Isothermal Titration Calorimetry in College Physical and Biochemical Chemistry Laboratory Courses

Abstract

Isothermal titration calorimetry (ITC) is a relatively new technique that is used to determine thermodynamic parameters for a reaction. ITC has become an essential tool in research and in the pharmaceutical industry. However, such an ultra‐sensitive calorimetry technique is absent in most undergraduate curriculums. Our institute implemented ITC in both Physical and Biochemistry laboratory courses in a sequential way. Students were first introduced to ITC and studied the reactions of EDTA to Mg2+ in the Fall semester. This exercise strengthened their understanding of the relationship among pKa of functional groups, pH of buffer, and protonation states of protein, which are commonly found in enzymatic catalysis. When paired with inferential questions, this exercise brought about effective critical thinking. In the following Spring semester, students performed ITC to determine the binding affinity of lysozyme to NAG3 and extended the measurement to a much weaker inhibitor, NAG, by a competitive binding assay. The enthalpy difference obtained from ITC was correlated to the number of hydrogen bonds formed, visualized by PyMol software. The assessment indicated that the successive ITC experiments in the Physical and Biochemistry labs served as an effective education tool. This work is supported in part by National Science Foundation and SIUE's Excellent in Undergraduate Education.