Metal-based affinity separation of alpha- and gamma-chymotrypsin and thermal stability analysis of isolates.
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.
Sagar
S L
SL
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA.
Beitle
R R
RR
Ataai
M M
MM
Domach
M M
MM
D002152:Calorimetry, Differential Scanning; D002846:Chromatography, Affinity / Q000379:methods*; D002918:Chymotrypsin / Q000302:isolation & purification* / Q000378:metabolism; D003300:Copper; D004795:Enzyme Stability; D006358:Hot Temperature; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D003300:Copper; D002918:Chymotrypsin