The stability of pyridine nucleotides

O H LOWRY, J V PASSONNEAU, M K ROCK
1961 Journal of Biological Chemistry  
Diphosphopyridine nucleotide and triphosphopyridine nucleotide when used in appropriate enzyme systems become exceedingly useful reagents for the measurement of almost any biochemical substance. A companion paper describes procedures for measuring each of the pyridine nucleotides, whether in the oxidized or reduced form, at concentrations as low as 1O-g M and in amounts as small as lo-l5 moles (1). To exploit this sensitivity it was necessary to know more about the stability of the oxidized and
more » ... reduced forms of the two nucleotides under a variety of conditions. It is well known that the reduced pyridine coenzymes may be destroyed by acid without damaging the oxidized forms (2, 3) and that, conversely, the oxidized nucleotides may be destroyed by alkali without the slightest loss of the reduced forms (3-6). This extreme difference in behavior is a key to their enormous analytical potential. This paper is intended as a practical statement of the range of conditions of temperature and pH value tolerated by the acid and basic forms. It can serve as a guide either for the destruction of the unwanted forms or for the preservation and storage of the desired forms. A particular problem dealt with, which is relevant to tissue analysis, is how to destroy diphosphopyridine nucleotidase without serious loss of the oxidized coenzymes. A second special problem encountered is how to destroy oxidized nucleotides and interfering enzymes in alkali, when blood is present, without loss of reduced coenzymes. STABILITY OF DPNH AND TPNH Acid Solutions-DPNH and TPNH are rapidly destroyed in acid (2,3) . The rate appears to be a linear function of hydrogen ion concentration (Fig. 1) . Bt 23" the first order rate constant for DPNH is approximately equal to 380 (H+) min-l (7). This means that 99% of a reduced nucleotide would be destroyed in 0.6 minute in 0.02 N HCl. Fig. 1 permits calculation of rates of destruction in the range from pH 2 to 6. At pH 7 the calculated rate of destruction at 23" is 0.2% per hour. The rates of DPNH destruction at 38", measured at pH 4.2 and 5.3 (acetate) and pH 7.1 (maleate), give a close fit to the line k = 1380 (H+) per minute or a rate 3.6 times that at 23". St 60" (Fig. 1) the rate is 6.0 times faster than at 38". These results indicate a temperature coefficient of 9% per degree from 23-60". TPNH has not been studied as extensively as DPNH but at 30" (pH 2 to 4.5) it was found to be destroyed 80% faster than DPNH. As noted by Winer and Schwert (8) , phosphate accelerates destruction of DPNH. At a phosphate concentration of 0.1 M the destruction rates were increased 3.5-fold at pH 6.2, and 7-fold
pmid:14466980 fatcat:yjz52o2gnzfsrdzxspmfgz662e