Thermodynamic Exploration of Eosin-Lysozyme Binding A Physical Chemistry and Biochemistry Laboratory Experiment

We have developed a modular pair of laboratory experiments for use both in undergraduate physical chemistry and biochemistry. Both laboratories examine the thermodynamics of the binding of a small molecule, eosin, to the protein lysozyme. The assay for binding is the quenching of lysozyme fluorescence by eosin through resonant energy transfer. In both experiments students measure fluorescence quenching at constant lysozyme concentration as a function of added eosin and determine the dissociation constant and ∆G for binding. In the physical chemistry experiment, students repeat the fluorescence measurements at several temperatures to determine the temperature-dependence of ∆G, and therefore ∆H and ∆S as well. Typical student results yield a dissociation constant, KD, of 22.7 ± 2.0 μM and a binding ∆G of -26.49 ± 0.22 kJ mol at 25 °C. ∆H and ∆S of binding are found to be -4.20 ± 0.80 kJ mol and 74.6 ± 2.7 J mol K, respectively. This pair of laboratories represents an interdisciplinary module in which students in different courses examine the same system, but from different perspectives and with different emphases.