Isolation and Characterization of the Major Gel Proteins in Human Semen, Semenogen I and Semenogen II

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Abstract
Semenogen I and semenogen II constitute the major gel-forming proteins in human semen. The gel proteins were rapidly solubized and separated from spermatozoa in ejaculates collected at pH 9.7 in buffer containing 4 mol/1 urea and dithiothreitol. This protected the semenogens from proteolytic degradation by prostate-specific antigen, and allowed their isolation by affinity chromatography on heparin-Sepharose. Semenogens I and II were almost selectively retained and eluted partially separated in 0.25 mol/1 NaCl. Further purification was achieved by chromatography on Superose. Approximately 10–20 mg semenogen I and 2–5 mg semenogen II were recovered from each sample with a purity exceeding 95% as judged by SDS/PAGE. The molecular mass of semenogen I (49 958 Da) and the major form of semenogen II (63539 Da) measured by mass spectrometry was consistent with the reported cDNA data. The occurrence of a second, larger form of semenogen II was due to asparagine-nked glycosylation. The amino-termini of the purified proteins were blocked, but digestion with pyroglutamate amino-peptidase enabled the identification of amino-terminal sequences consistent with the reported cDNA data. The amino acid compositions of the purified proteins were also consistent with those derived from cDNA data. The absorption coefficients (280 nm, 1%, 1 cm) for semenogens I and II were 5.5 and 5.4, respectively, and the isoelectric point was above pH 9.5 for both proteins.