Solubility-activity relationships in the inorganic salt-induced precipitation of α-chymotrypsin

Abstract

Inorganic salt-induced precipitation is a commonly employed protein separation/concentration technique that is potentially denaturing. The effects of precipitating environments on recoverable activity were investigated using α-chymotrypsin as a model protein. The following salts, in order of increasing denaturation potential and decreasing molal surface tension increment, were studied: Na 2SO 4, NaCl, NaBr, KBr, and KSCN. These salts span the lyotropic series. Solubility measurements gave salting-out constants that disagree with current salting-out theory. Salt-protein interactions and concomitant protein structural perturbations are the likely source of the discrepancies. Activity and active fraction measurements indicated that the fraction of precipitate that is active upon dissolution is a function of the salt molal surface tension increment, but not the salt concentration. The specific activity of soluble precipitate remained essentially constant regardless of salt concentration or type. A salting-out performance parameter was defined and calculations indicated that an optimum salt may exist for a particular protein. The recovered active fraction was not affected by varying the amount of salt added and, correspondingly, the amount of protein precipitated.