Amperometric assay of coenzyme‐dependent oxidoreductase enzymes in a flow‐through cell

Abstract

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.

Stability of catechol modified carbon electrodes for electrocatalysis of dihydronicotinamide adenine dinucleotide and ascorbic acid

“…There have been several recent attempts to couple the electrooxidation of the cofactor, dihydronicotinamide adenine dinucleotide (NADH), to the turnover of enzyme substrates (1) and to the analysis of enzyme or enzyme substrates (2,3) for clinical purposes. The potential of these attempts has not been fully realized because of problems associated with the electrooxidation of NADH, namely, the high overpotential and the lack of selectivity.…”

“…We previously reported the successful electrocatalysis of NADH oxidation using a catechol modified CME (10) where the peak potential, Ep, for the oxidation of NADH was lowered by ca. 0.3 V compared to a "bare" carbon electrode at pH 7. The characteristics of the cyclic voltammetric (CV) current-potential (i-E) waves, in the absence and presence of NADH, led to the proposal that the reaction sequence could be described by a surface ec catalytic mechanism: (2) where the bound catechol, IA, was oxidized to the quinone form, IB, which in turn oxidized NADH. Indeed as expected, the redox potential for the catalysis of NADH by the electrogenerated quinone from solution catechol and from immobilized catechol, for example, the 3,4-dihydroxybenzylamine (3,4-DHBA), were very similar.…”

Comparative Electrochemical Study of N‐, C‐terminal and Integral Centrin on Adsorption and Metal‐Binding Properties

“…We also explore the affinity of EoCen, C‐EoCen and N‐EoCen to electrode surface. The adsorption of proteins onto a GC electrode surface has been described by the Langmuir isotherm :…”

“…We also explore the affinity of EoCen, C-EoCen and N-EoCen to electrode surface. The adsorption of proteins onto a GC electrode surface has been described by the Langmuir isotherm [32][33][34]: (1) In which C (mol·dm À3 ) is the equilibrium concentration of the adsorbate in the bulk solution, G (mol·cm À2 ) is the amount of protein adsorbed (i. e., surface concentration), G max (mol·cm À2 ) is the maximum value of G (saturated surface concentration) and the parameter B ADS (dm 3 ·mol À1 ) reflects the affinity of the adsorbate molecules towards adsorption sites. Since R ct is directly propor- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 tional to G, substitution of R ct for G, and rearrangement of Eq.…”

Flow Injection Analysis of Reduced Nicotinamide Adenine Dinucleotide Usingβ-Naphthoquinone-4-sulfonateas a Mediator

“…5 ) Thus, the oxidation of NADH has been focused on mediator-linked oxidation instead of the direct oxidation of NADH at the electrode, and the electrochemical monitoring of the reduced mediator has been extensively used to determine the NADH and/or dehydrogenase substrate concentrations. Mediators such as phenazine methosulfate (PMS),6,7) 2,6-dichlorophenolindophenol (DCPIP),8.9) Bindschedler's Green (BG),1°) and Meldora's Blue (MB)l1) have been used and the characteristics of ideal mediators were reviewed recently.…”