Primary structures of two protamine 2 variants (St2a and St2b) from stallion spermatozoa

Protamines were extracted from stallion sperm cell nuclei, alkylated with iodoacetamide and separated by reversed-phase high-performance liquid chromatography. Two main components, protamine 1 and protamine 2, were obtained. The latter contains two subspecies, separable by acetic acid-urea-polyacrylamide gel electrophoresis. The primary structure of protamine 2a (St2a) was determined by analysis of fragments obtained from purified protamine 2 peak by thermolysin digestion. The digested peptides were separated by acetic acid-urea gel electrophoresis and, after electroblotting onto a polyvinylidene difluoride filter, their amino acid sequences were determined by pulse liquid peptide sequencing. The amino acid sequence of protamine 2b was predicted from the double sequence data of protamine 2 peak by eliminating the amino acid of St2a in each cycle. St2a and St2b were found to contain 62 and 58 amino acid residues, respectively, and they seem to be homologous with type 2 protamines from human and mouse spermatozoa.