Figure 9 Effect of hydration on the (a) phosphorescence emission intensity and (b) lifetimes of the six intrinsic tryptophans in hen egg white lysozyme powders. (From Ref. 57.)
dration over the range from 0.1 to 0.3 caused a nearly linear decrease in intensity and lifetime. The authors interpreted this effect as due to an increase in the internal mobility of the protein resulting from an increased ability to exchange internal hydrogen bonds for those with surface water molecules. Such behavior is expected for tryptophan phosphorescence, since studies of indole (58) and tryptophan (59) phosphorescence in solution indicate that the probe lifetime varies linearly with viscosity over several orders of magnitude (the lifetime of trypto-phan varies over 103-fold during a change in viscosity of over 104-fold).
The quenching of protein phosphorescence by water binding can also be viewed as the effect of water on the temperature of some internal dynamic transition that modulates protein mobility. Within this perspective, at h < 0.1 the experimental temperature (Texp) is lower than the onset temperature for the transition (Ttrans) and the phosphorescence intensity is high. At h > 0.1, however, Texp > Tttans and a novel quenching mechanism begins to operate, resulting in an increase in protein internal mobility and a decrease in phosphorescence intensity. Although there is a temptation to refer to this as a glass transition, there is no direct evidence that this softening transition corresponds to a change in the flow properties of the dry protein powder. A likely assignment could involve some as-yet-unidentified solid-solid transition within the glassy protein comparable to that seen in polymers. Since the entire issue of glassy states and glass transitions in proteins remains problematic, much additional work is necessary to illuminate this potentially important issue.
The use of tryptophan phosphorescence to study solid-state transitions in proteins shows great promise. The probe is nonperturbing (because intrinsic), exhibits a dynamic range of over three orders of magnitude (lifetimes in proteins range from less than 1 ms to 3 s), and has a long lifetime that has been shown in model studies to scale with solvent (matrix) viscosity (58, 59). Much additional research, however, is required to answer a number of outstanding questions. Precisely how does a change in viscosity modulate the tryptophan lifetime? Do molecular collisions per se quench the triplet state? Or must the collisions involve specific chemical groups? And, perhaps most importantly, do all molecular collisions or only specific sorts of molecular collisions at specific sites quench the triplet state? Gonnelli and Strambini (60), for example, have demonstrated that only the sidechains of histidine, tyrosine, tryptophan, cysteine, and cystine are efficient colli-sional quenchers of tryptophan phosphorescence in aqueous solution.
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