PURIFICATION AND CHARACTERIZATION OF THREE ALPHA-2-ANTIPLASMIN AND ALPHA-2-MACROGLOBULIN INACTIVATING ENZYMES FROM THE VENOM OF THE MEXICAN WEST COAST RATTLESNAKE (CROTALUS BASILISCUS)

摘要

Three distinct alpha 2PI (alpha 2-antiplasmin) degrading and alpha 2M (alpha 2-macroglobulin) inhibiting enzymes, named proteinase a, b and c, have been purified from the venom of Crotalus basiliscus (the Mexican west coast rattlesnake) by fast protein liquid chromatography (anion-exchange chromatography and gel filtration chromatography). SDS-PAGE revealed that proteinase a and b had similar mol. wts (approximately 23,500), whereas proteinase c displayed a mol. wt of approximately 24,200. Their isoelectric points were found to be acidic, ranging from pH 4.8 to 5.7. The proteinase activity of all three enzymes was inhibited in the presence of EDTA. Dependent on enzyme concentration, a progressive and catalytic inactivation of alpha 2PI was induced, leading to an almost complete loss of the plasmin inhibitory activity at a molar ratio of enzyme: alpha 2PI = 0.1 within 60 min. The ability of alpha 2M to protect the esterolytic activity of trypsin from inhibition by soybean trypsin inhibitor was only reduced at a molar ratio of enzyme:alpha 2M = 0.5, whereas no inactivation could be observed when the three venom proteinases were incubated with an excess of alpha 2M, suggesting that the inactivation occurred by complex formation but not by degradation of the intact alpha 2M molecule. In SDS-PAGE, inactivation of human alpha 2PI (mel. wt 68,000) correlated with the appearance of two cleavage products with an approximate mel. wt of 56,000 and 11,000, respectively. The three proteinases had no thrombin-like activity. Plasminogen and factor X were not activated and no platelet aggregation was induced. They degraded the A alpha- and B beta-chain of fibrinogen and showed plasma extravasation-inducing activity following intradermal injection into the abdominal skin. None of the enzymes showed any activity against a series of chromogenic p-nitroanilide substrates. [References: 47]