THE QUENCHING OF TYROSINE AND TRYPTOPHAN FLUORESCENCE BY H₂O AND D₂O*

Abstract

Abstract— The quantum yields and lifetimes of the fluorescence of tyrosine and tryptophan were determined in D2O‐H2O and glycerol‐H2O solvent mixtures of varying composition from 10 vol.% to 100% H2O at 15°C. Forboth amino acids the ratio of the quantum yields in D2O and H2O (i.e., qD/qH) was smaller than the ratio of the corresponding lifetimes (D/H). For tyrosine the ratio of the quantum yields in glycerol and H2O (qG/qH) was also smaller than the corresponding G/H ratio, but for tryptophan qG/gHG/H. The proximity of the q vs. plots for tyrosine in the two solvent mixtures indicates that at 15°C neither D2O nor glycerol, in the pure state or when diluted with H2O, quench tyrosine significantly. However, H2O quenches tyrosine by a dynamic process, which increases both the radiative and the nonradiative rate constant. The quenching action is attributed to a tyrosine‐H2O exciplex, whose formation is independent of bulk viscosity and dielectric constant. Unlike tyrosine, tryptophan is quenched weakly by D2O by a static process at 15°C (i.e., involving no change in), but H2O 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 D2O but shorter thanin H2O, 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.

On the permeability of water molecules across vesicular lipid bilayers

“…In contrast to the fluorescence spectra, the H-D exchange of the indole-ring imino-proton leads to rather small changes of the UV-absorption spectra for tryptophan molecules (Nakanishi et al, 1978). Though several explanations of the solvent-isotope effect on the quantum yields can be found in the literature (Stryer, 1966;Eisinger & Navon, 1969;Kirby & Steiner, 1970;McGuire & Feldman, 1973), it is felt that a clear-cut mechanism is still missing.…”

“…spectroscopists that among other fluorescent molecules indole derivatives show a solventisotope effect of the fluorescence quantum yields with q~i~2o > ~Heo (Stryer, 1966;Eisinger & Navon, 1969;Kirby & Steiner, 1970;McGuire & Feldman, 1973;ttoss, 1975). Chemical modifications at the indole-ring nitrogen have revealed that neither H-or Dbonds (the crystalline structure of tryptaminc HCI shows only a weak hydrogen-bond of the indole NH to a chloride ion 3.2fl, away (Wakahara, Fajiwara & Tomita, 1973)) nor the H-D exchange of the indole N-proton are solely responsible for the solvent-isotope effect of the fluorescence quantum yield (Eisinger & Navon, 1969).…”

Static and dynamic quenching of protein fluorescence. I. Bovine serum albumin

“…5 Fluorescence lifetimes were measured a t l5OC with a TRW nanosecond spectral source to a reproducibility of + 0.1 nsec, although the accuracy was probably -10%. A 270-nm interference filter (Optics Technology) was placed in the excitation path, and a 7-51 filter (Corning 5970) was used in the emission path.…”

“…Additional evidence for this conclusion is the complete insensitivity of the fluorescence lifetime and quantum yirld (Figure l a ) of BSA to a fivefold increase in solvent viscosity, i.e., 1.1-5.5 cp, produced by addition of glycerol to an aqueous solution up to 50% v/v, M hereas this solvent change increased both the quantum yield and the lifetime of free tryptophan about 40'%. 5 This lack of dependence on viscosity does not necessarily eliminate Class I1 as the proper designation for any fluorescent tryptophan in aqueous BSAd since it can be argued that the mean molecular diameter of glycer~l,~' 3.2 A, prevents its penetration into small hydrophilic crevices in which Class I1 residues would be expected to reside. Howcver, over the 0-.50% glycerol range there is a blue shift of 6 nm in the A, , , (Figurc Ib) of the BSA fluorescence (260-290-nm c.xcitation) , which is twice as large as one observes for free tryptophan over the same solvent composition range.…”

Photophysical Properties of Tyrosine and Its Simple Derivatives in Organic Solvents Studied by Time‐resolved Fluorescence Spectroscopy and Global Analysis¶

“…Studies of tyrosine spectral/photophysical properties in organic solvents or their mixtures with water have got little attention in spite of the similarities in the protein matrix environment and organic solvent. Absorption spectra and ultraviolet circular dichroism of tyrosine and its derivatives in organic solvents and/or their mixtures with water were studied by Chignell and Gratzer (35), Horwitz et al (36), Bailey et al (37), Schlessinger et al (38), Stricland et al (39) and Smith (40), whereas their photophysical properties were studied by Guzow et al (15), Noronha et al (26), Feitelson (41), Froehlich and Yeats (42) and McGuire and Feldman (43). The quenching of tyrosine fluorescence in water‐organic solvent mixture is attributed to a tyrosine‐water exciplex, the formation of which is independent of bulk viscosity and the dielectric constant (42, 43).…”