PHOSPHORYLATION OF HUMAN SPERM PROTAMINES HP1 AND HP2 - IDENTIFICATION OF PHOSPHORYLATION SITES

Human sperm is characterized by a high heterogeneity of its basic nucl ear protein complement of pro-protamines, protamines and histones. Thi s heterogeneity is increased by the persistence of phosphorylated prot amines in mature spermatozoa. Alkaline phosphatase treatment of whole protein indicated that protamines HP1 and HP2 were phosphorylated to v arious degrees. Presence of non-phosphorylated and phosphorylated prot amines HP1 and HP2 was further demonstrated by electrospray mass spect rometry. Phosphorylation sites of mono- and di-phosphorylated protamin e HP1 were identified by automatic Edman degradation of the protein af ter phosphoserine derivatization to S-ethylcysteine. In both phosphory lated forms, Ser-10 was found phosphorylated; in the di-phosphorylated form, Ser-8 was identified as the second site of phosphorylation. In protamine HP2, the unique site of phosphorylation (Ser-14) was located after limited acid hydrolysis of enzymic peptides and thin-layer elec trophoresis.