Role of the N-terminal region in phospholipases A 2 from Naja naja atra (Taiwan cobra) and Naja nigricollis (spitting cobra) venoms

Abstract

Role of the N-terminal region in phospholipases A 2 from Naja naja atra (Taiwan cobra) and Naja nigricollis (spitting cobra) venoms. Toxicon 26, 721 – 731, 1988. — The N-terminal α-amino groups of two phospholipases A 2 (PLA 2) from Naja naja atra and Naja nigricollis venoms were selectively modified with trinitrobenzene sulfonic acid, and the modified derivatives were separated by high performance liquid chromatography (HPLC). Trinitrophenylated (TNP) derivatives contained only one TNP group in the α-amino group of Asn-1 and showed a marked decrease in enzymatic activity. PLA 2 enzymes were cleaved with CNBr, and the N-terminal octapeptide was separated from the large C-terminal fragment by HPLC. Removal of the N-terminal octapeptide from PLA 2 enzymes caused a precipitous decrease in enzymatic activity. Enzyme immunoassay and double immunodiffusion revealed that the N-terminal octapeptide is one of the antigenic determinants of PLA 2 enzymes. The presence of dihexanoyllecithin influenced the interaction between PLA 2 enzymes and 8-anilinonaphthalene sulfonate (ANS), indicating that ANS-binding site of PLA 2 enzymes is at or near the substrate binding site. Modification of the N-terminal region perturbed the substrate binding and the binding ability for ANS. The modified derivatives retained their affinity for Ca 2+, indicating that the N-terminal region is not involved in Ca 2+-binding. A fluorescence study revealed that the α-amino group is near Trp residue(s) and that the N-terminal region is important for stabilizing the architectural environment of the Trp residue(s). The results, together with the proposal that Trp residues in PLA 2 enzymes are involved in substrate binding, suggest that the N-terminal region of PLA 2 enzymes is involved in substrate binding and in maintaining a functional active site.