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, primarily through its nonpolar anilino-naphthalene group. Such binding is followed by an increase in ANS fluorescence intensity, similar to that occurring 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, participate significantly in ANS-protein interaction. However, titration calorimetry has demonstrated that most ANS binding to a number of proteins occurs through electrostatic forces, in which ion pairs are formed between ANS sulfonate groups and cationic groups on the proteins (D. Matulis and R. E. Lovrien, Biophys. J., 1998, Vol. 74, pp. 1-8). Here we show by viscometry and diffusion coefficient measurements that bovine serum albumin and gamma-globulin, starting from their acid-expanded, most hydrated conformations, undergo extensive molecular compaction upon ANS binding. As the cationic protein binds negatively charged ANS anion it also takes up positively charged protons from water to compensate the effect of the negative charge, and leaves the free hydroxide anions in solution thus shifting pH upward (the Scatchard-Black effect). These results indicate that ANS is not always a definitive reporter of protein molecular conformation that existed before ANS binding. Instead, ANS reports on a conformationally tightened state produced by the interplay of ionic and hydrophobic characters of both protein and ligand.
Matulis
D
D
Biochemistry Department, University of Minnesota, St. Paul 55108, USA.
Baumann
C G
CG
Bloomfield
V A
VA
Lovrien
R E
RE
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013816:Thermodynamics; D005719:gamma-Globulins / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D005719:gamma-Globulins; D012710:Serum Albumin, Bovine; C027132:1-anilino-8-naphthalenesulfonate