A rapid and sensitive fluorometric method for the quantitative analysis of snake venom metalloproteases and their inhibitors

Metalloproteases are responsible for the hemorrhagic effects of many snake venoms and contribute to other pathways that lead to local tissue damage. Methods that quantify snake venom metalloproteases (SVMP) are therefore valuable tools in research on the clinical, physiological, and biochemical effects of envenomation. Comparative analysis of individual, population, and species differences requires screening of large numbers of samples and treatments, and therefore require a method of quantifying SVMP activity that is simple, rapid, and sensitive. This paper demonstrates the properties of a new fluorometric assay of SVMP activity that can provide a measure of metalloprotease activity in 1 h. The assay is reliable, with variation among replicates sufficiently small to reliably detect differences in between species (F-19.60 = 2924, p < 0.001), even for those venoms with low overall activity. It is also sensitive enough to detect differences among venoms using < 2 ng of whole venom protein. We provide an example use of this assay to detect the presence of natural SVMP inhibitors in minute samples of blood plasma from rock squirrels (S. variegatus), a natural prey species for North American rattlesnakes. We propose this assay is a useful addition to the set of tools used to characterize venoms, as well as high-throughput screening of natural or synthetic inhibitors, or other novel therapeutic agents against SVMP effects. (c) 2010 Elsevier Ltd. All rights reserved.