Reaction of Pyridine Nucleotides with 2,4-Dinitrophenylhydrazine

Abstract

WHILE attempting to isolate and identify pyridme nucleotide-dependent and enzymatically-formed aldehydes and α-keto acids as their 2,4-dinitrophenylhydrazone derivatives, it was noted that derivatives were formed which, when chromatographed on paper, were unlike known standards and gave blue colours when sprayed with alcoholic KOH. The reaction mixture in which this phenomenon was first observed was a control tube that contained: tris-HCl buffer, pH 8.5, 1,500 µmoles; nicotinamide-adenine-dinucleotide (NAD), 10.0 µmoles; and crude cell-free extract containing 1.2 mg protein in a total volume of 3.8 ml. The incubation was terminated by the addition of an equal volume of saturated 2,4-dinitrophenylhydrazine (DNPH) in 2 N HCl. The reaction mixture was centrifuged to remove precipitated protein and the supernatant fluid decanted into a clean test-tube and incubated overnight at 37° C. Further investigations revealed that the flocculent precipitate which formed was due to a reaction between NAD and DNPH. This product was purified by the usual extraction procedure including initial extraction with ethyl acetate to remove unreacted DNPH, neutral and acidic hydrazones; the latter were re-extracted into 10 per cent sodium carbonate, leaving unreacted DNPH and neutral hydrazones behind in the organic phase; the carbonate layer was acidified at 4° C and re-extracted with ethyl acetate. The ethyl acetate layer was evaporated and aliquots were used for spotting on to paper chromatograms.