A navel venombin B from Agkistrodon contortrix contortrix: Evidence for recognition properties in the surface around the primary specificity pocket different from thrombin

A novel thrombin-like enzyme (named contortrixobin) has been purified to ho mogeneity from the venom of Agkistrodon contortrix contortrix by affinity c hromatography on arginine-Sepharose, anionic exchange chromatography, and H PLC. The complete amino acid sequence has been determined by Edman degradat ion and by mass spectral analysis of peptides generated by enzymatic cleava ge, A microheterogeneity at the level of residue 234 has been detected, as demonstrated by peptides differing for the occurrence of Pro234 (similar to 85%) or Asp234 (similar to 15%). Contortrixobin (i) has six disulfide bend s whose sequence positions have been determined by mass spectrometry and (i i) does not contain carbohydrates in its structure. As expected, the 234 re sidue sequence of contortrixobin exhibits strong homology with snake venom serine proteases acting on either fibrinogen or other blood coagulation com ponents. The interaction of contortrixobin with chromogenic substrates indi cates a higher specificity for arginine over lysine in the primary subsite and a faster attack to ester than amides. The hydrolytic activity of contor trixobin is strongly inhibited by diisopropyl fluorophosphate and to a less extent by phenylmethylsulfonyl fluoride, benzamidine, and 4',6-diamidino-2 -phenylindole; hirudin (a specific alpha-thrombin inhibitor) as well as bas ic pancreatic trypsin inhibitor has a small effect on contortrixobin's cata lytic properties. Contortrixobin (i) preferentially releases fibrinopeptide B from human fibrinogen, (ii) activates blood coagulation Factors V and XI II with a rate 250-500-fold lower than human alpha-thrombin, and (iii) does not induce thrombocyte aggregation, intracytoplasmatic calcium ion increas e in platelets, and activation of Factor VIII. Evidence for biorecognition properties different from thrombin is also reported.