Cooperative Effects in Binding by Bovine Serum Albumin. I. The Binding of 1-Anilino-8-naphthalenesulfonate. Fluorimetric Titrations

The binding of anilinonaphthalenesulfonate by bovine serum albumin at various values of pH, temperature, and ionic strength has been studied by a fluorometric technique. Five moles of anilinonaphthalenesulfonate was bound/mole of serum albumin over the pH range 10-5.0. The equilibrium concentrations of free ligand, free protein, and complex were determined for values of the probability of binding from 0.05 to 0.95. The more complete titration curves obtained contained some 20 binary units of information. Abrupt changes in the apparent reaction order, similar to those demonstrated in the equilibrium of reduced diphosphopyridine nucleotide (DPNH) and beef muscle lactate dehydrogenase, were observed at pH 7, and a continuous increase in the reaction order with probability of binding was seen at pH 5. The results of pH 7, and probably also those at pH 5.0, are not expressible by an equation of Adair's type. We believe that, as in the case of lactate dehydrogenase, the results indicate the presence in the binding system of relaxation effects that are slow in comparison with the rate of dissociation of the ligand.