Fluorescence spectrophotometry of reduced phosphopyridine nucleotide in intact cells in the near-ultraviolet and visible region

Abstract

An apparatus is described with which it is possible to measure precisely fluorescence spectra, not only of clear solutions, but also of a scattering weakly fluorescent suspension of living cells or pieces of tissue, in the region 350–600 mω by excitation with the mercury line 313 mω and in the region 400–600 mω by excitation with 366 mω.

Fluorescence spectra of free DPNH, suspensions of baker's yeast, Photobacterium phosphoreum and Photobacterium splendidum, and the alga Chlorella were determined. When excited by carefully filtered light of 366 mω, all three species showed fluorescence spectra presumably caused mainly by reduced pyridine nucleotide. Measurements of the time course of the fluorescence of starved yeast upon addition of ethanol and glucose indicated that this fluorescence was for the main part caused by reduced phosphopyridine nucleotide.

The method appears to be more specific and more generally applicable than absorption spectrophotometry for the measurement of reduced pyridine nucleotide in vivo.