Clinical Chemistry, Vol 21, 884-887, Copyright © 1975 by the American Association for Clinical Chemistry

Molar Absorptivities of -NADH and -NAD at 260 nm

¹ Boehringer Mannheim GmbH, Biochemica Werk Tutzing, Forschungszentrum, 8132 Tutzing, Germany.

To determine the molar absorptivities of -NADH and -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 _-NADH³⁴⁰ = 6.22 x 10³ for -NADH at 260 nm and 25 °C, a molar absorptivity of 14.1 x 10³ liter · mol^-1 · cm^-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 -NAD at 260 nm and 25 °C was established to be 17.4 x 10³ liter · mol^-1 · cm^-1. The values observed are lower than those reported thus far.