Stability of Venom Collagenase Enzyme Activity and Effect of Order of Reactants with Inhibition by NNGH

Abstract

Earlier work had revealed complexities in enzyme inhibitor activity with snake venoms in reactions with a gelatin substrate (collagenase). To begin to clarify technical issues which could be in part related to enzyme stability or mechanism, baseline gelatinase activities of venom from three snake species: Agkistrodon contortrix contortrix, Crotalus atrox and Cerastes cerastes, were measured at room temperature using EnzChek® Gelatinase/Collagenase Kit E12055. A. c. contortrix and C. atrox were similar while C. cerastes venom showed the lowest baseline activity. Initial progress of reaction data with additional Selwyn plot analysis demonstrated enzymatic stability over a wide range of substrate concentrations and reaction times under the conditions tested. Next, because the order of reactants in enzyme inhibition reactions will affect results depending on the mechanism of inhibition, an hydroxamate inhibitor of interest was tested. The inhibitory effect of NNGH (N‐Isobutyl‐N‐(4‐methoxyphenylsulfonyl)glycyl hydroxamic acid) on venom gelatinase activity was not affected by enzyme pretreatment with inhibitor prior to the gelatinase reaction. These results will be useful to base later mechanistic studies to understand reaction kinetics of snake venom enzyme inhibition, which could lead to alternative treatment modalities of envenomation.