Sagar S L, Domach M M
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Bioseparation. 1995 Oct;5(5):289-94.
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.
结晶α-胰凝乳蛋白酶制剂被翻译后变体γ-胰凝乳蛋白酶污染。基于热分析，污染物可占制剂重量的5%-50%。这种污染可能会带来问题，因为这种丝氨酸蛋白酶具有商业和失活模型系统用途，并且活性测定可能检测不到污染物的存在。先前的工作表明，当加载到Cu(2+)-IMAC柱上时，简单的pH梯度洗脱可以分离两种胰凝乳蛋白酶；γ-胰凝乳蛋白酶先洗脱，表明其与固定化Cu2+的相互作用较弱。通过在有和没有Cu2+的情况下以及在不同pH值下进行差示扫描量热法(DSC)研究，进一步研究了赋予这些丝氨酸蛋白酶金属亲和力的分子特征。在固定金属浓度下，热稳定性对pH的依赖性揭示了稳定和不稳定金属结合事件之间的相互作用。结果与Cu(2+)-胰凝乳蛋白酶相互作用部分通过与含谷氨酸或天冬氨酸的螯合位点结合而发生一致。该位点的强度在两种胰凝乳蛋白酶中可能不同。
