Modification of tyrosine-3 (63) and lysine-6 of Taiwan cobra phospholipase A2 affects its ability to enhance 8-anilinonaphthalene-1-sulfonate fluorescence.

The Tyr-3 (63) and Lys-6 of Naja naja atra phospholipase A2 (PLA2) were modified with p-nitrobenzenesulfonyl fluoride and 4-chloro-3,5-dinitrobenzoate, respectively. Although the ability of the Lys-modified derivative to enhance the ANS fluorescence was lower than that observed with native PLA2, the ANS-binding affinity of the Lys-modified derivative was similar to that of the native enzyme. Modifications on Tyr-3 or/and Tyr-63 of PLA2 resulted in the complete loss of its ability to enhance the ANS fluorescence. Nevertheless, the extent of O-sulfonylation of Tyr residues was not effectively reduced by the addition of ANS. This suggests that Tyr-3 and Tyr-63 interact with the bound ANS in an aromatic ring-stacking manner. Alternatively, the incorporation of a carboxydinitrophenylated group on Lys-6 may notably perturb the nonpolarity of the ANS-binding site.