Isolation and partial characterization of a prothrombin-activating enzyme from the venom of the Australian rough-scaled snake (Tropidechis carinatus).
A prothrombin activator from the venom of Tropidechis carinatus has been isolated by means of gel filtration and benzamidine-based affinity chromatography, a novel use of the latter technique. Two bands possessing prothrombinase activity were obtained from the affinity chromatography procedure and designated A1 and A2. The bulk of the enzyme activity was recovered in peak A2 which represented 27-31% of the starting activity and a 14-16-fold purification. The venom contained, in total, around 5% by weight of the two isoforms of the prothrombin activator. The two fractions were electrophoretically similar on polyacrylamide electrophoresis, migrating with a mol. wt of 64,500 under native conditions and as a single band of 41,500 under reducing conditions. The prothrombinase was dependent on factor Va, phospholipid and calcium ions for its activity and is, thus, a member of the type II class of prothrombinases requiring such co-factors. The enzyme did not possess any phospholipase activity nor did it cleave the substrates N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), N-benzoyl-L-tyrosine ethyl ester (BTEE), azocollagen or azocasein, indicating a lack of amidolytic, esterolytic and broad-spectrum protease activity.
Marsh
N A
NA
School of Life Science, Queensland University of Technology, Brisbane, Australia.
Fyffe
T L
TL
Bennett
E A
EA
D000818:Animals; D001315:Australia; D001777:Blood Coagulation / Q000187:drug effects*; D002118:Calcium; D002850:Chromatography, Gel; D003029:Coagulants / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa; D006801:Humans; D010743:Phospholipids; D012910:Snake Venoms / Q000737:chemistry* / Q000201:enzymology / Q000494:pharmacology*; D013925:Thromboplastin / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*
D003029:Coagulants; D010743:Phospholipids; D012910:Snake Venoms; D013925:Thromboplastin; D015951:Factor Xa; D002118:Calcium