Computer-assisted indirect coulometric titrations of biological redox components

Abstract

The optically coupled, indirect coulometric titration (ICT) method has been applied to assist in the characterization of several redox biocomponents such as heme proteins. This method has provided rapid and repetitive evaluation of redox stoichiometry and energeties with a high degree of precision. The usual manual ICT procedure requires operator control of the repetitive incremental additions of electrochemical charge, the on/off of the magnetic stirrer between such additions, and the recording of optical spectra after each addition. The sequence and timing of events in the above procedure can be fixed which then lends itself ideally to computer control. In this paper, a computer-controlled ICT intsrumentation is described. The hardware and software developed for the computerization permit versatility of displaying ICT data in various formats, i.e., conventional absorbance or derivative spectra and dual wavelength or differences between dual-wavelength spectra. Also, absorbance at a given wavelength could be obtained from the spectra as a function of the incremental addition of charge. The three-dimensional plots of spectral absorbance as a function of wavelength and incremental charge could also easily be obtained. To demonstrate the capability and versatility of the computer-controlled instrumentation, the ICT of the redox components, cytochrome c and cytochrome c oxidase, and of intact mitochondria is presented.