基于金属的α-和γ-胰凝乳蛋白酶亲和分离及分离物的热稳定性分析

Metal-based affinity separation of alpha- and gamma-chymotrypsin and thermal stability analysis of isolates.

作者信息

Sagar S L, Beitle R R, Ataai M M, Domach M M

机构信息

Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA.

出版信息

Bioseparation. 1992;3(5):291-6.

PMID:1369428

Abstract

Chymotrypsin preparations are contaminated by autolysis products and other post-translational products derived from the zymogen. Some prevalent contaminants are difficult to detect with activity assays and molecular weight separation. However, our differential scanning calorimetry (DSC) studies of alpha-chymotrypsin have revealed that gamma-chymotrypsin is a common contaminant in commercial preparations, and the alpha- and gamma-species differ significantly in thermal stability. Thus, DSC analysis provides a sensitive and rapid means to assess the homogeneity of preparations. Moreover, free metal ion binding studies conducted indicate that the alpha- and gamma-species differ substantially in the number of metal binding sites and metal affinity. Therefore, to attempt to repurify a commercial preparation of alpha-chymotrypsin, a resolubilized sample of alpha-chymotrypsin was subjected to immobilized metal (Cu+2) affinity chromatography with pH elution and the fractions were subjected to DSC analysis. The process successfully removed the majority of the contaminating gamma-chymotrypsin.

摘要

胰凝乳蛋白酶制剂被自溶产物和其他源自酶原的翻译后产物污染。一些常见的污染物难以通过活性测定和分子量分离来检测。然而，我们对α-胰凝乳蛋白酶的差示扫描量热法（DSC）研究表明，γ-胰凝乳蛋白酶是商业制剂中的常见污染物，并且α型和γ型在热稳定性上有显著差异。因此，DSC分析提供了一种灵敏且快速的方法来评估制剂的纯度。此外，进行的游离金属离子结合研究表明，α型和γ型在金属结合位点数量和金属亲和力方面有很大差异。因此，为了尝试对商业α-胰凝乳蛋白酶制剂进行再纯化，将重新溶解的α-胰凝乳蛋白酶样品进行固定化金属（Cu²⁺）亲和色谱法，采用pH洗脱，然后对各馏分进行DSC分析。该过程成功去除了大部分污染性γ-胰凝乳蛋白酶。