Purification and characterization of two high molecular weight hemorrhagic toxins from Crotalus viridis viridis venom using monoclonal antibodies.
Two high mol. wt hemorrhagic toxins were purified from Crotalus viridis viridis venom using HPLC anion exchange and monoclonal antibody affinity chromatography following initial separation by HPLC with a preparative DEAE column. The fraction from the initial column having the highest hemorrhagic activity was used to immunize BALB/c mice for production of monoclonal antibodies. One of the monoclonal antibodies specifically recognized a single protein band of the immunizing fraction in native polyacrylamide gel electrophoresis. This monoclonal antibody was purified and conjugated to activated membranes for affinity purification of the antigen from the hemorrhagic fraction. Proteins eluted from the affinity membranes showed three bands in SDS-PAGE with apparent mol. wts of 68,000, 62,000 and 30,000. The 68,000 and 62,000 mol. wt proteins were further purified using an analytical DEAE column, and purity was demonstrated by the presence of a single band in SDS-PAGE and a single precipitation arc against antibodies to Crotalus viridis viridis venom in immunoelectrophoresis. The 68,000 (pI of 8.5) and 62,000 (pI of 4.1) proteins are highly hemorrhagic metalloproteinases which cleave N,N-dimethylcasein and fibrinogen. Both are glycoproteins with the same galactose-beta (1-4)-N-acetylglucosamine side-chains linked to asparagine residues. Treatment of both toxins with glycosidase F blocked their hemorrhagic activities. Oxidation of the sugar moieties by periodate inhibited the hemorrhagic activity of the 62,000 toxin, but not of the 68,000 mol. wt toxin.
Department of Physiological Sciences, Oklahoma State University, Stillwater 74078.
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000918:Antibody Specificity; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D004492:Edetic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006470:Hemorrhage / Q000139:chemically induced*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010504:Periodic Acid / Q000494:pharmacology; D014118:Toxins, Biological / Q000302:isolation & purification* / Q000633:toxicity
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D014118:Toxins, Biological; D010504:Periodic Acid; D004492:Edetic Acid; C009288:metaperiodate; D006026:Glycoside Hydrolases