Excited State Proton Transfer Reaction as a Probe for the Microenvironment of a Binding Site of Bovine Serum Albumin: Effect of Urea

Abstract

The dynamics of proton dissociation from 2-naphthol 6,8-disulfonate (NSOH) in its first excited singlet state has been studied in the microenvironment of the binding sites of a protein, bovine serum albumin (BSA), using time-resolved fluorimetry. In concentrated salt solution the dissociation is slowed down as an exponential function of activity of water in the solution. This kinetic parameter has been used to probe the microenvironment of the binding sites of bovine serum albumin at which NSOH is bound. The dissociation of the proton in water is a very fast reaction, k‘12 = 7.2 × 109 s-1, but upon binding to BSA the rate of proton dissociation is slowed down significantly to 2.0 × 109 s-1. This slow dissociation rate constant suggests a strong interaction of the water molecules with the inner walls of the cavity. On addition of urea k12 increases to 2.5 × 109 s-1 because of increased availability of water molecules to hydrate the dissociated proton. The ability of the water molecules to hydrate the dissociated proton in the site is equivalent to a homogeneous salt solution with activity, a(H2O) ≅ 0.67, but in the presence of urea, the activity of the water molecules in the binding site is 0.78.