Determination of the molar absorptivity of NADH

Abstract

The molar absorptivity of NADH at 340 nm has been determined by an indirect procedure in which high purity glucose is phosphorylated by ATP in the presence of hexokinase, coupled to oxidation of the glucose 6 phosphate by NAD+ in the presence of glucose 6 phosphate dehydrogenase. The average value from 85 independent determinations is 6317 liter mol-1 cm-1 at 25 °C and pH 7.8. The overall uncertainty is -4.0 to +5.5 ppt (6292 to 6352 liter mol-1 cm-1), based on a standard error of the mean of 0.48 ppt and an estimate of systematic error of -2.6 to +4.1 ppt. Effects of pH, buffer, and temperature on the molar absorptivity are also reported.