Purification, sequencing and characterization of single amino acid-substituted phospholipase A₂ isozymes from Trimeresurus Gramineus (green habu snake) venom

T. Fukagawa, T. Nose, Y. Shimohigashi, T. Ogawa, N. Oda, K. Nakashima, C.-C. Chang and M. Ohno. Purification, sequencing and characterization of single amino acid-substituted phospholipase A₂ isozymes from Trimeresurus gramineus (green habu snake) venom. Toxicon 31, 957-967, 1993.-Two phospholipases A₂ named PLA₂-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA₂-I and II reported previously [Oda et al. (1991) Toxicon 29, 157; Fukagawa et al. (1992) Toxicon 30, 133]. Their isoelectric points were determined to be about 4.5. PLA₂-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA₂-I. The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins. Both proteins consisted of 122 amino acid residues. When compared with PLA₂-I, PLA₂-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn. PLA₂-IV also showed a single substitution from Ala to Asp at position 72. It was inferred that these amino acid substitutions between PLA₂-I and PLA₂-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently. The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca²⁺-binding loop, was conserved in both PLA₂-III and IV as in PLA₂-I. There was no significant difference in the dissociation constants (4.3-5.2 × 10-4 M) for Ca²⁺ between these PLA₂s and Tyr-28-containing PLA₂s. These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA₂s to Ca²⁺.