Binding of the fluorescent dye 8-anilinonaphthalene 1-sulfonic acid to the native and pressure dissociated beta 2-dimer of tryptophan synthase from Escherichia coli.

Abstract

The beta 2-dimer of tryptophan synthase from Escherichia coli exhibits weak binding of 8-anilinonaphthalene-1-sulfonic acid (ANS). Titrating the dye at 0.2 mM concentration with the apo-beta 2-dimer at atmospheric pressure causes increased fluorescence emission at 480 nm (lambda exc = 380 nm), corresponding to unspecific binding of the ligand to hydrophobic residues. Increasing hydrostatic pressure affects ANS binding. Up to 700 bar, a sigmoidal increase of ANS fluorescence reflects an increase in hydrophobic surface area, probably caused by subunit dissociation. At approximately 1 kbar, a maximum is reached; beyond this value, pressure competes with ligand binding causing fluorescence emission to be decreased again. Pressure release leads to a drastic fluorescence enhancement, ascribed to ANS binding to the partially and reversibly denatured enzyme. Plotting the total fluorescence enhancement vs. pressure yields a profile which parallels the pressure dependent dimer in equilibrium monomer transition monitored by subunit hybridization (T. Seifert, P. Bartholmes, and R. Jaenicke, Biochemistry, in press).