Kinetic Studies of Proton Transfer in the Microenvironment of a Binding Site

Abstract

Excitation of 8‐hydroxypyrene 1,3,6‐trisulfonate to its first electronic singlet state converts the compound from weak base (pK°= 7.7) into a strong acid (pK^*= 0.5). The dissociation of the proton in water or dilute salt solution is a very fast reaction, k₁₂= 1 × 10¹⁰ s⁻¹. In concentrated salt solutions the dissociation is slowed as an exponential function of the chemical activity of the water in the solution. This kinetic parameter has been used to gauge the properties of the microenvironment of the binding sites of bovine serum albumin at which this compound is bound. Time‐resolved flurometry reveals two distinct steps:a rapid dissoiciation of the proton with τ=300± 40 ps which lasts ∼ 0.5 ns, followed by a slower reaction with τ= 3.3 ns. The first rapid phase represents proton dissociation taking place in the binding site. From the rate constant k= 3.3 × 10⁹ s⁻¹ we estimate that the ability of the water molecules in the site to hydrate the ejected proton is equivalent to a salt solution with water activity of 0.85. The slow phase represents the escape of the proton from the binding site. The rate of the escape, 1.4 × 10⁸ s⁻¹, is significantly slower than diffusion‐controlled dissociation. It is concluded that the shape of the site or its lowered proton conductivity do not allow a rapid escape of the proton to the bulk. Still it should be remembered that the escape of the proton is 10⁵–10⁶‐times faster than a typical turnover of an enzyme.