The fluorometric measurement of pyridine nucleotides.

Measurement of DPNH’ or TPNH by absorption at 344 rnp has been a standard procedure for many years and has been used to study innumerable enzyme reactions. In spite of the proved value of this calorimetric method, there may be advantages in fluorometric methods for the pyridine nucleotides. First, it has been found practicable with a fluorometer to determine separately oxidized and reduced pyridine nucleotides. As a result, the formation of a little DPN+ from a large amount of DPNH may be easily measured, and vice versa. Second, each determination may be made accurately at a nucleotide concentration as low as 10m8 M, i.e. at a concentration only a thousandth of that required for ordinary calorimetry at 340 ml.c. Third, the useful range of sensitivity is about 2000-fold, which is much greater than that obtained with calorimetry. Since fluorometry may be performed with as little as 0.01 ml. (2), it is possible to measure lo-la mole of either oxidized or reduced pyridine nucleotide. Amounts of DPN+ or DPNH as great as 10-l’ mole or over may be measured rapidly and easily in a volume of 1 ml. The fluorometric methods, because of their sensitivity, have been of considerable utility for quantitative histochemical purposes. With the addition of appropriate accessory enzymes, almost any enzyme reaction may be made to produce an oxidation of DPNH or TPNH or a reduction of DPN+ or TPN+. The basic method for DPN+ (TPN+) is that of Kaplan et al. (3). The method has merely been changed slightly to increase reproducibility and to provide for destruction of DPNH which otherwise contributes some fluorescence. The method has also been studied further to establish some