Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Abstract

Two phospholipases A2 (PLA2) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA2 were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca 2+; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca2+. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA2 could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modifiedN. naja atra PLA2 exhibited the same Ca2+-induced difference spectra as that of native PLA2, indicating that the Ca2+-binding ability of Tyr-63-modifiedN. naja atra PLA2 was not impaired. However, Tyr-3-modified PLA2 and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca2+, revealing that the Ca2+-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca2+ binding. AtpH 8.0, both native PLA2 enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca2+, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.