Oliver H. Lowry

[111] Measurement of pyridine nucleotides by enzymatic cycling

Publisher Summary This chapter describes the measurement of pyridine nucleotides by enzymatic cycling. The coenzyme to be determined is made to catalyze an enzymatic dismutation between two substrates. After several thousand cycles, one of the products is measured. The nucleotides during cycling are used at concentrations well below their Michaelis constants and consequently, reaction rates are proportional to nucleotide concentrations. It is found that as pyruvate formation during cycling is not strictly linear with DPN concentration, standards of DPN + are included which increase in steps of two to cover the range of assay. Blanks and standards are carried through the entire procedure and are treated as nearly as possible to the samples being analyzed. The cycling procedure is not as flexible as that for TPN, owing primarily to a fall-off in rate as pyruvate accumulates. The TPN cycle is proportional within a 100-fold range of coenzymes with the same cycling reagent. It is found that the DPN cycle is proportional without too much departure from linearity over a 20-fold range.