Immunological properties of the fibrinolytic enzyme (fibrolase) from southern copperhead (Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography.
An antibody to the fibrinolytic enzyme in southern copperhead venom was produced by immunizing rabbits with chromatographically purified enzyme. The antibody was purified from rabbit blood by ammonium sulfate fractionation and protein-A affinity chromatography. The purified antibody reacted only with the fibrinolytic enzyme in southern copperhead venom as demonstrated by immunodiffusion and immunoelectrophoresis. Western immunoblotting revealed that several snake venoms, including Agkistrodon piscivorus conanti, Crotalus atrox, Crotalus basiliscus basiliscus, and Bothrops asper, cross-reacted with the antibody to varying degrees. However, Deinagkistrodon acutus showed no cross-reaction. Immobilized antibody has been used, in combination with molecular sieve chromatography, to purify the fibrinolytic enzyme from southern copperhead venom. In this two-step purification procedure, the enzyme was purified in good yield within two days. The specific activity of the enzyme purified by the immunoaffinity chromatography procedure is comparable with that of enzyme purified by a four-step chromatographic procedure. The mol. wt of the purified enzyme is approximately 23,000-24,000 as determined by SDS-PAGE. Interestingly, the enzyme purified by this two-step immunoaffinity chromatography procedure possesses virtually no hemorrhagic activity.
Chen
H M
HM
Department of Biochemistry, University of Southern California, School of Medicine, Los Angeles 90033.
Guan
A L
AL
Markland
F S
FS
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D002846:Chromatography, Affinity; D003429:Cross Reactions; D003435:Crotalid Venoms / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006470:Hemorrhage / Q000139:chemically induced; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D008666:Metalloendopeptidases / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D011817:Rabbits; D012910:Snake Venoms / Q000276:immunology; D013045:Species Specificity
C031442:Agkistrodon venoms; D002364:Caseins; D003435:Crotalid Venoms; D012910:Snake Venoms; C026018:azocasein; D008666:Metalloendopeptidases; C058789:fibrolase