Dehydrogenases optical assay

Fig. 23. Optical assay of a dehydrogenase. The change in absorption is plotted against time. With more enzyme used, the reaction runs faster.

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. 

Numerous methods based on different analytical principles have been offered for the measurement of dehydrogenase reactions. Most reliable and simple are spectrophotometric procedures ( optical test ) founded on the assay of one of the participating coenzymes (or substrates). 

Fig. 7. Enzyme-coupled assay in which the hydrolase-catalyzed reaction releases acetic acid. The latter is converted by acetyl-CoA synthetase (ACS) into acetyl-CoA in the presence of (ATP) and coenzyme A (CoA). Citrate synthase (CS) catalyzes the reaction between acetyl-CoA and oxaloacetate to give citrate. The oxaloacetate required for this reaction is formed from L-malate and NAD in the presence of L-malate dehydrogenase (l-MDH). Initial rates of acetic acid formation can thus be determined by the increase in adsorption at 340 nm due to the increase in NADH concentration. Use of optically pure (Ry- or (5)-acetates allows the determination of the apparent enantioselectivity i app i81)-...

Both are abundant in skeletal muscle, myocardium, liver, and erythrocytes, so that hemolysis must be avoided, and in serum they may be assayed spectrophotometrically by their conversion of phosphate-buffered pyruvate to lactate (R6, W16) or oxalacetate to malate (S25) at the expense of added NADH2, when the rate of decrease of optical density at 340 m x thus measmes the serum activities of the respective enzymes. Recently, however, the reverse reaction has been found best for serum lactic dehydrogenase assay (A2a). In conventional spectrophotometric units the normal ranges are 100-600 units per ml for lactic dehydrogenase (W16) and 42-195 xmits per ml for malic dehydrogenase (S25) as before, one conventional spectrophotometric unit per ml = 0.48 pmoles/ minute/liter (W13). 

The catalytic detection of ammonium ions has not been extensively investigated in contrast with the large variety of potentiometric and amperometric chemical sensors and optical sensors described in the literature [236], Similarly, the detection of ammonia in air has merited diflierent approaches in the field of chemical sensors. Screen-printed electrodes modified with Meldola s Blue and covered with a polycarbonate membrane constitute the basis of the catalytic detection of NHj. The measurement is based on the electrocatalytic reduction of NADH upon addition of glutamate dehydrogenase to a stirred solution containing NADH, 2-oxoglutarate and ammonium ions. The rate of current decrease (nA s ), measured at 50 mV, correlates to the concentration of ammonium ions in the sample. Recoveries of ammonium ions in spiked pond and tap waters at the level of 0.1 ppm are close to 100%, which demonstrates the feasibility of this assay for the detection of ammonium ions in waters [237],... 

After the addition of 30 pL of TetraColar One reagent, 300 pL of media mentioned above are incubated under 5% C02-air at 37°C for 2h. The optical density at 450 nm of the media containing the water-soluble tetrazolium salt produced by the enzyme reaction is read, and the cell number in cultures is measured by the modification (WST-8 method) of the MTT assay for mitochondrial dehydrogenase activity in viable cells [27]. 

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