EXCITED-STATE PROTON-TRANSFER REACTION AS A PROBE FOR THE MICROENVIRONMENT OF A BINDING-SITE OF BOVINE SERUM-ALBUMIN - EFFECT OF UREA

The dynamics of proton dissociation from 2-naphthol 6,8-disulfonate (N SOH) in its first excited singlet state has been studied in the microe nvironment of the binding sites of a protein, bovine serum albumin (BS A), 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 w hich NSOH is bound. The dissociation of the proton in water is a very fast reaction, K-12' = 7.2 x 10(9) s(-1), but upon binding to BSA the rate of proton dissociation is slowed down significantly to 2.0 x 10(9 ) s(-1). This slow dissociation rate constant suggests a strong intera ction of the water molecules with the inner walls of the cavity. On ad dition of urea k(12) increases to 2.5 x 10(9) s(-1) because of increas ed availability of water molecules to hydrate the dissociated proton. The ability of the water molecules to hydrate the dissociated proton i n the site is equivalent to a homogeneous salt solution with activity, (a)(H2O) congruent to 0.67, but in the presence of urea, the activity of the water molecules in the binding site is 0.78.