Collaborative Research Project (Protease inhibitors in opossum serum) Snakes have an arsenal of venom metalloproteinases(SVMP) that create emergencies when humans are envenomated; however, certain animals have a natural resistance to snake venoms due to snake venom metalloproteinase inhibitors (SVMPI). Both the SVMP and SVMPI are molecules that could have important applications in medicine. Thus, the study of their inhibition mechanism could also improve our understanding of other matrix metalloproteinases in humans. Many animal matrix metalloproteinases (MMP) such as type IV collagenase are involved in extracellular matrix digestion, which is a critical component of many normal physiological processes (e.g., wound healing, tissue remodeling, and differentiation) and pathological conditions (e.g. tumor cell invasion, metastasis, and arthritis). Purified SVMPI (DM43 in opossum serum) neutralizes certain SVMP found in snake venoms (fibrinogenolytic activities of bothrolysin and jararhagin) but crude opossum serum neutralizes all hemorrhagic activity in venoms tested thus far. The goals of this project are to: (1) determine how many different SVMPI are found in opossum serum, (2) determine the mechanism of neutralization, (3) measure the kinetics of neutralization, (4) determine if SVMPI prevents metastasis of cancer, and (5) clone SVMPI. The substrate specificity of SVMP will be determined by the cleavage of B chain insulin. Mass spectrometry will be used to determine the cleavage site on B chain insulin. The majority of experiments performed with the Biacore systenwill be geared toward measuring the kinetics and affinity of binding interactions. Measurements of onrates (ka) and off-rates (kd) of protein interactions will be measured. These values are directly related to equilibrium binding affinities (KD). The Biacore system provides the only way to measure the concentration of binding protein, rate of binding, affinity, and specificity of binding in real time.
