1-anilino-8-naphthalene sulfonate as a protein conformational tightening agent

1-Anilino-8-naphthalene sulfonate (ANS) anion is conventionally considered to bind to preexisting hydrophobic (nonpolar) surfaces of proteins, primari ly through its nonpolar anilinonaphthalene group. Such binding is followed by an increase in ANS fluorescence intensity, similar to that occuring when ANS is dissolved in organic solvents. It is generally assumed that neither the negative sulfonate charge on the ANS, nor charges on the protein, part icipate significantly in ANS-protein interaction. However, titration calori metry has demonstrated that most ANS binding to a number of proteins occurs through electrostatic forces in which ion pairs are formed between ANS sul fonate groups and cationic groups on the proteins (D. Matulis and R. E. Lov rien, Biophys. J., 1998, Vol. 74, pp. 1-8). Here we show by viscometry and diffusion coefficient measurements that bovine serum albumin and gamma-glob ulin, starting from their acid-expanded, most hydrated conformations, under go extensive molecular compaction upon ANS binding. As the cationic protein binds negatively charged ANS anion it also takes up positively charged pro tons from water to compensate the effect of the negative charge, and leaves the free hydroxide anions in solution thus shifting pH upward (the Scatcha rd-Black effect). These results indicate that AWS is not always a definitiv e reporter of protein molecular conformation that existed before ANS bindin g. Instead, ANS reports on a conformationally tightened state produced by t he interplay of ionic and hydrophobic characters of both protein and ligand . (C) 1999 John Wiley & Sons, Inc.