8-Anilinonaphthalene 1-Su lfon ic Acid to the Native and Pressure D issociated /f2-Dimer o f Tryptophan Synthase from Escherichia coli

The /?2-dimer o f tryptophan synthase from Escherichia coli exhibits weak binding o f 8-anilinonaphthalene-lsulfonic acid (ANS). Titrating the dye at 0.2 mM concen­ tration with the apo-/?2-dimer at atmospheric pressure causes increased fluorescence emission at 480 nm (/.exc = 380 nm), corresponding to unspecific binding o f the ligand to hydrophobic residues. Increasing hydrostatic pressure affects AN S binding. Up to 700 bar, a sigmoidal increase o f ANS fluorescence reflects an increase in hydrophobic surface area, probably caused by subunit dissociation. At ~ 1 kbar, a maximum is reached; beyond this value, pressure com petes with ligand binding causing fluorescence emission to be de­ creased again. Pressure release leads to a drastic fluorescence enhance­ ment, 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 ^ monomer transition monitored by subunit hybridization (T. Seifert, P. Bartholmes, and R. Jaenicke, Biochemistry, in press).