Concentration jump experiments for the precise determination of rate constants of reverse reactions in the millisecond time range.

Abstract

Low dissociation or reverse rate constants of single-step or multistep complex formation equilibria are usually obtained with reduced precision from standard stopped-flow binding experiments by determination of the intercept of the concentration dependence of k(obs). Large and fast concentration jumps, based on two different step-motor-driven mixing setups, are performed with 60-300-fold dilutions that allow the precise, convenient, and independent determination of dissociation rate constants in the range of approximately 0.1-100 s(-1) in a single stopped-flow dissociation experiment. A theoretical basis is developed for the design and for the evaluation of such dilution experiments by considering the rebinding occurring during dissociation. The kinetics of three chemical systems are investigated, the binding of Mg2+ to 8-hydroxyquinoline as well as of Ca2+ and K+ to the cryptand [2.2.2], by carrying out standard stopped-flow binding as well as dissociation experiments employing various dilution factors. The advantage of the dilution method for investigating chemical and biological systems is emphasized.