The essentiality of His-47 and the N-terminal region for the binding of 8-anilinonaphthalene-1-sulfonate with Taiwan cobra phospholipase A2

Abstract

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to Naja naja atra phospholipase A2 (PLA2) as well as the enhancement of ANS fluorescence of the PLA2-ANS complex decreased with increasing pH in a pH range from 3 to 9. These pH-dependent curves can be well interpreted as the perturbation of an ionizable group with pK value of 5.8, which was assigned as His-47 in the active site of PLA2. The ionizable group with pK 5.8 was no longer observed after methylation of His-47, supporting the idea that the pH dependence of ANS binding arose from an electrostatic interaction between His-47 and the bound ANS. Removal of the N-terminal octapeptide of PLA2 caused a precipitous drop in the capability of PLA2 for binding with ANS and enhancing ANS fluorescence, reflecting that the integrity of the N-terminal region was essential for maintaining the hydrophobic character of the ANS-binding site. However, the nonpolarity of the ANS-binding site in the N-terminus-removed derivative was still partially retained at low pH, but was completely lost at high pH. Evidently, the N-terminal region plays a more crucial role in ANS binding at high pH than at low pH. These results indicate that hydrophobic interaction as well as electrostatic interaction are involved in the binding of ANS to PLA2, and that the relative contributions of both interactions in ANS fluorescence enhancement may be different under different pH.