PURIFICATION AND CHARACTERIZATION OF A KININ-RELEASING AND FIBRINOGEN-CLOTTING SERINE PROTEINASE (KN-BJ) FROM THE VENOM OF BOTHROPS-JARARACA, AND MOLECULAR CLOSING AND SEQUENCE-ANALYSIS OF ITS CDNA

Two forms of a proteinase, KN-BJ 1 and 2, were purified to homogeneity from the venom of Bothrops jararaca. In SDS/PAGE reduced KN-BJ 1 and 2 migrated as single bands with molecular masses of 38 kDa and 39 kDa. The two enzymes have similar N-terminal amino acid sequences and spec ific activities on synthetic chromogenic substrates, and both release bradykinin fi om bovine low-molecular-mass kininogen. KN-BJ 1 and KN-B J 2 clot fibrinogen with specific activities of 245 NIH U/mg and 219 N IH U/mg, releasing only fibrinopeptide A. The amidolytic, kinin-releas ing and coagulant activities are inhibited by phenylmethylsulfonyl flu oride, demonstrating that KN-BJ is a serine proteinase. Benzamidine de rivatives, which are competitive inhibitors of trypsin-like proteinase s. also inhibited the amidolytic activity of KN-BJ. A cDNA clone (HS10 4, 2.2 kb) has been isolated from a cDNA library of B. jararaca venom glands with an ORF of 771 bp. The deduced amino acid sequence contains segments that are identical to the sequences of the N-terminus and th ree tryptic peptides of KN-BJ 2. Therefore, the cDNA is believed to re present the gene of KN-BJ 2. The deduced amino acid sequence indicates that KN-BJ 2 is synthesized as a prezymogen of 257 amino acids with a putative signal peptide of 18 amino acids and an activating peptide o f six amino acid residues. The sequence of 233 amino acids representin g the mature enzyme exhibits high similarity to sequences of serine pr oteinases isolated from crotalid venoms.