Absorption spectra of NAD+

Figure 7-9. Absorption spectra of NAD and NADH. Densities are for a 44 mg/L solution in a cell with a 1 cm light path. NADP and NADPH have spectrums analogous to NAD and NADH, respectively.

Figure 15-2 Absorption spectra of NAD+ and NADH. Spectra of NADP+ and NADPH are nearly the same as these. The difference in absorbance between oxidized and reduced forms at 340 nm is the basis for what is probably the single most often used spectral measurement in biochemistry. Reduction of NAD+ or NADP+ or oxidation of NADH or NADPH is measured by changes in absorbance at 340 nm in many methods of enzyme assay. If a pyridine nucleotide is not a reactant for the enzyme being studied, a coupled assay is often possible. For example, the rate of enzymatic formation of ATP in a process can be measured by adding to the reaction mixture the following enzymes and substrates hexokinase + glucose + glucose-6-phosphate dehydrogenase + NADP+. As ATP is formed, it phosphorylates glucose via the action of hexokinase. NADP+ then oxidizes the glucose 6-phosphate that is formed with production of NADPH, whose rate of appearance is monitored at 340 nm.

Absorption spectra of NAD+ (NADP" ") and NADH (NADPH). At 340 nm, the reduced coenzymes (NADH or NADPH) show significant absorbance, whereas the oxidized forms (NAD or NADP" ") show negligible absorbance. Thus, many enzymatic reactions can be monitored at 340 nm if the reaction is directly or indirectly dependent upon a dehydrogenase reaction involving a nicotinamide adenine dinucleotide as a coenzyme. 

Pig. 1. UV-visible absorption spectra of NAD(P)H rubredoxiii oxidoreductase. The solid line... 

Figure 2 UV absorption spectra of NAD, NADP, and their reduced forms used for photometry in enzymatic glucose assays.

The basicity of pyridinyl radicals is of interest since some pyridinyl radicals react with one another in an acid catalyzed reaction (sect. 4.4). The absorption spectra of radicals generated from the pyridinium ion by pulse radiolysis in aqueous solution at different pH values allow the evaluation of the basicity of the radicals 97,98) I jjg Qf protonated form of l-methyl-3-carbamidopyridinyI radical, (CONHj) ), is 1.43 the protonated radical has alsorption maxima at 3(X) nm and 440 nm, at somewhat longer wavelengths than those for the unprotonated radical at 280 nm and 420 nm. The nicotinamide adenine dinucleotide radical (NAD ) has a pK, of 0 or less, with a shift in absorption maximum due to protonation being observed only in 1.5 M HCIO4. 

Dehydrogenase Reactions. The reduced (NADH) and oxidized (NAD+) forms of nicotinamide adenine dinucleotide exhibit marked differences in their ultraviolet absorption spectra and are, therefore, widely used for following the course of dehydrogenase reactions. The ultraviolet absorption spectra for NAD" " and NADH are given in Figure 22.4. NAD has negligible absorption at 340 nm while NADH has an absorption maximum, and so it is a simple matter to monitor the increase or decrease in NADH concentration. 

The classic cytochrome h, expressed sometimes as cytochrome hn, b-561, or b-562, has a single symmetric a band at 561-562 nm in the reduced minus oxidized difference spectrum at room temperature at liquid nitrogen temperature (77°K) it has an a band at 558-559.5 nm, a /3 band at 529 nm, and a Soret band at 428 nm. Cytochrome b is readily reduced by succinate and NAD-linked substrate in both coupled and uncoupled mitochondria. This cytochrome is associated with complex III (ubiquinone-cytochrome c reductase) 24). Figure 1 shows the absorption spectra of reduced cytochrome b at different temperatures between liquid helium and room temperature 26). 

Figure 16.2 Absorption spectra of the oxidized and reduced forms of the nicotinamide adenine nucleotides (NAD and NADP)...

When the substrate or the reaction product or both has a characteristic absorption spectra in the visible or ultraviolet ranges, it is possible to calculate the correction factor by measurement of the optical density variations in identical experimental conditions at two selected wavelengths. For example at 334 nm, NAD+ does not show any absorption, whereas... 

At 334 nm NAD(P)H is near its maximum absorption, and Hb02—Hb spectra are in the vicinity of an isosbestic point (eJSP2 - e m = 1.3). Therefore, from measurements at 436 and 334 nm, it is possible to simultaneously determine both the formation or disappearance of NAD(P)H and the oxygen consumption (Figure 7). This procedure permits rapid and accurate estimation of mitochondrial oxidative phosphorylation, or of hydroxylation reactions that occur in liver microsomes... 

Proteins that have tightly bound cofactors, such as heme proteins, photosynthetic reaction centers and antenna proteins, flavoproteins, and pyridoxal phosphate- and NAD-dependent enzymes, provide a variety of chromophores which have absorption bands in the visible and UV region. The CD bands associated with the chromophoric groups are frequently quite intense, despite the fact that the isolated chromophores are achiral in many cases, and therefore have no CD, or are separated from the nearest chiral center by several bonds about which relatively free rotation can occur, and therefore have only weak CD. The extrinsic or induced CD observed in the visible and near-UV spectra of the proteins can provide useful information about the conformation and/or environment of the bound chromophore, which usually plays a critical role in the function of the protein. 

Pileni and co-workers [411] also used normal micelles with Cu(DS)2 and with or without NaDS solubilized in an aqueous solution. Sodium borohydride, NaBH Cu(DS)2 = 2, was added to the solution after deaeration the reactions took place in a glove box. At [Cu(DS)2] = 1.2 x 10 M, pure Cu particles formed below this, absorption spectra indicated copper oxide as the major phase. Above the concentration of Cu(DS)2 = 1.2 x 10" M, both spherical and elongated particles were obtained. The peak size, as a function of the relative concentrations of the two surfactants, changed from 2 nm to -6 nm. 

Several enzymatic methods use the coenzyme nicotinamide adenine dinucleotide in its oxidized (NAD ) and reduced (NADH) forms, which makes a very useful reversible system as they are involved in several enzymatic reactions and have rather different absorption spectra (Figure 3). For example, glucose in blood serum can be determined with glucose dehydrogenase according to reaction [I] by monitoring the absorbance increase of NADH at 340 nm ... 

Nicotinamide adenine dinucleotide phosphate, which occurs in oxidized (NADP+) and reduced (NADPH) forms with markedly different absorption spectra and an absorptivity similar to NAD can also be used for enzymatic analysis. Thus, Mg in serum can be measured by the oxidation reaction of isocitrate with NADP+, catalyzed by isocitrate dehydrogenase (IDH) (eqn [HI]) ... 

The complexation of PABA with nucleotides was further studied using polarization modulated infrared reflection absorption spectroscopy (PM-IRRAS). PM-IRRAS spectra of PABA films exhibit all the characteristic vibrations of polyaniline and boronic acid (Figure 3.27) [93, 94]. After complexation with NAD+ (Figure 3.27, b) and NADH (Figure 3.27, c), the disappearance of the free B-OH group vibration at 986 cm and increase in the intensity of the asymmetric B-O bond vibration at 1330 cm indicate the formation of boronate ester. The new vibrations at 1080 and 1470 cm have been attributed to ribose and adenine moieties, respectively [153]. The vibrations at 1218 (NAD+), 1245... 

Ultraviolet (UV) and visible spectra, also known as electronic spectra, involve transitions between different electronic states. The accessible regions are 200-400 nm for UV and 400-750 nm for visible spectra. The groups giving rise to the electronic transitions in the accessible regions is termed chromophores, which include aromatic amino acid residues in proteins, nucleic acid bases, NAD(P)H, flavins, hemes, and some transition metal ions. Two parameters characterize an absorption band, namely the position of peak absorption Wmax) and the extinction coefficient (e), which is related to concentrations of the sample by the Beer-Lambert law ... 

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