Absorption Spectrum of NADH

Students should receive a stock solution of NADH with a concentration equal to 5 mg ml-1. Since the molecular weight of NADH is 709, the concentration of the stock 

The absorption spectrum of NADH should be obtained by adding 10 pt 1 of the stock solution to 1.2 ml of phosphate buffer (0.1 M, pH 7.5). The concentration of NADH stock solution should be calculated at 340 nm using an e equal to 6200 M-1 cm-1. 

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NAD+/ NADH ratio, redox potential 767 NADH (NADH + H+ NADH2) 765-771 absorption spectrum of 768 modification in acid 780 NADH-X 780... 

Fig. 14. Absorption spectrum of NADPH-treated minus NADH-treated complex...

Fig. 14. Absorption spectrum of NADPH-treated minus NADH-treated complex I. Conditions complex I, 6 mg protein/ml of 0.66 M sucrose containing 50 mAf Tris-HCl (pH 8.0), 1 mM histidine, and 0.25% (v/v) Triton X-100. The sample cuvette was treated with 200 iiM NADPH, and the reference cuvette with 100 /lAf NADH. Dashed line, untreated complex I in both cuvettes. From Hatefi and Hanstein (,80).

NADH-cytochrome P-450 reductase was purified as a single polypeptide chain with a molecular weight of 51 kDa. The absorption spectrum of the purified protein showed typical flavoprotein absorption maxima at 370 and 446 run, and a shoulder at 475 nm. Consistent with the detection of the flavin prosthetic group, the purified NADH-... 

Fig. 35. Absorption spectrum of octane fraction after 5 h incubation in argon atmosphere. Medium 10" M FeEP, 2 x 10" M TMQ, 2 x 10 M NADH, 20 mM tris-HCl, pH 7.5, 1) before incubation with NADH, 2) after incubation with NADH [56]...

Fig. 2.4 The spectrum of bacterial luminescence measured with B. harveyi luciferase, FMN, tetradecanal and NADH, in 50 mM phosphate buffer, pH 7.0, at 0°C (dashed line from Matheson et al., 1981) and the absorption and fluorescence emission spectra of LumP (solid lines) and Rf-LumP (dotted lines) obtained from P. leiog-natbi, in 25 mM phosphate buffer, pH 7.0, containing 1 mM EDTA and 10 mM 2-mercaptoethanol, at room temperature (from Petushkov et al, 2000, with permission from Elsevier). LumP is a lumazine protein, and Rf-LumP contains riboflavin instead of lumazine in the lumazine protein. Fluorescence emission curves are at the right side of the absorption curves.

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 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.

The stability of EH2 is very species dependent. All of the above results refer to the pig heart enzyme and, where tested, to other mammalian species. It was initially reported that no long wavelength absorption was observed upon reduction of E. coli enzyme with NADH 109), but reduction by 1 equivalent of NADH or dihydrolipoamide leads to the formation of 25% of the maximal 2-electron-reduced species 108) and similar results are obtained with the Azotobacter enzyme 114)- That this species is the catalytically important one in the E. coli enzyme as well as in the mammalian enzyme has also been demonstrated 50). Reduction with dihydrolipoamide in the rapid reaction spectrophotometer at 2° results in the full formation of EH2 followed by the slow k = 13 min, 1 mAf dihydrolipoamide) four-electron reduction. The spectrum of EHa generated in this way is shown in Fig. 7 and is identical with that of the pig heart enzyme. The 2-electron-reduced form, EHj of lipoamide dehydrogenase of spinach 99) may be somewhat unstable however, spectrally it is difficult to distinguish between instability and formation of the EHa-NADH complex (see above) on the basis of available spectral data. Either phenomenon could lead to inhibition by excess NADH. In glutathione reductase it is possible that the complex can be rapidly reoxidized by glutathione 53). 

Fia. 9. Spectral characteristics of the soluble NADH dehydrogenase (1.6 mg/ml) isolated from complex I. Traces 1, spectrum of oxidised enzyme 2, NADH-reduced enzyme 5, dithionite-reduced enzyme 4, flavin contribution to 1 after destruction of iron-sulfur chromophore with sodium mersalyl 3, iron-sulfur contribution to l obtained by subtraction of 4 from 1 6, 4 plus dithionite showing that after destruction of the iron-sulfur chromophore with mersalyl and reduction of flavin with dithionite the enzyme has no absorption in the visible region. From Hatefl and Stempel (40). 

Membranes prepared from T. acidophilum oxidize succinate, malate, lactate, and NADH, consuming oxygen in the process, but only contain a single cytochrome. It appears to be a b-type cytochrome based on its absorption spectrum and the solubility of the heme in acid-acetone [126]. Since aeration during growUh affects the cytochrome composition in T. acidophilum, failure to detect a more complex cytochrome pattern may reflect the level of aerobiosis during growth. 

The binding of the nonfunctional part of the coenzyme can be studied by measuring the absorption spectra of the complex against those of the isolated components. The formation of enzyme—coenzyme complexes changes the intramolecular interactions the intensity of these interactions can be measured by comparing the spectra of dihydro coenzyme analogues and the mononucleotides. Against NADH the spectrum of an equimolar mixture of dihydronicotinamide mononucleotide and adenosine monophosphate shows rises at 335 nm and 260 nm and a minimum at 280 nm. The peaks at 320 nm and... 

The equilibrium and rate constants for NADH binding to the three isozymes EE, ES, and SS of the horse enzyme have been determined 305). Differences in binding to the two types of chains were found both for the binding strength and the pH dependence. Changes in the absorption spectrum 306,307), the fluorescence polarization spectrum (305), the optical rotatory dispersion spectrum 309), and the effect of DzO on the fluorescence spectrum 310) have been studied for the binary enzyme coenzyme complexes compared to the free molecules. 

Reduction of the enzyme-bound FAD was observed on adding sodium dithionite at pH 10.5 (Figure 3). FAD reduction was also observed on adding NADH both in the presence of imidazoleacetate and in its absence (Figure 3). An absorption spectrum characteristic of the semiquinoid form of FAD appeared when the enzyme was half reduced with sodium dithionite but not with NADH. 

The reduced dinucleotide, carba-NADH, has an electronic absorption spectram with maxima at 260 nm and 360 nm. It exhibits a broad fluorescence emission at 456 nm, typical of a dihydropyridine. The fluorescence excitation spectrum of carba-NADH shows the expected maxima at 360 nm as well as a second, weaker excitation at 260 nm, due to intramolecular energy transfer from the adenosyl ring (9, 10). The similarities in the ratios of the 260 nm excitation to the longer wavelength excitation between carba-NADH and NADH indicate a similar amount of fluorescence energy transfer, and therefore a similar amount of stacked conformer present in solution. Thus, by this spectroscopic criteria, carba-NADH adopts a conformation in solution indistinguishable from that of NADH. 

The change in structure when NAD is reduced or oxidized is reflected in an alteration of its ultraviolet spectrum. The reduced form has an absorption maximum at 340nm, while the oxidized form has little absorption at this wavelength (Figure 16.2). This forms the basis for assays of biological oxidation/reduction reactions where alteration in the absorption at 340 nm can be related to changes in the concentration of NADH. 

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