Using differential scanning calorimetry to elucidate metal-protein binding sites in alpha- and gamma-chymotrypsin.

Crystalline alpha-chymotrypsin preparations are contaminated by the post translational variant, gamma-chymotrypsin. The contaminant can account for 5-50 weight percent of the preparation based on thermal analysis. Such contamination can be problematic because this serine protease has both commercial and deactivation model system utility, and the presence of the contaminant may not be detectable by activity assays. Prior work has shown that simple pH gradient elution can separate the two chymotrypsins when loaded to a Cu(2+)-IMAC column; gamma-chymotrypsin eluted first indicating that its interaction with immobilized Cu2+ is weaker. The molecular features that endow these serine proteases with metal affinity has been investigated further by performing differential scanning calorimetry (DSC) studies in the presence and absence of Cu2+, and at different pH values. The dependence of thermostability on pH for fixed metal concentration reveals an interplay between stabilizing and destabilizing metal binding events. The results are consistent with Cu(2+)-chymotrypsin interaction occurring, in part, through binding to a glutamate- or aspartate-containing chelation site. The strength of this site may differ in the two chymotrypsins.