The Quenching of Tyrosine and Tryptophan Fluorescence by H₂o and D₂o*

The quantum yields and lifetimes of the fluorescence of tyrosine and tryptophan were determined in D 2 O‐H 2 O and glycerol‐H 2 O solvent mixtures of varying composition from 10 vol.% to 100% H 2 O at 15°C. Forboth amino acids the ratio of the quantum yields in D 2 O and H 2 O (i.e., q D / q H ) was smaller than the ratio of the corresponding lifetimes ( D / H ). For tyrosine the ratio of the quantum yields in glycerol and H 2 O ( q G / q H ) was also smaller than the corresponding G / H ratio, but for tryptophan q G / g H G / H . The proximity of the q vs. plots for tyrosine in the two solvent mixtures indicates that at 15°C neither D 2 O nor glycerol, in the pure state or when diluted with H 2 O, quench tyrosine significantly. However, H 2 O quenches tyrosine by a dynamic process, which increases both the radiative and the nonradiative rate constant. The quenching action is attributed to a tyrosine‐H 2 O exciplex, whose formation is independent of bulk viscosity and dielectric constant. Unlike tyrosine, tryptophan is quenched weakly by D 2 O by a static process at 15°C (i.e., involving no change in), but H 2 O quenches tryptophan much more efficiently by a dynamic process, which involves the nonradiative rate constant, but not the radiative constant. These results are explained on the basis of electrostatic complexation of the ammonium group to the carbon atom adjacent to the ring nitrogen with a lifetime which is longer thanin D 2 O but shorter thanin H 2 O, with solvent reorientation possibly also being an important factor in the quenching. This explanation is consistent with the fact that concentrated (8 M ) urea increases q andof aqueous tryptophan ˜ 15–20%, while guanidine hydrochloride (6.4 M ) has the opposite effect, i.e., it decreases q and t of tryptophan ˜ 15–20%, and with the fact that neither 8 M urea nor 6.4 M guanidine hydrochloride affects any fluorescence parameter of tyrosine at all.