NADH assay

Kurkijarvi et al. were the first to demonstrate the feasibility of seg-mented-flow bioluminescence assays by use of a bioreactor packed with bacterial bioluminescent enzymes immobilized on Sepharose 4B [60]. The packed glass colunrn used was placed in front of the photomultiplier tube of a luminometer. The luminescence signal obtained was linearly related to the NADH concentration from 1 pmol to 10 nmol for sample volumes of 2-20 pL. In the region of 400 NADH assays could be performed with a single enzyme column, with no appreciable change in sensitivity or accuracy. However, problems arising from packing or disruption of the matrix were encountered after 4 days of intensive use. 

Luminescence has been used in conjunction with flow cells to detect electro-generated intennediates downstream of the electrode. The teclmique lends itself especially to the investigation of photoelectrochemical processes, since it can yield mfonnation about excited states of reactive species and their lifetimes. It has become an attractive detection method for various organic and inorganic compounds, and highly sensitive assays for several clinically important analytes such as oxalate, NADH, amino acids and various aliphatic and cyclic amines have been developed. It has also found use in microelectrode fundamental studies in low-dielectric-constant organic solvents. 

The physicochemical properties of the reactants in an eiKyme-catalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the abihty of a substrate or product to absorb hght. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb hght at a wavelength of 340 run, whereas their oxidized forms NAD(P) do not (Figure 7—9). When NAD(P)+ is reduced, the absorbance at 340 run therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, for a dehydrogenase that catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 run will be observed. In each case, the rate of change in optical density at 340 nm will be proportionate to the quantity of enzyme present. 

Very recently, a sandwich assay for prostatic acid phosphatase antigen was carried out using two cascaded enzyme reactions to provide amplification of the immunochemical event. In one format, an optical readout was used whereby a forma-zan dye was generated by reaction of a dye precursor and NADH generated from the second enzyme cycle. In the electrochemical format, the NADH generated in the second enzyme cycle was used to reduce Fe(CN) to FeCCN) " which was then detected amperometrically. While the use of Fe(CN) in ECIA has appeared in the... 

Levels of a number of metabolites as well as a number of enzymes in body fluids are indicative of disease conditions. Many of the enzymatic reactions mentioned above have been used in solution clinical assays as well as in test strips.446,497-508 512-515 Assays for hydrogen peroxide and the enzyme peroxidase using NADH and a tetrazolium salt have been de-scribed.509,5io Assays of exogenous substances (e.g., drugs or their metabolites) also utilize this chemistry. The determination of alcohol using alcohol dehydrogenase is an example.511 As mentioned above, the assay of enzyme levels can also be achieved using tetrazolium salts.516-520... 

Recent European patents,645,646 drawing on much earlier work647 649 describe an assay for NADH based on its ability to reduce naphthotri-azolium salts (236) to the azo dye (237) in close similarity to tetrazolium salts. 

Reisinger, C., van Assema, F., Schuermann, M. et al. (2006) A versatile colony assay based on NADH fluorescence. Journal of Molecular Catalysis B-Enzymatic, 39, 149-155. 

Oldfield s work confirmed the complete pathway and identified all the intermediates via an exhaustive experimental scheme, which included whole cell assays, cell-free extracts of IGTS8 as well as extracts from clones containing individual genes expressed in Escherichia coli. The need for NADH was clearly demonstrated in cell-free assays by amendment of NADH. The experimental evidence for involvement of FMN in the pathway was demonstrated [53,66,67],... 

Blum L.J., Gautier S.M., Coulet P.R., Continuous-flow bioluminescent assay of NADH using a fiber-optic sensor, Anal. Chim. Acta 1989 226 331. 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

As only the soluble hydrogenase utilized NADH, in vivo assays could be applied to investigate this activity further. Hydrogen-driven MMO activities were measured to obtain information on the in vivo function of this hydrogenase. The apparent Ks for hydrogen was again 0.8 mM in both assays. Maximal rates of MMO activities were 140 nmol min 1... 

The reaction mixture for a coupled assay includes the substrates for the initial or test enzyme and also the additional enzymes and reagents necessary to convert the product of the first reaction into a detectable product of the final reaction. The enzyme aspartate aminotransferase (EC 2.6.1.1), for instance, results in the formation of oxaloacetate, which can be converted to malic acid by the enzyme malate dehydrogenase (EC 1.1.1.37) with the simultaneous conversion of NADH to NAD+, a reaction which can be followed spectropho-tometrically at 340 nm ... 

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

TK activity was determined by a spectrophotometric assay at 340 nm. In 0.5 mL of tris buffer (50 mM) pH 7.6, were added 50 pL of L-erythrulose from a 1 m stock solution in water (0.05 mmol), 25 pL of o-ribose-5-phosphate from a 160 him stock solution in water (4.0 pmol), 5 pC of ThDP from a 21 mM stock solution in water (0.1 pmol), 10 pL of MgCl2 from a 50 mM stock solution in water (0.5 /rmol), 10 pL of NADH from a 14 him solution in water (0.14 /imol), alcohol dehydrogenase (12 units). 

Figure 5. Ultraviolet spectrum of NAD+ and NADH. Note that the absorption band centered at 340 nm serves as a valuable way to assay many dehydrogenases as well as other enzymes that form products that can be coupled to NAD+ reduction or NADH oxidation.

Figure 2. Illustration of the importance of the choice of reaction conditions on the determination of initial velocity. Shown are four conditions applied to examine the rate behavior of Escherichia coli NAD+-dependent coenzyme A-linked aldehyde dehydrogenase (Reaction NAD+ + CoA-SH + Acetaldehyde = NADH + Acetyl-S-CoA + H+). All assay mixtures contained enzyme, 0.5 mM NAD+, 8 /jlW CoA-SFI, 16 mM acetaldehyde, and 22.5 mM Tris buffer at pFI 8.1. (a) Time-course observed when enzyme was added to the standard assay (b) time-course observed when enzyme was added to standard assay augmented with 10 mM 2-mercaptoethanol (c) time-course observed when enzyme was first preincubated for 15 min with 8 /jlW CoA-SH, 16 mM acetaldehyde, 10 mM 2-mercaptoethanol, and 22.5 mM Tris buffer at pH 8.1, and the reaction was initiated by addition of NAD+ (d) time-course observed when enzyme was preincubated with lOmM 2-mercaptoethanol for 15 min andthen addedtostandard assay augmented with 10 mM 2-mercaptoethanol. The data are most compatible with the idea that the enzyme has an active-site thiol group that must be reduced to express full catalytic activity during assay.

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