The interaction of azo-dyes with proteins. I. Binding of dyes with hydroxyl and sulphonate substituents to Bovine Serum Albumin

Abstract

Equilibrium dialysis experiments on aqueous solutions of p-hydroxyazobenzene (I), azobenzene-p-sulphonic acid (II), and p-hydroxyazobenzene-p'-sulphonic acid (III) in the presence of bovine serum albumin show that each of these azo compounds is bound by the protein. At 28 °C the interactions between protein and dye are of about equal extent for (II) and (III) whereas at 2 °C (III) is bound more strongly than (II). At both temperatures (I) is bound less strongly than either (II) or (III). Buffered solutions of these dyes show ultraviolet spectral changes in the presence of the serum albumin which presumably are also related to the interactions between dye and protein. However, these spectral shifts do not directly correlate with the relative strengths of binding for the three dyes as measured by equilibrium dialysis. This lack of correlation is ascribed to the fact that spectral changes may occur in bands associated with transitions to locally excited states which may not necessarily contain the binding site(s) through which interaction occurs with the protein. This emphasizes that spectral changes must be used cautiously as a measure of the interactions between proteins and organic molecules. The nature of these interactions for (I), (II), and (III) is discussed.