Purification and Properties of a Proteinase Inhibitor from Chicken Seminal Plasma1

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Abstract
A low molecular weight, acid stable proteinase inhibitor similar to those found in mammalian species was identified in chicken seminal plasma. The inhibitor was purified to a specific activity of 250 units/mg by means of affinity chromatography on insolubilized trypsin, gel filtration and cation exchange. Ion exchange resulted in partial resolution of 2 active forms which probably represent native and modified versions of the same inhibitor. The amino acid composition of the chicken inhibitor was similar to those reported for mammalian seminal plasma proteinase inhibitors. A molecular weight of 6,100 was obtained by gel filtration under denaturing conditions and by calculation from the amino acid composition. The inhibitor was capable of inhibiting trypsin, acrosin and plasmin, but not chymotrypsin. Formation of the inactive proteinase-inhibitor complex was completed within minutes. A value of 7.5 x 10-9 M was found for the dissociation constant of the trypsin-inhibitor complex. The acrosin-inhibitor complex was more resistant to competitive displacement of the inhibitor by benzoyl-arginine ethyl ester than was the trypsin-inhibitor complex. Benzoyl-arginine-p-nitroanilide caused little competitive displacement of inhibitor from the trypsin-inhibitor complex and appeared to be superior to benzoyl-arginine ethyl ester as a substrate for routine inhibitor assays. The physiological function of the proteinase inhibitor in chicken seminal plasma may be related to the inactivation of acrosin released from dead or damaged spermatozoa.