INTERACTION OF ALPHA-CHYMOTRYPSIN WITH THE FLUORESCENT-PROBE 1-ANILINONAPHTHALENE-8-SULFONATE IN SOLUTION

The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to a-chymotrypsin (α-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to α-CHT at a site different from either the active site of α-CHT or the 2-p-toluidinylnaphthalene-6-sulfonate binding site. The binding constant of Ans is about the same (104 M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to a-CHT. The fluorescence enhancement due to binding of Ans to α-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-α-CHT complex decreases appreciably, and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-α-CHT can be interpreted in terms of a pH-dependent equilibrium between α-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site. © 1979, American Chemical Society. All rights reserved.