THE PEPTIDE-BOND QUENCHES INDOLE FLUORESCENCE

The effect of the peptide bond on protein fluorescence is an important unresolved question in tryptophan photophysics. Definitive evidence f or the peptide group as a weak quencher of indole fluorescence was obt ained from solute quenching studies with a series of model compounds. Two amides are required for detectable quenching of 3-methylindole flu orescence and the quenching rate depends on the distance between amide s. The bimolecular rate constants k(q) of malonamide, N-acetylasparagi ne, N-acetylglycinamide, and N-acetylglutamine are 33 x 10(7), 8.8 x 1 0(7), 6.6 x 10(7) and 2.2 x 10(7) M(-1) s(-1), respectively. Transient absorption and temperature dependence of the fluorescence lifetime me asured in the absence and presence of quencher gave strong circumstant ial evidence for electron transfer as the quenching mechanism. Triplet yields were measured for five indole derivatives using transient abso rption. Intersystem crossing rates were calculated from triplet yield and fluorescence lifetime data. The intersystem crossing rate k(isc) v aries from 2.1 x 10(7) s(-1) for 3-methylindole to 7.6 x 10(7) s(-1) f or 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 de cay rate for 3-methylindole, indole, N-acetyltryptophanamide, and N-me thylindole, confirming that intersystem crossing in indoles is indepen dent of temperature in aqueous solution. The temperature dependence of the fluorescence lifetime of 3-methylindole was determined in the pre sence of N-acetylglycinamide, ethyl acetate, and GdCl3. Two separate A rrhenius 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/mo l, 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.