Intramolecular Transfer of Electronic Energy in Dihydro Diphosphopyridine Nucleotide

Abstract

DIHYDRO diphosphopyridine nucleotide shows blue-white fluorescence (λ_max. = 468 mµ) in water solution¹ with a quantum yield² of 2 per cent on excitation by mercury 366 mµ radiation. Several reduced N-alkyl nicotinamides show fluorescence almost identical in spectrum and quantum yield with that of dihydro diphosphopyridine nucleotide. The excitation spectrum of the fluorescence³ of these reduced N-alkyl nicotinamides (Fig. 1) shows complete coincidence with the fractional absorption spectrum over the whole range of wave-lengths explored (220–410 mµ). The fluorescence-excitation spectrum of water solutions of dihydro diphosphopyridine nucleotide at pH 7–9 shows a further strong band with a maximum at 260 mµ, a region where 80–90 per cent of the absorption is due to the purine part of the molecule. From these observations it appears that 30 per cent of the photons absorbed by the adenine appear as nicotinamide fluorescence. The integrity of the molecule is required for this energy transfer to show itself: after a solution of dihydro diphosphopyridine nucleotide at pH 8.3 has been acted upon by nucleotide pyrophosphatase⁴ the 260 mµ band of the fluorescence-excitation spectrum disappears, the resulting spectrum becoming similar to that of the reduced N-alkyl nicotinamides. The enzyme hydrolyses the P—O—P linkage and causes the purine and nicotinamide nucleotides to become independent molecules, which in a 9 × 10⁻⁶ M solution, the concentration of the experiments, are at an average distance of 300 A. of each other. Solutions of dihydro diphosphopyridine nucleotide in propylene glycol show no detectable 260 mµ band in the fluorescence-excitation spectrum, while solutions in 60 per cent sucrose in water show a 260 mµ band the intensity of which is four-fifths of that in pure water.