THE EXTINCTION COEFFICIENTS OF THE REDUCED BAND OF PYRIDINE NUCLEOTIDES

Abstract

Warburg and Christian (1) observed that the reduced forms of diphosphopyridine nucleotide (DPN) and triphosphopyridine nucleotide (TPN) have absorption bands with maxima at 340 rnp, whereas the oxidized forms have no absorption at this wave-length. Application of this observation to t.he quantitative determination of the pyridine nucleotides and of substrates which can be brought into stoichiometric reaction with them has been hampered by the lack of reliable extinction coefficients for these substances. The published values for DPN, recently reviewed by Drabkin (2), vary from 4.78 X lo6 to 6.28 X lo6 sq. cm. X mole-*. In the case of those values which were determined by calculation from the absorption of a given quantity of nucleotide which was assumed to be pure, it may be presumed that the purest samples yielded the highest values, although no good criterion of purity is available. The highest value thus far reported? 6.28 X 106, was obt’ained by Ohlmeyer (3) for a sample of isolated reduced DPN. In the case of TPN there is considerably less information, although the molecular extinction coefficient has been reported to be the same as for DPN (4). Precise values for the extinction coefficients can be determined with pyridine nucleotide preparations which are not necessarily pure by the use of pure substrates in reactions which are essentially complete. Thus, with an excess of pyridine nucleotide over substrate under suitable conditions the change in absorption would be due to the reaction of a quantity of nucleotide equivalent to the added substrate. Such determinations have been made with pyruvic acid, acetaldehyde, and isocitric acid. For the reactions,