Use of NAD P in Enzyme Assays

Use of NAD(P) in Enzyme Assays The reduced coenzymes have an absorption maximum at 340 nm, whereas the oxidized coenzymes do not. This is widely exploited to provide sensitive and specific methods for determining a variety of analytes using purified NAD (P)-linked enzymes and following the change in absorption at 340 nm as the coenzyme is either reduced or oxidized by the substrate. 

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. 

Occasionally, one can increase the Ae by utilizing alternative substrates. For example, 3-acetyl-NAD or thio-NAD can often be used with NAD -dependent dehydrogenases. Note however that an alternative substrate may change the kinetic mechanism, as compared to that observed with the naturally occurring substrate. Alternative substrates are of particular value when the normal substrate(s) and product(s) do not efficiently absorb UV or visible light. For example, many p-nitroaniline or p-nitrophenyl derivatives have proved to be quite useful in enzyme assays because they exhibit intense absorption around 410 nm. 

Numerous procedures have been used to assay aminotransferase activity. Spectrophotometric methods may use an auxiliary dehydrogenase enzyme, so that the oxo acid product is reduced at the expense of NAD(P)H (e.g.. Hatch, 1973 Achituv and Bar-Akiva, 1976 Huang et al., 1976). Alternatively, one can measure the absorbance of a product directly (Kanamori and Matsumoto, 1974), or after formation of a spectrophotometrically detectable chemical derivative (Forest and Wightman, 1972a Matherton and Moore, 1973). Radiocarbon assays (e.g., Hiller and Walker, 1961 Abbadi and Shannon, 1969 Givan al., 1970 Kirk and Leech, 1972), are sometimes used in... 

NADH and NADPH, the respective reduced forms of NAD and NADP, are highly fluorescent hence, all NAD" "- and NADP -dependent reactions involved in enzymatic assays can be monitored fluorimetrically with a sensitivity higher than those of absorptiometric techniques by two or three orders of magnitude. Reactants must be used at much lower concentrations, and detection limits of 10 moll or even lower can be achieved. Numerous methods for enzyme determination involve the use of substrates that are transformed into highly fluorescent products. For instance, p-hydroxyphenyl-acetic acid has been used widely as a substrate for the determination of several oxidases such as glucose oxidase and xanthine oxidase. 

The main steps of the activity gel assay are outlined in Table 1. The in situ detection of poly(ADP-ribose) polymerase activities includes gel electrophoresis in SDS, renat-uration of proteins with appropriate buffers, incubation of the intact gel with [ P]-NAD, removal of nonincorporated precursor by TCA, washing, and autoradiography. The method was developed by using extracts and partially purified fractions of the poly(ADP-ribose) polymerase. The catalytic peptides of the enzyme are identified as activity bands and their Mj. determined by referring to protein markers [1]. 

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