Human protamine P2 was purified to homogeneity by solubilizing whole spermatozoa in guanidinium . HC1 containing 2-mercaptoethanol, alkylating the resulting protamine thiols with vinylpyridine, removing acid-insoluble material by acid dialysis and using CM-cellulose chromatography to remove non-protamine basic proteins and separate protamines P1 and P2. The P2 preparation contained two components, P2a and P2b, which were sequenced completely without being separated. The peptides obtained from thermolysin and endoproteinase Lys-C digestions were purified by reverse-phase high-pressure liquid chromatography and sequenced using a gasphase sequencer. P2a contains 57 amino acids and has a relative molecular mass of 7636 while P2b contains 54 amino acids, which are identical to residues 4-57 of P2a, and has a relative molecular mass of 7242. Protamine P2a is approximately 50% homologous with human protamine PI. The amino acid sequence of P2a is: In a previous paper [I 31 we reported the complete amino acid sequence of human protamine PI whch was found to be quite homologous to the single protamine in bull [I], ram [2] and boar [3] as well as to mouse protamine 1 [14]. In this paper we present the complete amino acid sequences of two forms of the second major human protamine, P2. MATERIALS AND METHODS MaterialsThe materials used have been described previously [13] Methods Two purification methods were used to isolate human protamine P2. The first method was the modified procedure of Kolk and Samuel [9] which was used to obtain pure protamine PI [13]. Whole spermatozoa from 8 -10 fertile donors were washed, dissolved in 6M guanidinium . HC1 containing 2-mercaptoethanol and alkylated with vinylpyridine. The resulting solution was dialyzed against 2% HCl and the dialysate supernatant was fractionated on a Bio-Rex 70 ionexchange column. The second method was the same as the first except that CM-cellulose [I51 was used in place of the Bio-Rex 70 in the ion-exchange step. CM-cellulose (CM-52, Whatman) was equilibrated with 5% guanidinium . HCI in 50 mM lithium acetate buffer at pH 5.0 and packed into a 1 .0-cm-diameter column on top of a 0.5-cm bed of Sephadex G-10 to a height of 8 cm. The acid dialysate supernatant was mixed with 2.5 g guanidinium . HC1, diluted to 50 ml with lithium acetate buffer and run into the column under gravity at room temperature. Only the very basic protamines bound to the CM-cellulose and they were eluted with a linear gradient of 5 -15% guanidinium . HC1 in lithium acetate buffer in a total volume of 200ml. The fractions under each 230-nm absorbance peak were pooled, exhaustively dialysed against 1% acetic acid in Spectrapore 3 tubing, dried down in a vacuum centrifuge, redissolved in 0.2 M acetic acid and stored at -20 "C. Amino acid analysis, automated protein sequencing and reverse-phase peptide HPLC were performed as described previously [13].Protamine P2 was digested with thermolysin by dissolving 18 nmol in 0.2 ml0.5% ammonium bicarbonate, adding 2 pg thermolysin and incubating the solu...

The endogenous pool sizes of 17 amino acids were measured directly in samples of mouse eggs, 8-cell embryos and blastocysts by estimation of the fluorescent product of the reaction of o-phthalaldehyde and primary amines. Taurine, glycine, alanine, glutamate and aspartate were detected at high levels. During the transition to the blastocyst, most amino acid pools increased 2-3-fold, but the taurine and glycine pools decreased to about 50 and 10%, respectively, of the egg value. The amino acid distribution in cumulus masses was similar to that of the egg and embryo samples but different from that of serum.

Human sperm protamines have been extracted from spermatozoa pooled from several donors, converted to their S-pyridylethylated derivatives and resolved into two major components, P1 and P2, by Bio-Rex 70 chromatography. Protamine P1 was further purified by Bio-Gel P-10 chromatography and sequenced directly on a gas phase protein sequencer for 43 residues. To complete the sequence, P1 was cleaved at methionine 36 and the C-terminal tetradecapeptide was purified by h.p.l.c. and sequenced completely. The 50 residue sequence is: (sequence see text) This sequence has a calculated molecular weight of 6674 and is homologous with four other published mammalian protamine sequences.

Two mouse protamines, denoted as P1 and P2, have been purified directly from mature sperm nuclei and characterized as distinct polypeptide species. The complete primary structure of P2 was determined by peptide sequencing analyses. P1 and P2 were purified by a sequence of cation-exchange chromatography on Bio-Rex 70 and permeation chromatography on Bio-Gel P10, both in the presence of guanidine hydrochloride. Biochemical analyses demonstrate P1 has a molecular weight of 7400 and is characterized by the presence of arginine, cysteine, lysine, and tyrosine. By contrast, P2 is unusual in containing an abundance of arginine, histidine, lysine, and cysteine, but no tyrosine. The primary structure of P2 was determined from the sequencing of overlapping, high-pressure liquid chromatography purified peptides generated by thermolysin and endoproteinase Lys-C digestions and by chemical cleavage at each of four serine residues. Sequence analyses have demonstrated that P2, with a molecular weight of 8841, contains 62 amino acids, in the sequence NH2-Arg-Gly-His-His-His-His-Arg-His-Arg-Arg-Cys- Ser-Arg-Lys-Arg- Leu-His-Arg-Ile-His-Lys-Arg-Arg-Arg-Ser-Cys-Arg-Arg-Arg-Arg-Arg-His-Ser- Cys-Arg - His-Arg-Arg- Arg-His-Arg-Arg-Gly-Cys-Arg-Arg-Ser-Arg-Arg-Arg-Arg-Arg-Cys-Arg-Cys-Arg- Lys-Cys - Arg-Arg- His-His-COOH. Thus, the primary structure includes six clusters of arginine and histidine, distributed throughout the polypeptide, each ranging from five to eight amino acids in length. Sequence comparisons of mouse and human protamines by the Dayhoff program have revealed greater homology exists between human P2 and mouse P2 than within the P1 family from the two mammalian species.