Isolation and identification of a snake venom metalloproteinase inhibitor from California ground squirrel (Spermophilus beecheyi) blood sera.

California ground squirrels (Spermophilus beecheyi) show blood-based defenses to a variety of toxins in the venom of the Northern Pacific rattlesnake (Crotalus oreganus oreganus). In this study we demonstrate the presence of an effective snake venom metalloproteinase inhibitor (SVMPI) in S. beecheyi. The blood sera of California ground squirrels were effective at reducing the metalloproteinase activity of Northern Pacific (C. o. oreganus) and prairie rattlesnake (Crotalus viridis viridis) venoms by over 75%, significantly more than its ability to reduce the activity of western diamondback rattlesnake venom. We used anion exchange and affinity chromatography to isolate this protein from the blood sera of S. beecheyi. This SVMPI had a molecular mass of 108.3 kDa and a pI of 5.1. The IC(50) of this inhibitor against whole venom from C. o. oreganus was determined to be 3.14 × 10(-8) M. Subsequent LC MS/MS analysis of a CNBr/tryptic digest of the inhibitor yielded multiple internal peptide sequences. These sequences showed homology to three other known mammalian plasma proteins: inter-α trypsin inhibitor, and two hibernation-associated proteins, HP25 and HP27. The presence of SVMPI in S. beecheyi blood sera is consistent with the resistance of these animals to venom-induced hemorrhage and tissue damage, and consistent with the protective factors conferring venom resistance in other mammals. However, the variety of SVMPI identified to date from mammalian taxa suggests that different species have converged on neutralization of venom metalloproteinase activity as a key step in venom neutralization.

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.

California ground squirrel (Spermophilus beecheyi) blood sera inhibits crotalid venom proteolytic activity.

Some California ground squirrels (Spermophilus beecheyi) show limited necrosis following envenomation by northern Pacific rattlesnakes (Crotalus viridis oreganus). This study demonstrates that S. beecheyi blood sera inhibits venom proteases. Sera from rattlesnake-abundant habitats inhibited C. v. oreganus venom more effectively than venom from two allopatric rattlesnake species, C. v. viridis and C. atrox, suggesting evolutionary specialization. The pattern of inhibition among squirrel populations corresponds best with history of rattlesnake predation, in contrast to current rattlesnake density.