Purification, characterization, crystallization and preliminary X-ray diffraction of acuthrombin-B, a thrombin-like enzyme from Agkistrodon acutus venom.
Acuthrombin-B, a thrombin-like enzyme from Agkistrodon acutus venom, has been isolated and purified to homogeneity by ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephacryl S-100 and fast performance liquid chromatography on DEAE-8HR. The protease is an acid protein (pI 6.0) consisting of two non-identical polypeptide chains (14.4 and 16 kDa) and there is no disulfide bond between the subunits. Its molecular weight is 27 kDa as estimated by gel filtration on Sephacryl S-100. The protease has arginine-esterase activity and hydrolyzes synthetic substrates such as p-toluenesulfonyl arginine methyl ester and alpha-N-benzoyl-L-arginine amide ethyl ester, and shows clotting activity with human fibrinogen, rabbit citrated plasma and human citrated plasma in vitro. The specific activity with human fibrinogen was estimated to be 230 NIH units mg-1. The protease is considered as a serine-type protease and contains metal ion(s) to some extent, as indicated by the fact that its clotting and arginine-esterase activities could be completely inhibited by PMSF and partially inhibited by the chelating agent EDTA, while the thrombin inhibitor heparin had no effect on its clotting activity towards rabbit citrated plasma. Acuthrombin-B crystals with a resolution limit of 2.06 A were obtained by conventional hanging-drop vapour diffusion. The crystals belong to space group P21 with unit-cell parameters a = 34.97, b = 53.58, c = 67.88 A, beta = 98.89 degrees and contain one molecule per asymmetric unit.
D017836:Agkistrodon; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D003435:Crotalid Venoms / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011487:Protein Conformation; D011817:Rabbits; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology