On the Problem of Acid-labile Triphosphopyridine Nucleotide in Biological Material

Abstract

TPN+ which was 4 times higher than that obtained by the usual extraction procedure with high acid concentrations. They concluded that this addit.ional TPN+ was destroyed in the strong acid ext,raction and that therefore in liver tissue the total content of TPNf + TPNH is higher, and the ratio of TPNH to TPN+ lower, than reported by earlier investigators (3-6). Burch, Lowry, and Von Dippe (1) used the common method consisting of extraction of the tissue first and then assay of the pyridine nucleotides. The reduced pyridine nucleotides were measured after alkaline extraction and the oxidized forms after acid extraction. Each extraction must preserve the corresponding oxidation-reduction form of the pyridine nucleotides or else apparent deviations of the total pyridine nucleotide content will be observed. The extraction method of Burch, Lowry, and Von Dippe (1) was examined with a new enzymatic assay of reduced pyridine nucleotides in alkaline extracts (7) and a method for the assay of both reduced and oxidized pyridine nucleotides in one extract (8,Q). Upon extraction with perchloric acid, TPNH and DPNH are quantitatively degraded to adenosine 5’.diphosphate ribose 2’.phosphate and adenosine 5’-diphosphate ribose, respectively. These degradation products and t,he unaffected oxidized pyridine nucleotides are separated by ion exchange chromatography and measured by their absorption at 265 mp. By this method, it can be decided whether the acid-labile TPN+ of Burch et al. (I) really occurs, or whether it is due to conversion of TPNH to TPN during the inactivation of the tissue in the course of extraction. In the first case, the total amount of TPN+ + TPNH assayed should be higher in the weak acid ext.ract than in the strong acid extract, whereas in the second case it should be the same in both extracts.