Radioimmunoassay Study of Immunologic Changes Associated with the Conversion of Rabbit Testicular Proacrosin to Acrosin

This study was made to determine the degree of immunologic similarity or dissimilarity between rabbit proacrosin and acrosin and thereby to elucidate the extent of molecular change that occurs when proacrosin is converted to acrosin. A highly sensitive and specific radioimmunoassay for rabbit acrosin was developed and used to quantify the immunologic reactions. Testicular proacrosin was purified, but not to homogeneity, by sequential acid extraction, gel filtration, cation exchange and Concanavalin A chromatography. Acrosin immunogen was purified to homogeneity by subjecting activated proacrosin to affinity chromatography on CH-Sepharose benzamidine. A monospecific antiserum against acrosin was obtained from female rabbits following immunization with 500 µg doses of TLCK (tosyl-lysine-chloromethyl-ketone) treated acrosin 6 weeks apart. A labeled antigen (specific activity 40 µCi/µg) for the radioimmunoassays was obtained by iodination of TLCK treated acrosin by the chloramine T method. All subsequent immunologic tests were performed in the presence of 0.05 M benzamidine. Qualitative indications of immunologic dissimilarity between proacrosin and acrosin were obtained in Ouchterlony tests where proacrosin gave no precipitin band while acrosin gave a strong band against the antiacrosin serum. For the radioimmunoassays, serial dilutions of proacrosin, representing progressive stages in the activation, were used to generate dose response curves with the antiacrosin serum. Calculations based on the 50% displacement values obtained from these curves showed that the cross reaction between unactivated proacrosin and antiacrosin antibody was only 1.8% of that obtained with fully activated proacrosin (acrosin) and the same antibody. The results indicate that a major conformational change occurs when proacrosin is activated to acrosin.