Molar absorptivities of beta-NADH and beta-NADPH.

Abstract

Re-investigating the accuracy of the commonly used values for molar absorptivities (epsilon) of beta-NADH and beta-NADPH at Hg 334, Hg 365, or 340 nm, we obtained the following results: The maximum of absorbance of NADH is shifted from about 340 nm at 0 degrees C to about 338.5 nm at 38 degrees C; the corresponding maxima of NADPH are located at about 0.5-nm longer wavelengths. In addition, the absorption curves of both coenzymes broaden with increasing temperature. For these reasons, the epsilon-values of NADH and NADPH are generally different from each other, and are temperature-dependent. Only at 334 nm are they almost identical and nearly independent of temperature. Therefore this wavelength is recommended for precise measurements. The epsilon-values of these coenzymes are influenced by ionic strength and pH. To determine the absolute values of the molar absorptivities, we performed the glutamate dehydrogenase or lactate dehydrogenase assay with carefully purified 2-oxoglutaric acid or pyruvic acid in the presence of excess coenzyme. The purity of the substrates was checked through differential scanning calorimetry, moisture analysis, gas-liquid chromatography, gas chromatography in combination with mass spectrometry, and nuclear magnetic resonance spectroscopy. The epsilon-values observed under the various conditions are about 1-7% higher than those currently used.

A Complex of 2‐Hydroxyisocaproyl‐Coenzyme A Dehydratase and its Activator from Clostridium difficile Stabilized by Aluminium Tetrafluoride‐Adenosine Diphosphate

“…The decrease in the absorbance of NADH was followed at 340 nm (e = 6.3 mm À1 cm À1 ). [29] 2-Hydroxyisocaproyl-CoA dehydratase (1.5 nmol) and 1.0 nmol activator were mixed with 50 mm Mops pH 7.0, 10 mm dithiothreitol, 1 mm dithionite, 1 mm MgCl 2 , 1 mm ATP, 10 mm AlF 3 and 100 mm KF in a total volume of 50 mL. After 1 h incubation under anoxic conditions at room temperature, the mixture was centrifuged at 13 000 g for 10 min to remove a minor precipitate.…”

Structure‐guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+

“…Measurements of the pH‐dependence of enzyme activity was carried out at 37 °C in 0.2 m potassium phosphate buffer (pH 5.5–8.5). The molar extinction coefficient of NADPH was corrected for pH effects as previously described [41]. Turnover numbers were calculated on the basis of one active site per 42 kDa subunit.…”

In vitro studies on reductive vinyl chloride dehalogenation by an anaerobic mixed culture

“…Hydrogenase activity was measured in cell extracts by using a photometric assay with methyl viologen as the electron acceptor (modified from the assay described in references 5 and 7). ATPase activity was measured by using a coupled photometric enzyme assay with pyruvate kinase and lactate dehydrogenase (49,56). Malate dehydrogenase activity was measured by a photometric assay as described by Stams et al (43).…”