Molar absorptivities of β NADH and β NADPH

Abstract

The authors re investigated the accuracy of the commonly used values for molar absorptivities (ε) of β NADH and β NADPH at Hg 334, Hg 365, or 340 nm. The maximum of absorbance of NADH is shifted from about 340 nm at 0 °C to about 338.5 nm at 38 °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 ε 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 ε values of these coenzymes are influenced by ionic strength and pH. To determine the absolute values of the molar absorptivities, the glutamate dehydrogenase or lactate dehydrogenase assay was performed 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 ε values observed under the various conditions are about 1-7% higher than those currently used.