Molar absorptivities of beta-NADH and beta-NAD at 260 nm.
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.
D002852:Chromatography, Ion Exchange; D004099:Dihydroxyacetone Phosphate / Q000032:analysis; D005993:Glycerolphosphate Dehydrogenase; D005994:Glycerophosphates / Q000032:analysis; D007770:L-Lactate Dehydrogenase; D007773:Lactates / Q000032:analysis; D008433:Mathematics; D009243:NAD / Q000378:metabolism*; D011773:Pyruvates / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet
D005994:Glycerophosphates; D007773:Lactates; D011773:Pyruvates; D009243:NAD; D004099:Dihydroxyacetone Phosphate; D005993:Glycerolphosphate Dehydrogenase; D007770:L-Lactate Dehydrogenase