Isolation and properties of a blood coagulation factor X activator from the venom of king cobra (Ophiophagus hannah).
A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.
Lee
W H
WH
Kunming Institute of Zoology, Chinese Academy of Sciences, Yunnan, P. R. China.
Zhang
Y
Y
Wang
W Y
WY
Xiong
Y L
YL
Gao
R
R
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001779:Blood Coagulation Factors / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004546:Elapid Venoms / Q000201:enzymology*; D017815:Elapidae; D004591:Electrophoresis, Polyacrylamide Gel; D005170:Factor X / Q000187:drug effects*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011817:Rabbits; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D001779:Blood Coagulation Factors; D004546:Elapid Venoms; D015842:Serine Proteinase Inhibitors; D005170:Factor X; D001120:Arginine; C009727:benzoylarginine ethyl ester; D012697:Serine Endopeptidases; D002118:Calcium