Purification and partial chemical characterization of a glycoprotein with antifertility activity from human seminal plasma.
The preparation of a highly purified antifertility factor from human seminal plasma is described, and some of its biochemical properties have been determined. Purification was achieved by ultracentrifugation of particle-free human seminal plasma, followed by chromatography of the precipitate using carboxymethyl cellulose, concanavalin A, and Sepharose 6B columns. An occasional contaminant was further removed by preparative isoelectric focusing. During the purification procedures, the activity of the fractions was monitored by mixing them with capacitated mouse spermatozoa for 20 min before adding an aliquot to intact mouse oocytes, and determining fertilization after 24 h. The I50 (amount causing a 50% reduction in fertilization as compared to the control) of the final purified factor was 45 micrograms protein. Purity was established by standard and sodium dodecyl sulfate disc gel electrophoresis, isoelectric focusing, high-pressure liquid chromatography, and sedimentation analysis. These methods, as well as Sepharose gel filtration, were also used for the molecular weight estimations; good agreement was obtained between the various techniques. The factor appears to be a glycoprotein with a molecular weight of about 200,000. It consists of two subunits with molecular weights of about 125,000 and 72,000 and s-20,w of 6.2 s and 4.3 s. The factor contains relatively high amounts of aspartic acid and glutamic acid residues as well as leucine and serine, but only small amounts of tryptophan and no methionine was detected. The carbohydrate fraction is particularly rich in galactose and N-acetylgalactosamine but also contains mannose and N-acetylglucosamine and small amounts of fucose and sialic acid.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D005306:Fertilization / Q000187:drug effects; D006023:Glycoproteins / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D008297:Male; D008970:Molecular Weight; D029584:Prostatic Secretory Proteins; D011487:Protein Conformation; D011506:Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D012661:Semen / Q000032:analysis*; D029589:Seminal Plasma Proteins
D000596:Amino Acids; D002241:Carbohydrates; D006023:Glycoproteins; D029584:Prostatic Secretory Proteins; D011506:Proteins; D029589:Seminal Plasma Proteins; C043076:beta-microseminoprotein