THE FLUOROMETRIC MEASUREMENT OF PYRIDINE NUCLEOTIDES

Abstract

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 340ml.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.