Isolation and characterization of nuclear basic protein (protamine) from boar spermatozoa

Abstract

1. Sperm nuclei were isolated and purified from boar semen by a procedure involving differential solubilization of sperm tail and acellular materials by brief exposure to reducing reagent in the presence of cationic detergent, and sedimentation through 60% sucrose. The weight ratio of DNA: RNA:total protein:protamine in this preparation was 1.00:0.02:1.05:0.75, and the molar ratio of phosphorus to arginine was 1.12. 2. Boar protamine was extracted with cold acid from ethanol precipitate of reduced and carboxymethylated nuclei in 6 M guanidine hydrochloride and purified by ion-exchange chromatography on CM-cellulose. The molecular weight of the protamine was estimated to be 6600 by the gel filtration method. The protamine consisted of a single amino terminus alanine and either half-cystine or arginine as carboxy terminus, and was composed of Thr, Ser 3, Pro 2, Ala 2, Val 2, Ile, His, Haif-cystine 9–10 and Arg 26. 3. Chymotryptic digestion gave rise to a single amino-terminal peptide Ala-Arg-Tyr, and two carboxy-terminal peptides, Thr-Val-Ile-Arg-Cys-Arg 2Cys and Thr-Val-Ile-Arg-Cys-Arg 2, which confirmed the heterogeneity of the protamine at the carboxyterminal end.