Purification and partial characterization of proteinase inhibitors of equine seminal plasma

SUMMARY The aims of the study were 1/to isolate and identify equine seminal plasma proteinase inhibitors, 2/to evaluate their inhibitory potential, and 3/to test a correlation between protein concentration in seminal plasma supernatant (obtained after precipitation with 36% ammonium sulfate) and stallion sexual maturity. Seminal plasma proteins obtained from six stallions were chromatographed in a Superose 12 (FPLC system) column followed by C 18 HPLC reverse-phase. Inhibition of trypsin amidase activity was evaluated in the collected fractions. Active proteins with a molecular mass of 6.3-7.0 kDa were identified using mass spectrometry. The older stallions showed a reduction in total seminal plasma protein concentration, but had similar concentrations of proteinase inhibitors (0.28+0.10 mg/ml) in seminal plasma supernatant. Different proteinase inhibitor isoforms were found in semen of all stallions which suggests that the isoforms may be used as biomarkers of individual animals.