The effect of viscosity on the accessibility of the single tryptophan in human serum albumin.

When reactions take place with one of the reactants tied to protein matrix, movements along the reaction coordinate towards the transition state can become coupled to structural fluctuations of the protein matrix. This investigation aims to test the assumptions underlying the arguments supporting such a coupling. A coupling is allowed only if the activation barrier is high and broad enough as shown to be the case for the proton catalyzed isotope exchange at Trp-63 of lysozyme. In the present investigation the activation barrier for the same reaction has been lowered radically in an effort to show that the coupling, as measured by the dependence of rate on solution viscosity, will diminish and ideally vanish, despite the unchanged effects of cosolvents on the chemical activities of all the reactants. The isotope exchange rate at the indole nitrogen of the single tryptophan residue of human serum albumin was measured with UV. This residue is rigidly held to the protein surface and the solvent access, although restricted, corresponds to a partially exposed residue. As a consequence, the isotope exchange rates and the bimolecular quenching rate of fluorescence by acrylamide, also measured, are high. The experiments were carried out at pH 5.2 where the molecule is in the N-form and the exchange is catalyzed by OH- ions. The activation energy of the hydroxyl catalyzed reaction is 22 kJ lower than for the proton catalyzed process. Under these conditions the exchange rate is viscosity independent both in the case of glycerol and in ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)