The Application of a Snake Venom Anticoagulant Enzyme in Burn Therapy.
Final rept. 1 Jul 76-1 Jul 77,
UNIVERSITY OF SOUTHERN CALIFORNIA LOS ANGELES SCHOOL OF MEDICINE
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Crotalase, the thrombin-like enzyme from Eastern diamondback rattlesnake Crotalus adamanteus, has been purified to homogeneity. This was shown by chromatographic and electrophoretic patterns of the enzyme. A single band was observed on SDS polyacrylamide gel electrophoresis. The molecular weight was estimated to be about 33,000 by gel filtration on Sephadex G-100 and SDS polyacrylamide gel electrophoresis. The molecular weight determined from amino acid composition was shown to be 30,334. However, the estimated molecular weight contributed by carbohydrates 2013 gave us an overall value of 32,347. Analytical isoelectric focusing of crotalase showed five bands. This heterogeneity of enzyme may be due to differences in sialic acid content of the different forms of the enzyme. Upon treatment of the enzyme with neuraminidase to remove the sialic acid a single band was obtained by isoelectric focusing, supporting our contention that the enzyme is homogeneous and that the five bands represent different levels of sialylation of the enzyme. This heterogeneity may have been an artifact of preparation. Fibrinolytic activity of crotalase was shown to be an inherent property since the ratio of fibrinogenolytic activity to that of fibrinolytic activity remained constant throughout the purification procedure. Crotalase hydrolysed a chromogenic substrate S-2238 used for thrombin assay this activity could be used as a method for estimation of crotalase.