A novel nonhemorragic protease from the African puff adder (Bitis arietans) venom.
A nonhemorrhagic proteinase B-20 from the venom of Bitis arietans has been purified to apparent electrophoretic homogeneity by chromatography on Sephadex G-100, Q-Sepharose, and CM-cellulose. It has a molecular weight of 20 k Da as determined by size exclusion chromatography on Sephadex G-100 and migrated as a single 20-k Da band on SDS polyacrylamide. It has an optimum pH of 6-8 and is inactive at pH 4.0. EDTA and 1,10-phenanthroline strongly inhibited the enzyme suggesting it is a metalloenzyme. Also it is inhibited by antipain but is unaffected by trasylol, antitrypsin, and pepsptatin. Colombin, an identified active component of Aristolochia albida used in the treatment of snake poisoning, did not inhibit the protease activity. It lost over 90% of its activity in the presence of 0.5 microM Hg(2+) but the inhibition was completely blocked in the presence of 10 microM mercaptoethanol implicating sulfhydryl groups in the catalytic entity of the protein. The activity was also inhibited competitively by glutathione and cysteine with inhibition binding constants K(i) of 240 and 40 microM, respectively. The enzyme is unaffected by several divalent cations but activated by 1 mM Fe(3+). It had a prolyl endopeptidase and thermolysin-like activity. The enzyme displayed a fast acting alpha-fibrinolytic and delayed gamma-fibrinolytic activity when tested on human fibrinogen. The relevance of these findings is discussed.
Nok
A J
AJ
Department of Biochemsitry, Ahmadu Bello University, Zaria, Nigeria. jandrew@mail.skannet.com
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D005340:Fibrinogen / Q000737:chemistry; D005342:Fibrinolysis / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry / Q000633:toxicity*; D008670:Metals / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014757:Viper Venoms / Q000201:enzymology*
D008670:Metals; D011480:Protease Inhibitors; D014757:Viper Venoms; D005340:Fibrinogen; D010450:Endopeptidases; C445564:B-20 protease, Bitis arietans; D008666:Metalloendopeptidases