names should not begin with a number nor should they contain any hyphens periods or spaces E OneInj003 sim E OneInjoo4 sim
132. the Fitting Session dialog box to apply mathematical constraints to the fitting parameters We mention this subject only in passing for a detailed discussion see page 59 and the appendix To hold a parameter constant The Vary column in the Fitting Session dialog box contains three checkboxes one associated with each fitting parameter Ifa box is check marked Origin will vary that parameter during the fitting process in order to achieve a better fit To hold a parameter constant during iterations click in the box to remove the checkmark from the checkbox Fitting Parameters Text To copy and paste the fitting parameters to the DeltaH window Once you have a good fit click on the Done button and the fitting parameters will be automatically pasted into a text window named Results Log and to the DeltaH window in a text label This label is a named object called Fit P that is linked to the fitting process through Origin s label control feature For more information see the Origin User s Manual or for online help right click anywhere in the text label and select Label Control then press the F1 key Data Rnahhh NDH Model OneSites Chi 2 DoF 2856 N 1 02 0 0016 K 5 54E4 1 1E3 AH 1 361E4 29 8 AS 22 0 Position and format this label just as you want the fitting parameters to appear When you paste the fitting parameters they will replace the Fit Parameters label but retain its position and style Origin will use any te
133. to show that at half saturation the dominant molecular forms were the macromolecule with either two or no ligands attached with very little of the singly liganded form Negative cooperativity can be more easily detected from binding studies since there will be two different phases occurring the strong binding of the first ligand and the weaker binding of the second The Origin file protb dh shows integrated heat data on a macromolecule with two identical sites If you have not done so yet click on Done in the Fitting Sessions window to exit Then select File New Project or click on the New Project button to Open a new project To open protb dh click on the Read Data button in the RawITC window then select Area Data dh from the File of Type drop down box Go to the C Origin70 Samples sub folder and double click on protb dh MAU130010 Rev D 63 ITC Tutorial Guide m t 2 5 2 x g Since there are clearly two phases to this binding isotherm it exhibits negative cooperativity Before fitting edit the concentrations for this data as follows e Click on the Concentration button in the DeltaH window e Enter the following values in the dialog box 20 7 mM ligand in the syringe 0 494 mM macromolecule in the cell 4 ul injection volume 1 32 ml cell volume e Click OK Notice that the Y axis automatically rescales in accordance with the changes you made Be sure to check the ITC menu to see that L
134. to the Y position on the graph where you wish that point to be after displacement click on it and hit enter The entire data set will be translated on the Y axis by that amount Subtracting Reference Data Additional Topics In the previous example the sample injection data and reference injection data matched precisely This may not always be the case however Your reference data may have a different number of injections than your sample data or the injection time spacing may differ between the two runs You will see below how to deal with these situations In the following example you will open two ITC raw ITC data file series one containing the sample data and one containing the reference data You will then plot the area data for each data file series and subtract reference data from sample data Begin by opening a new project Select File New Project and click OK MAU130010 Rev D Lesson 4 Analyzing Multiple runs and subtracting Reference To open both sample and reference raw data files e Click on the Read Data button in the RawITC window and select ITC Data it from the Files of type drop down list e Double click on Rnahhh in the File Name list located in the Origin70 samples subfolder The Rnahhh ite file opens The data are integrated normalized and the area data plots in the DeltaH window e Return to the RawITC window and repeat the above steps to open the Febuf10 ite data file e The Febuf
135. ton to enter the Advanced Mode of the Fitter From the Fitting Session window select Options Control to open the Control Parameters dialog box Edit this dialog box to specify several quantitative properties as described below of the fitting procedure These properties directly affect the way the fitter performs iterations The Control Parameters Dialog Box NHonLinear Curve Fitting Control Parameters Piel x Categor Function Acton Options Scripts Amam Tolerance 0 05 Mas Humber of Iterations 30 Parameters Significant Digits Masimum Minimum l Fixed delta Perel etal Datasets pfit feothhd rndh Weighting Method No weighting Scale Errore with sgrtireduced chi 2 feotfo4_mt feotto4_ xt feothhd inj feothh4 dh Dependent War Weighting Select dependent variable for whch to set weighting Basic Mode method Eo The Tolerance Text Box MicroCal has preset this value to be 0 05 but you may type a new value for the tolerance in this text box When you click n Iter in the Fit Session dialog box this causes the fitter to try to perform at most n Levenberg Marquardt LM iterations If the relative change of the value of chi square between two successive iterations is less than the value in the Tolerance text box less than n iterations are performed If you want the fitter to perform still more iterations click on either the n Iter or the 1 Iter button in the Fitting Session dialog box The val
136. ubtract a constant from Rnahhh_ ndh e Select the Data Reader tool El from the toolbox The pointer changes to a cross hair e Click the mouse on several different data points in the Febuf10_ndh data series in the DeltaH window each time noting the Y value that appears in the Data Display FebufiO_NDH E Rnahhh WOH One Set of Sites Two Sets of Sites Data Dinas Sequential Binding Sites Competitive Binding Ne of inj e Using these Y values figure a rough average for the data For this example let s say the average Y value is 350 See Calculating a Mean Value for Reference Data starting on page 103 for a method to quickly calculate a mean of the data Note that the Data Reader tool shows values in calories while the Y axis in this graph shows values in kcal This is because the Y axis in the DeltaH plot window template is factored by a value of 1000 See Lesson 6 for more about factoring e Select Simple Math from the Math menu The Math on between Data Set dialog box opens e Select Rnahhh_ndh from the Available Data list then click on the uppermost gt button e Rnahhh_ndh copies to the Y1 text box Rnahhh_ndh also appears next to Y Y indicates the name of the data set into which the resulting data will be copied Click in the Y2 text box and type 350 at the insertion point Click in the operator box and type at the insertion point 38 MAU130010 Rev D Le
137. ue 100 is specified as n in the Max Number of Iterations text box see below MAU130010 Rev D o3 ITC Tutorial Guide 60 The Max Number of Iterations Drop down List Specify the value for the maximum number of iterations performed when the n Iter button is clicked on in the Fitting Session dialog box This has been preset by MicroCal to be 100 but the user may change this number to be effective during a session of Origin by entering a new value in the text box However the value will be reset to 100 after exiting Origin The Derivative Delta Group This group determines how the fitter will compute the partial derivatives with respect to parameters for ITC fitting functions during the iterative procedure If the Fixed Delta check box is unchecked recommended for ITC users then the actual value of Delta derivative step size for a particular parameter is equal to the current value of the parameter times the value specified in the Delta text box The Maximum and Minimum text boxes specify the limiting values of the actual Delta in case a parameter value becomes too large or too small MicroCal has preset the Delta to be 06 with the limiting 30 maximum to be 5 x 10 and the minimum to be 5x 10 If your fit curve is not converging well you may want to try a different value for the Delta for ITC users this is typically a larger value e g 0 07 0 08 etc The new value will be valid for the current session of Origin
138. urately When binding constants significantly exceed 10 M instrument sensitivity becomes challenged as concentrations are lowered to the point where quantitative measurements of the binding constant would be possible On the other hand binding constants substantially in excess of 10 M can be measured quantitatively if such strong binding ligands are studied in competition with a second ligand which binds competitively but more weakly to the same binding site 112 MAU130010 Rev D Appendix Equations Used for Fitting ITC Data Competitive binding studies are carried out using the strong binding ligand A as the injectant with the solution in the cell containing the second competitive ligand B as well as the binding protein P or other target molecule This system then has two equilibria which are displaced with each injection 1 e A P S PA KoL P A B P S PB kad PILS The value of Kg and AHg for the competing ligand are first measured in a conventional ITC experiment and these parameter values are entered as known parameters when determining K from results of the competition experiment For the competition experiment the total concentration of competing ligand B tot should be selected such that K K B tot 10 10 M where K3 is the estimated value of Ky The detailed equations used in the fitting model for competitive binding are found in a paper by Sigurskjold Sigurskjold B W 2000 Analytical
139. ure If your own data files are generated with the Omega DOS based data collection programs you must open them via the Read Data Omega Data 1 procedure Refer to Lesson 1 for more information about data import Opening and Analyzing Previous Versions of Origin ORG Documents Shortcut Saved Origin documents or projects org or opj may be opened from explorer by double clicking on the file name To open a previous version of an Origin document project select File Open This menu command opens the Open dialog box Select Old version ORG from the List Files of type drop down list Select the desired file from the list box and click Open to close the dialog box and open the document You may then make formatting changes and print the graph If you wish to analyze the previous version of the document you must update the document to version 7 0 To update a previous version of Origin Document project select File Update to Origin 7 0 Interface All templates will be updated to new templates compatible with version 7 0 You may then analyze the data with version 7 0 Please note when you update the Origin templates to Origin 7 0 most of the text labeling on the graphs will be lost MAU130010 Rev D J ITC Tutorial Guide including the fitting parameters If you want to save the old fitting parameters text you must copy the text before you update to Origin 7 0 To copy the fitting parameter text or any other text right
140. v D 81 ITC Tutorial Guide Using Macromolecule Concentration rather than n as a Fitting Parameter In some instances you may know the value for the stoichiometric parameter n from independent studies but are not able to come up with an accurate estimate for macromolecule concentration M Sigurskjold Altman amp Bundle 1991 Eur J Biochem 197 239 246 Using Origin it is an easy matter to determine M along with the correct binding constant and heat of binding from curve fitting The procedure 1s as follows 1 Guess at the macromolecular concentration and enter this incorrect concentration M into the Concentration dialog box 2 Select the model for curve fitting and proceed to find the best fit in the usual way The values which you obtain for the binding constant and heat of binding will be correct since these depend only on the accuracy of the ligand concentration However the best value which appears for the stochiometric parameter n will be incorrect since you wish to assign this yourself and after making the correct assignment n to determine the actual M 3 Once curve fitting is completed you may calculate the correct M which is equal to the incorrect concentration M times the ratio n n You may satisfy yourself that the above procedure is correct by calling the RNAHHH ITC data into Origin do curve fitting using the correct concentration and record the best values of parameters n K and H as the corre
141. xact font placement and size is guaranteed at some sacrifice to screen appearance since the printer driver fonts must be scaled to fit their positions on the page this will not harm the appearance of true vector fonts This is a slow process and screen refresh speed suffers as a result Thus reserve the Print View mode for previewing your work prior to printing Origin automatically changes to Print View mode when graphics are exported to another application and when printing The view mode automatically returns to the selected view mode after the operation is complete Page View provides faster screen updating than Print View but does not guarantee exact text placement on the screen unless you are using typeface scaling software such as Adobe Type Manager Use Page View mode until your application is ready for printing or copying to another application Change to Print View mode to check object placement before exporting copying or printing 6 MAU130010 Rev D Getting Started Window View expands the page to fill up the entire graph window Labels buttons or other objects in a graph window that reside in the gray area of the page are not visible in Window View mode Draft View has the fastest screen update of the four view modes In Draft View the page automatically sizes to fill the graph window This is a convenient mode to use when you are primarily interested in looking at on screen data Note that view mode will not affe
142. xt label named Fit P to display the fitting parameters To name a text label click on the label once to select it select Format Label Control and enter a name in the Object Name text box in the Label Control dialog box MAU130010 Rev D Lesson 1 Routine ITC Data Analysis and Fitting To format the fitting parameters text e Right click anywhere in the text box and select Properties item from the drop down menu The Text Control dialog box will appear allowing you to format the fitting parameters text Text Control E4 Background Shadow M Use System Font T Center Multi Line jo 7 Rotate deg I White Out Cancel Table 4 Size 14 T Apply formatting to all labels in layer Set Default Tp Arial E Black N Bl z ul T Data Rnahhh HDH Hodel OneSites Chi 2856 23 H 1 023 86 661628 K 5 543E4 1665 giDJH 1 361E4 29 83 4q D S 22 01 B F Data Rnahhh MOH odel Onesies hi 2856 23 1 0235 0 001626 Ss 4SE4 1065 1 361E4 2905 22 01 The Text Control dialog box is in three sections The upper section contains various formatting options The middle contains the text box where the desired text with formatting options are entered The lower view box provides a WYSIWYG What You See Is What You Get display of the text entered into the middle text box Hint Press the F1 key while the Text Control dialog box is open for Online help and a thorough description of text formattin

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