74] The fluorometric determination of mitochondrial adenine and pyridine nucleotides

Abstract

Publisher Summary This chapter describes the fluorometric determination of mitochondrial adenine and pyridine nucleotides. Determination of the concentrations of oxidized and reduced diphospho- and triphosphopyridine nucleotides, and adenine nucleotides, can only be accomplished by rapidly terminating mitochondrial reactions with acid or alkali followed by extraction and assay of the various forms of the nucleotides. Any one of a number of commercial fluorometers may be conveniently used for the measurement of reduced pyridine nucleotide fluorescence. A sample is irradiated by an intense source of monochromatic light, for example the 365 mμ emission line from a mercury lamp. The emitted fluorescent light is detected by a photomultiplier after transmitting through a secondary filter such as a Wratten 2C. The content of ATP in acid extracts of mitochondria may be determined by two alternate methods. Both methods utilize coupled enzymatic systems ultimately involving the reduction or oxidation of pyridine nucleotide. The content of DPN and TPN in the neutralized perchloric acid extracts can be determined by measuring the increase in fluorescence associated with reduction of the pyridine nucleotides. The concentrations of DPNH and TPNH in neutralized alkaline extracts of mitochondria can be determined fluorometrically by measuring the decrease in fluorescence associated with the enzymatic oxidation of the reduced pyridine nucleotides.