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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.

A study of the effect of varying ionic strength on the glucose-induced quenching of tryptophan fluorescence of hexokinase isoenzymes A(P-I) and B(P-II) was carried out at pH 8.3 and pH 5.5. At p/ 8.3 both isoenzymes gave apparently linear Scatchard-type data plots even with protein concentrations and ionic strengths for which both dimeric and monomeric forms of hexokinase coexist in signiciant amounts. Taking inco account a 1% accuracy in the experimental measurements, we concluded that the intrinsic dissociation constants K(M) and K(D), for the binding of glucose to the monomeric and dimeric forms of HkB, are within a factor of two of each other, i.e. K(D)/K(M) less than or equal to 2. The values of K(M), estimated from the apparent K, were so greatly influenced by ionic strength that it is clear that it is meaningless to compare K(M) and K(D) values measured at different ionic strengths as has been done in the literature. Curvature in the pH 5.5. fluorescence-quenching plots for relatively low ionic strengths demonstrates cooperativity for glucose-binding to the dimer, positive for HkA but negative for HkB. In contrast, the binding is relatively non-cooperative at high ionic strength at this pH. These results were attributed to the well known effect of salt-neutralization of side chain electrical charges on the flexibility and compactness of proteins.

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