1-Anilino-8-Naphthalene Sulfonate Anion-Protein Binding Depends Primarily on Ion Pair Formation

Abstract

The ANS − (1-anilino-8-naphthalene sulfonate) anion is strongly, dominantly bound to cationic groups of water-soluble proteins and polyamino acids through ion pair formation. This mode of ANS − binding, broad and pH dependent, is expressed by the quite rigorous stoichiometry of ANS − bound with respect to the available summed number of H + titrated lysine, histidine, and arginine groups. By titration calorimetry, the integral or overall enthalpies of ANS − binding to four proteins, bovine serum albumin, lysozyme, papain, and protease omega, were arithmetic sums of individual ANS −–polyamino acid sidechain binding enthalpies (polyhistidine, polyarginine, polylysine), weighted by numbers of such cationic groups of each protein (additivity of binding enthalpies). ANS − binding energetics to both classes of macromolecules, cationic proteins and synthetic cationic polyamino acids, is reinforced by the organic moiety (anilinonaphthalene) of ANS −. In a much narrower range of binding, where ANS − is sometimes assumed to act as a hydrophobic probe, ANS − may become fluorescent. However, the broad overall range is sharply dependent on electrostatic, ion pair formation, where the organic sulfonate group is the major determinant of binding.