Foundations of Biomedical Science Exam
This figure represents an isothermal titration calorimetry experiment. Protein X and ligand Y interact inside a calorimeter chamber. The experiment is done like this:
The heat released can be measured by the area of each one of the peaks shown in the upper panel. Calorimeters come with software that does this automatically for you and generate curves like that shown in the lower panel. The software corrects for the small dilution of X in the chamber (after 13 injections the volume has increased from 200 to 213 ?l assuming that nothing gets removed). Therefore, the Molar Ratio (mols of Y/mol of X in the chamber) is an accurate number. The analysis software extracts several parameters from these data: ?H, ?S, K (equilibrium constant), and some estimate of the stoichiometry of the reaction.
Your job is to replace the instrument’s software with your understanding of the analysis methodology. In other words, you must calculate the thermodynamic numbers associated with this reaction. You can do this using Excel or a piece of software available for download from the University (Prism). Because you need to plug in some numbers, there is a piece of freeware that is very useful for the digitation of plots (Plotdigitizer: http://plotdigitizer.sourceforge.net). If you don’t want to use Plotdigitizer, you can always extract the data using a ruler and measuring distances to the X and Y axes.
These are your tasks:
1. Transform the lower graph into a classical binding isotherm and plot Bound [Y] vs Free [Y] (or Bound [Y] vs log Free [Y]).
2. Calculate the equilibrium constant and the binding stoichiometry.
3. If the stoichiometry is not 12, please discuss your result.
4. Calculate the ?H and the ?S of the reaction. The SIGNS are important.
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