The structural elements of phospholipase A2 affecting the enhancement of 8-anilinonaphthalene-1-sulfonate fluorescence.

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to Naja naja atra and Bungarus multicinctus phospholipase A2 (PLA2) as well as the ANS fluorescence enhancement of PLA2-ANS complexes were pH-dependent. However, the pH-dependent curves and the ionizable groups perturbed by the binding of ANS were essentially different for the two PLA2 enzymes. Methylation of His residue or removal of the N-terminal octapeptide made the ANS fluorescence of PLA2-ANS complexes become less sensitive to pH changes. Moreover, the N-terminus-removed derivatives showed the same fluorescence-pH profiles. These results, together with the findings that the His residue and the N-terminal residues might locate around the ANS-binding site, suggested that the His residue and N-terminus of the two PLA2 enzymes may interact electrostatically with the bound ANS, and that ionization of these residues should appreciably affect the ANS fluorescence.