Biology of Reproduction, Vol 25, 197-201, Copyright © 1981 by Society for the Study of Reproduction

Improved Method for the Preparation and Purification of Boar m-Acrosin

¹ Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110
² Department of Urology, Washington University School of Medicine, St. Louis, Missouri 63110

Boar m-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 m-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 m-acrosin by completely suppressing the conversion of m-acrosin into lower molecular weight forms. As a consequence, the overall yield of m-acrosin from proacrosin has been improved 2.5-fold compared with previously reported methods, and sufficient quantities of boar m-acrosin can now be prepared so that future in vitro characterization studies can be undertaken.

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ACKNOWLEDGMENTS The authors are grateful to Dr. Billy Day for providing the boar semen. Research supported by grants from the Rockefeller Foundation and NIH (HD 12863, HD 09422, and HD 00296).