Chemical modification of the histidine residue in basic phospholipase A2 from the venom of Naja nigricollis.
Phospholipase A2 from Naja nigricollis venom was separated into three fractions by chromatography on a column of CM-Sephadex C-25. The pI values of fractions CMS-5, CMS-6 and CMS-9 were determined to be 7.6, 8.3 and 10.6, respectively. Fraction CMS-9 was further purified on a DEAE-Sephacel column and the homogeneity was verified. The specific activity of CMS-9 was found to be 1300 units per mg and lethal toxicity 0.3 mg per kg mouse. The most basic and toxic fraction, CMS-9, was subjected to chemical modification with p-bromophenzcyl bromide. The enzyme lost both the enzyme activity and lethal toxicity, however, the antigenicity remained unchanged. Although both 8-anilinonaphthalenesulfonate and Ca2+ showed pronounced protection on the inactivation process, the mechanism of 8-anilinonaphthalene-sulfonate protection is different from that of Ca2+. Amino acid analysis showed that only one (His-47) out of three histidine residues was modified. Although both native and His-modified CMS-9 were perturbed by the presence of Ca2+, the modified enzyme lost the characteristic tryptophan blue shift suggesting that the modified enzyme is unable to exert a charge effect upon Ca2+ binding in the vicinity of the tryptophan group. Scatchard plots revealed only one type of binding sites for 8-anilinonaphthalenesulfonate in the presence of Ca2+. On the other hand, the modified enzyme lost the ability to bind 8-anilinonaphthalene. It is suggested tentatively that the hydrophobic pocket in which 8-anilinonaphthalenesulfonate is bound may be the site of the enzyme that interacts with phospholipid.
D000098:Acetophenones / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004546:Elapid Venoms / Q000032:analysis*; D006639:Histidine; D051379:Mice; D010740:Phospholipases / Q000037:antagonists & inhibitors*; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D054467:Phospholipases A2; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000098:Acetophenones; D000596:Amino Acids; D000817:Anilino Naphthalenesulfonates; D004546:Elapid Venoms; D006639:Histidine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; C006821:4-bromophenacyl bromide; D002118:Calcium