Isolation and characterization of a thrombin-like enzyme from the venom of the snake Bothrops insularis (jararaca ilhoa).
A thrombin-like enzyme (I-SII-R) from the venom of the Brazilian snake Bothrops insularis (jararaca ilhoa) was purified to homogeneity by gel filtration on Sephadex G-150 followed by precipitation of contaminating proteins with half-saturated NaCl and two further gel filtrations under the same conditions. I-SII-R showed specific activities of 820 clotting units/mg protein and 530 TAME units/mg protein (a 23-fold purification) and a single precipitation band against antibothropic horse antiserum by immunoelectrophoresis, as well as a single band by PAGE and by SDS-PAGE with beta-mercaptoethanol. The estimated molecular weight was 45,000, of which the neutral sugars account for 9900, or 22%. Its amino acid composition, which accounts for a minimum mol. wt of 33,900 is as follows: Asx35, Thr18, Ser17, Glx15, Pro29, Gly22, Ala27, Val21, Cys18, Met7, Ile19, Leu25, Tyr11, Phe11, His7, Lys8, Arg17, Trp5. When compared to thrombin, I-SII-R is relatively stable at 45 degrees C and 60 degrees C, pH 7.4, but is inhibited by heparin at a higher rate than thrombin. Both enzymes hydrolyze the alpha and beta chains of fibrinogen and lose more than 95% of their clotting and esterase activities when treated with phenylmethylsulphonyl fluoride. In contrast to thrombin, I-SII-R does not activate factor XIII.
Departamento de Bioquímica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brasil.
H S
HS
Selistre
J R
JR
Giglio
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D003435:Crotalid Venoms / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005176:Factor XIII / Q000032:analysis; D005340:Fibrinogen / Q000378:metabolism; D007122:Immunoelectrophoresis; D012967:Sodium Dodecyl Sulfate; D013696:Temperature; D013917:Thrombin / Q000032:analysis / Q000302:isolation & purification*
D000596:Amino Acids; D002241:Carbohydrates; D003435:Crotalid Venoms; D012967:Sodium Dodecyl Sulfate; D005340:Fibrinogen; D005176:Factor XIII; D013917:Thrombin