The role of ground state complexation in the electron transfer quenching of methylene blue fluorescence by purine nucleotides.

The effect of three purine nucleotides on the fluorescence of methylene blue in aqueous buffer has been investigated. Guanosine-5'-monophosphate (GMP) and xanthosine-5'-monophosphate cause fluorescence quenching while adenosine-5'-monophosphate causes a red shift in the fluorescence maximum. All three nucleotides form ground state complexes with the nucleotides as indicated by absorption spectroscopy. The fluorescence changes at nucleotide concentrations less than 30 mM are best described by a static mechanism involving the formation of non-fluorescent binary and ternary complexes in competition with dimerization of the dye. Quenching of the fluorescence decay (tau = 368 ps) at high GMP concentrations (10-100 mM) occurs at the rate of diffusion. The mechanism of fluorescence quenching may involve electron transfer within the singlet excited dye-nucleotide complex although published values of the oxidation potentials of various purine derivatives would suggest that all three nucleotides should cause quenching. Evidence for electron transfer was obtained from flash photolysis experiments in which 100 mM GMP was found to cause the appearance of a long lived transient species absorbing in the region expected for semimethylene blue.