The Chronoamperometric Determination of Homogeneous Small Molecule-Redox Protein Reaction Rates

An essentially new application of chronoamperometry is presented for the determination of homogeneous second-order rate constants for the reactions between small molecule reductants and redox proteins. The first part of the work is a comparison between stopped-flow kinetics and chronoamperometric kinetics for the reaction of ferrous-EDTA with horse cytochrome c. The reaction was demonstrated to be first order in both ferrous-EDTA and cytochrome c and the effect of ionic strength was also studied. All of the chronoamperometric results compared well with the stopped-flow work which had been done previously. Chronoamperometry was then used to study several other reactions which have not been previously examined, including the reaction of ferrous-diethylenetriamine pentaacetic acid with cytochrome c. The reaction was slower than the ferrous-EDTA reaction but was more sensitive to ionic strength because of the greater charge (−3) on the complex. The second study was the reaction of ferrous-EDTA with Rhodospirillum rubrum cytochrome c2 as a function of ionic strength. This novel application of chronoamperometry to small molecule-redox protein reactions represent a new and relatively easy alternative to anaerobic stopped-flow kinetics