Haid E, Lehmann P, Ziegenhorn J
Clin Chem. 1975 Jun;21(7):884-7.
To determine the molar absorptivities of beta-NADH and beta-NAD at 260 nm, we purified the reduced form of the coenzyme by repeated anion-exchange chromatography. A NADH preparation was so obtained for which the 260 nm/340 nm absorbance ratio was 2.265. When calculated with epsilon 340 beta-NADH = 6.22 times 10-3 for beta-NADH at 260 nm and 25 degrees C, a molar absorptivity of 14.1 times 10-3 liter - mol minus 1 - cm minus 1 resulted from this quotient. By use of the lactate dehydrogenase or glycerol-3-phosphate dehydrogenase assay, respectively, and referring to the new absorption coefficient for NADH at 260 nm, the molar absorptivity of beta-NAD at 260 nm and 25 degrees C was established to be 17.4 times 10-3 liter - mol minus 1 - cm minus 1. The values observed are lower than those reported thus far.
为了测定β-NADH和β-NAD在260nm处的摩尔吸光系数，我们通过反复的阴离子交换色谱法纯化了辅酶的还原形式。由此获得了一种NADH制剂，其260nm/340nm吸光度比值为2.265。当根据25℃下β-NADH在260nm处的ε340β-NADH = 6.22×10⁻³进行计算时，该商得出的摩尔吸光系数为14.1×10⁻³升·摩尔⁻¹·厘米⁻¹。分别使用乳酸脱氢酶或甘油-3-磷酸脱氢酶测定法，并参考260nm处NADH的新吸收系数，确定25℃下β-NAD在260nm处的摩尔吸光系数为17.4×10⁻³升·摩尔⁻¹·厘米⁻¹。观察到的值低于迄今为止报道的值。
