Amino Acids, Amines and Peptides of Ram

Epididymal semen was obtained from rams with a fistula present in the vas deferens (Tadmor & Schindler, 1966). It was deproteinized immediately, and the protein-free clear supernatant evaporated to dryness in vacuo at a temperature not exceeding 55\s=deg\C. The residue was taken up with about 10 ml water, and the solution thus obtained used for determination of both amines and amino acids. For the determination of amino acids, the solution was once more dried in vacuo, and the residue taken up with 3 ml 75% ethanol. Two-dimensional chromatograms of two samples were run, using 0\m=.\2ml of the final solution, according to Mizell & Simpson (1961). For the determination of the amines, the solution was extracted by the method of Yoshinaga, Itoh, Ishida, Sato & Wada (1961), slightly modified (Neumark, 1964). After three extractions with ethyl acetate (50 ml each time), the combined material was evaporated to dryness in vacuo, the residue taken up with 1 ml absolute methanol, and the solution thus obtained used for chromatography. Descending chromatograms were run using two 0\m=.\4ml samples; the solvent mixture used was composed of n-butanol : glacial acetic acid: water (12:3:5). The dried chromatograms were treated with a solution of ninhydrin (0\m=.\2 g/100 ml acetone) containing 10% (v/v) glacial acetic acid. The sheets were dried at 110\s=deg\C for 4 min. For the determination of peptides, the epididymal semen was deproteinized by saturation with ammonium sulphate. After removal of the precipitate, the supernatant was dialysed against constantly flowing distilled water for 36 hr. The non-dialysable material was evaporated in vacuo, and the residue taken up with 75% ethanol. The solution thus obtained served for chromatography. About 2 ml was applied to a paper sheet and chromatographed in one direction. The different peptides were spotted by developing small reference strips of the chromatogram. The corresponding areas in the main chromatogram