Abstract
Two proteinases (proteinases I and II) have been purified from Crotalus adamanteus venom to the stage of electrophoretic homogeneity and proteinase II has been crystallized. The proteinase differ slightly in molecular weight and amino acid composition. Both are metalloenzymes requiring Zn2+ or Ca2+, or both; neither requires thiol compounds for activation. The proteinases are free of esterolytic activity against benzoly-L-arginine ethyl ester and benzoyl--tyrosine ethyl ester. Proteinase II cleaves the oxidized B chain of insulin at the bonds Phe1-Val2, His5-Leu6, His10-Leu11, Ala14-Leu15, Leu15-Tyr16, and Tyr-16-Leu17. Digestion of polylsine and polyarginine by proteinase II liberates products ranging from dodecapeptides to hexapeptides. Proteinases I and II catalytically inactive human plasma alpha 1-proteinase inhibitor (54,000 daltons). Electrophoretic analysis of the reaction of proteinase II with alpha 1-proteinase inhibitor reveals that an inactivated inhibitor species of 50,000 daltons is formed, and a peptide of 4,000 daltons is released. The gradual disappearance of the native inhibitor results in the corresponding loss of inhibitory activity against trypsin and chymotrypsin.
Highlights
Venoms from snakes of the families Crotalidae and Viperidae are known to possess a variety of proteinases which react with plasma proteins [28]
The results reported in the present paper extend the interaction of venom proteinases to another class of plasma proteins, namely, the plasma proteinase inhibitors
The inactivation of alPI by venom proteinase II proceeds in a fashion similar to that reported for papain and alPI [8]
Summary
Materials-Venom of Crotalus adamanteus (Lot CAGSO-1Z) was obtained from Miami Serpentarium, hide powder azure was from. Substrate Specificity-The oxidized B chain of insulin (50 mg) was incubated with proteinase II (0.5 mg) for 16 h at 37°C in 0.01 M bicarbonate, pH 10, and the reaction was stopped by addition of 0.5 ml of glacial acetic acid. Aliquots (35 al) were withdrawn at various times; a portion of this (5 ~1) was added to 5 ~1 of buffer containing 0.01 M EDTA to end the reaction, and assayed for inhibitory activity against trypsin [21] or chymotrypsin [22]. The heavy solution contained 6.5 ml of acrylamide/bisacrylamide (60%/3%), 6.5 ml of 0.25 M Tris-HCl, pH 8.0, 0.2% SDS, 40% sucrose, and 20 ~1 of Temed. Stacking solution was formed from 1 ml of the light gel solution, 4 ml of electrophoresis buffer (0.125 M Tris-HCl, pH 8.0, containing 0.1% SDS), 10 al of. Using human serum albumin, ovalbumin, soybean trypsin inhibitor, pancreatic trypsin inhibitor (Kunitz), and oxidized B chain of insulin as standards
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