The effect of the peptide bond on protein fluorescence is an important unresolved question in tryptophan photophysics. Definitive evidence for the peptide group as a weak quencher of indole fluorescence was obtained from solute quenching studies with a series of model compounds. Two amides are required for detectable quenching of 3-methylindole fluorescence and the quenching rate depends on the distance between amides. The bimolecular rate constants k q of malonamide, N-acetylasparagine, N-acetylglycinamide, and N-acetylglutamine are 33 × 107, 8.8 × 107, 6.6 × 107, and 2.2 × 107 M-1 s-1, respectively. Transient absorption and temperature dependence of the fluorescence lifetime measured in the absence and presence of quencher gave strong circumstantial evidence for electron transfer as the quenching mechanism. Triplet yields were measured for five indole derivatives using transient absorption. Intersystem crossing rates were calculated from triplet yield and fluorescence lifetime data. The intersystem crossing rate k isc varies from 2.1 × 107 s-1 for 3-methylindole to 7.6 × 107 s-1 for indole. The peptide group does not change the value of k isc of 3-methylindole. The sum of the radiative and intersystem crossing rates is equal to the temperature-independent portion of the fluorescence decay rate for 3-methylindole, indole, N-acetyltryptophanamide, and N-methylindole, confirming that intersystem crossing in indoles is independent of temperature in aqueous solution. The temperature dependence of the fluorescence lifetime of 3-methylindole was determined in the presence of N-acetylglycinamide, ethyl acetate, and GdCl3. Two separate Arrhenius terms were resolved for water quenching and solute quenching. The activation energies for solute quenching by N-acetylglycinamide, ethyl acetate, and GdCl3 are 2.5 ± 0.3, 0.0, and 6.0 ± 0.5 kcal/mol, respectively. For intramolecular quenching by the peptide bonds in N-acetyltryptophanamide, the activation energy is 3.2 ± 0.3 kcal/mol. The strategy of using the temperature dependence of the fluorescence lifetime to calculate the rates of individual nonradiative processes is discussed.

Indoles undergo two isotopically sensitive temperature-dependent fluorescence quenching processes: solvent quenching and excited-state proton transfer. Fluorescence quantum yields of simple indoles in protium and deuterium solvents were measured in the absence and presence of glycine. Photochemical H-D exchange was monitored by NMR and mass spectrometry.Although the fluorescence quantum yield and lifetime of 2-methylindole show large deuterium isotope effects in aqueous solutions, photochemical H-D exchange was not detected after extensive irradiation, whereas, H-D exchange is readily observed for 2-and 3-methylindole in solutions containing glycine. Stern-Volmer plots of glycine quenching data give bimolecular rate constants kq from (0.5-3) X 108 M™1 11s™1 for indoles in water. The kq values of 2-and 3-methylindole are faster in protium than in deuterium solvents. The isotope effect on kq implicates excited-state proton transfer in the collisional quenching mechanism.This contrasts with iodide quenching which has no isotope effect on kq. A glycine derivative lacking the ammonium protons, iV,iV,7V-trimethylglycine, does not quench indole fluorescence. The intermolecular excited-state reaction of 2-and 3-methylindole with 0.3 M glycine-d5 in 50% D20/CD30D induces H-D exchange at three ring carbons. In 2-methylindole the exchange is fastest at C3 and occurs with similar rates at C4 and C7 on the indole ring. The temperature dependence of 3-methylindole fluorescence in 0.5 M glycine was also determined. The large difference in temperature dependence for solvent quenching and glycine quenching causes curvature in the Arrhenius plot. The frequency factor A2 = 7.2 X 1010 s™1 and activation energy E2* = 3.6 kcal/mol for glycine quenching are similar to the values for intramolecular excited-state proton transfer in tryptamine.Possible mechanisms for the excited-state proton transfer reaction and the implications of this reaction for tryptophan fluorescence in proteins are discussed.

No abstract

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.