Ronald E. McNair Scholars Summer Research Institute; Center for Academic Enrichment and Outreach
University of Nevada, Las Vegas; Center for Academic Enrichment and Outreach
Las Vegas (Nev.)
The binding affinity between an enzyme and its substrate is often dependent on the pH of the local environment. Knowing the pH at which reduced glutathione (GSH) binds with the highest affinity to the enzyme glutathione S-transferase (GST) is useful for determining the optimal pH for purification of GST-fusion proteins during GST-affinity chromatography. In this study, GST of the species Schistosoma japonicum was purified, quantified, and utilized to study its binding interaction with GSH at pH 6.5 and 8.5 via isothermal titration calorimetry (ITC). After protein expression, extraction, and purification, the GST concentration was quantified using QubitTM fluorometry. Thermodynamic properties and a dissociation constant (KD) for each experiment were obtained utilizing the MicroCal PEAQ-ITC Analysis Software for the binding of GSH to GST at pH 6.5 and 8.5. Statistical analysis of the technical replicate data was performed to obtain an average and standard deviation of the KD at each pH point. The results indicate a statistically significant difference (p
Glutathione; Glutathione S-transferase; Binding affinity; Dissociation constant; pH dependence; Isothermal titration calorimetry; Thermodynamics
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Renshaw, C. P.,
Gary, R. K.
Quantifying the Differences in Binding Affinity of Reduced Glutathione for Glutathione S-Transferase at pH 6.5 and 8.5 Using Isothermal Titration Calorimetry.
Available at: https://digitalscholarship.unlv.edu/mcnair_posters/95