Bombardment of bovine spermatozoa with glass beads and subsequent extraction in phosphate buffered sodium chloride yielded soluble sperm-specific proteins (approximately 10% yield) sufficient for physicochemical analysis.
Moving boundary electrophoresis revealed three sperm-specific components with mobilities of 2·0, 3·8 and 5·1×10-5 cm2 volt-1 sec-1 at pH 8·6 in barbital (μ = 0·10). The proportions of these components were 20, 50 and 30%, respectively. Sedimentation velocity ultracentrifugal analysis revealed at least three sedimentation gradients. The two major gradients had S20 values of 1·7 and 12·6. The third exhibited polydispersion. Diffusion coefficients of 4·2 and 10·2×10-7 cm2 sec-1 were calculated for two sperm-specific antigens by an agar-gel-diffusion method.
Three protein fractions were isolated by chromatographic elution from diethylaminoethyl (deae) cellulose with an ionic gradient. The first protein fraction eluted was not bound to the deae cellulose. It migrated toward the cathode in agar-gel electrophoresis at pH 8·4, and was firmly bound to carboxymethylcellulose at pH 6·0, 7·6 and 11·0. A second protein fraction from the deae cellulose appeared just after the ionic gradient commenced, and a third protein fraction appeared when the ionic gradient approached 0·2 m-sodium chloride.