ABSTRACT
The proteolytic activity of hemorrhagic proteinase IV isolated from timber rattlesnake (Crotalus horridus horridus) venom was resistant to inactivation by trypsin, pronase and the proteolytic IIt fraction isolated from timber rattlesnake venom. SDS-polyacrylamide gel electrophoresis of the hemorrhagin incubated alone and with the three proteinases revealed that the addition of trypsin or the IIt fraction caused little apparent degradation of the hemorrhagin, whether or not the samples were reduced prior to electrophoresis. SDS electrophoresis of the hemorrhagin after incubation with pronase revealed a single band of 28,000 apparent molecular weight (as compared to 52,000 for the original hemorrhagin) if the samples were not reduced prior to electrophoresis, and a single band of 17,000 if reduced. If the hemorrhagin was reduced and alkylated, it was much more susceptible to hydrolysis by all three proteinases.
Subject(s)
Crotalid Venoms/analysis , Endopeptidases/metabolism , Animals , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Pronase , TrypsinABSTRACT
A protein isolated from timber rattlesnake (Crotalus horridus horridus) venom by ion-exchange and high-pressure liquid chromatography is hemorrhage inducing and lethal to mice (LD50 of 10 micrograms/g of body weight). It is a Ca2+- and Zn2+-containing proteinase and has the ability to hydrolyze hide powder azure. Atomic absorption spectroscopy shows 2.5 Ca2+ and 1 Zn2+ per protein monomer. The proteinase activity is destroyed by incubation with disulfide-reducing agents and by dialysis against ethylenediaminetetraacetate. Coincident with the loss of proteinase activity is a corresponding loss of lethal and hemorrhagic activities, suggesting that all three are related. Attempts to replace the metals and restore activity have been unsuccessful. Amino acid analysis and isoelectric focusing reveal that this component is an acidic protein (pI = 5.1) containing about 20 disulfide bonds and 507 residues. Reduction of one disulfide bond per molecule decreases proteinase activity by 50% while reduction of eight disulfide bonds decreases activity by 80%. Loss of hemorrhagic activity parallels the decrease in proteinase activity.
Subject(s)
Crotalid Venoms/toxicity , Endopeptidases/isolation & purification , Hemorrhage/chemically induced , Amino Acids/analysis , Animals , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Endopeptidases/metabolism , Endopeptidases/toxicity , Kinetics , Lethal Dose 50 , MiceABSTRACT
Loxosceles reclusa venom apparatus extract, toxin, cephalothorax and abdomen extracts were tested for six activities. Protease, lipase, nonspecific hydrolase and direct hemolytic activities were found primarily in abdomen extracts while sphingomyelinase activity appeared predominantly in the toxin. Appreciable complement-mediated hemolysis was not observed.
