Rattlesnake envenomation is common in certain regions of the United States and Central and South America. Due to the widespread use of antivenom, envenomation seldom results in death; however, most bites do give rise to severe local hemorrhage and necrosis. This pathology often results in dysfunction or loss of the affected area. The general goal of this proposal is to gain an understanding of the mechanism of action of the hemorrhage-producing toxins isolated from rattlesnake venoms by investigation of the structure and substrate specificities of these hemorrhagic-proteolytic toxins. This data will serve as a foundation to design specific inhibitors of these toxins. Work will continue on the amino acid sequence analyses of the hemorrhagic toxins isolated from the venom of Crotalus atrox in order to ascertain whether primary structural homology exists among the toxins and/or with other proteolytic enzymes. This information will give some indication as to the class of proteolytic enzymes to which these toxins belong. Amino acid sequence analyses will be performed using the current methodologies available in our laboratory. We will continue to elucidate the peptide bond specificities that these toxins have for their proteolytic function. Peptide substrates with known sequences and rationally designed synthetic peptides will be subjected to digestion by the toxins and the sites and kinetics of cleavage analyzed so that the substrate binding sites of the toxins may be characterized. These studies will allow us to carry on our design improvements of synthetic peptides modified such that they may function as competitive inhibitors of the toxins. As in the past, these peptide inhibitors will be synthesized via the solid-phase method. The syntheses will involve such techniques as the use of stereoisomers, amino acids with modified side chains, backbone length variations, functional group substitution, and site directed irreversible alkylation as a means to give rise to specific competitive inhibitors for each toxin. The synthetic peptides will be assayed for their effectiveness as inhibitors using in vivo hemorrhagic assays and standard proteolytic assays. It is our hope that this research will lead to more effective methods of crotalid bite treatment such that local tissue damage may be minimized. Furthermore, the knowledge gained about the design of peptide inhibitors for these proteolytic enzymes may be useful in pathologies where other proteolytic enzymes are implicated.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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University of Virginia
Schools of Medicine
United States
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