Improved method for the preparation and purification of boar m alpha-acrosin.

Abstract

Boar m0-acrosin has been prepared and purified in high yield starting with a partially purified preparation of proacrosin. The simple, three step procedure included 1) selective and quantitative conversion of proacrosin into m0-acrosin at pH 8 using excess leupeptin, a highly effective acrosin inhibitor; 2) dissociation of a tightly acrosin-bound protein by denaturation in 6 M guanidine hydrochloride at pH 3; and 3) gel filtration over Sephadex G-100 superfine resin. The enzyme so obtained appeared homogeneous as judged by SDS-polyacrylamide disc gel electrophoresis, and had, under reducing conditions, an apparent molecular weight of 49,000. These methods allowed for the quantitative conversion of proacrosin into m0-acrosin by completely suppressing the conversion of m0-acrosin into lower molecular weight forms. As a consequence, the overall yield of m0-acrosin from proacrosin has been improved 2.5-fold compared with previously reported methods, and sufficient quantities of boar m0-acrosin can now be prepared so that future in vitro characterization studies can be undertaken.