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

Molar Absorptivities of beta-NADH and beta-NAD at 260 nm

Erich Hald 1, Paul Lehmann 1, and Joachim Ziegenhorn 1

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

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 egrbeta-NADH340 = 6.22 x 103 for beta-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 beta-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.

Submitted on January 27, 1975
Accepted on February 27, 1975






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Copyright © 1975 by the American Association for Clinical Chemistry.