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

Low dissociation or reverse rate constants of single-step or multistep comp lex formation equilibria are usually obtained with reduced precision from s tandard stopped-flow binding experiments by determination of the intercept of the concentration dependence of k(obs). Large and fast concentration jum ps, based on two different step-motor-driven mixng setups, are performed wi th 60-300-fold dilutions that allow the precise, convenient, and independen t determination of dissociation rate constants in the range of approximatel y 0.1-100 s(-1) in a single stopped-flow dissociation experiment, A theoret ical basis is developed for the design and for the evaluation of such dilut ion experiments by considering the rebinding occurring during dissociation. The kinetics of three chemical systems are investigated, the binding of Mg 2+ 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 expe riments employing various dilution factors. The advantage of the dilution m ethod for investigating chemical and biological systems is emphasized.