We have demonstrated that a simple electrochemical cell can serve as a detector of NADH concentration in a flow system thereby providing an assay technique for NADH dependent enzymes. When this is applied to NADH produced by enzymatic reaction, then a reproducible measure of enzyme activity is obtained. This method of enzyme activity assay is applicable to a number of oxidoreductase enzymes which employ NAD+ or NADP+ as coenzymes to achieve substrate modification. The presence of electroactive species in samples of human serum has proved a serious problem in the electrochemical analysis of serum activity. These species produce a large background anode current at the anode voltage appropriate for NADH oxidation. The presence of this high current limits the usefulness of amplification of the current output to detect small changes in NADH concentration.
SummaryGraphites of different manufacture and origin exert positive but different accelerations on the rate of oxidation of NADH to NAD' in aqueous solution; different proportions of the oxidized form of NAD retain enzymatic activity depending on the nature of the graphite. Oxidative pretreatment of the graphite surfaces increases the rates of NADH oxidation, but subsequent silanization of the surfaces to attach alkylamine groups causes the rates to decrease. The experimental results suggest the presence of at least two types of sites on graphite surfaces: one very reactive site which produces a high percentage of an enzymatically inactive reaction product of NADH and is itself deactivated during the course of reaction, and another type of site which promotes the oxidation of NADH to enzymatically active NAD+ in high yields.
Electron spin resonance (esr) spectra have been obtained for the anion radicals of 1,2,3,4-tetrakis(trifluoromethyl)-l,2-di- (CF3C) 2(PCF3)2, and l,2,3,4,5-pentakis(trifluoromethyl)-l,2,3-triphosphacyclopent-4-ene, (CF3C)2(PCF3)3, prepared by electrolytic reduction at low temperature. The observed 19F and 31P hyperfine coupling constants indicate that the unpaired electron is localized mainly in the carbon-carbon double bond and that little delocalization to the phosphorus atoms occurs.Reduction of the two compounds with alkali metals led to anion radicals of decomposition products of the parent phosphines. Attempts to generate anion radicals from the cyclopolyphosphines, (CF3P)4 and (CH3P)5, or 3,4-bis(trifluoromethyl)-l,2-dithietene, (CF3C)2S2, were unsuccessful.
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