Abstract
Structural and dynamical features of bovine γ-crystallin tryptophan residues were investigated by phosphorescence measurements at 77 and 293 K. The low temperature phosphorescence spectra and lifetimes of calf γ-II, III, and IV crystallins did not reflect heterogeneity among the γ-crystallins. The 0-0 bands were all at 414±1 nm and the emission lifetimes were all single-exponential with lifetimes of 5·1, 5·3 and 5·3±0·3 sec, respectively. In contrast, phosphorescence measurements at room temperature were sensitive to subtle differences in exposure, accessibility, and flexibility of γ-crystallin tryptophan residues. Thorough deoxygenation allowed for measurement of the normally-quenched room-temperature phosphorescence, and we report the first native phosphorescence measurements of lens crystallins at ambient temperature. The emission maxima for γ-II, III and IV were 446, 442, and 440±2 nm, respectively. The intensity decay curves were all non-single exponential, and the decays were fit to a sum of two exponentials with lifetimes of 9·1 and 93 msec (γ-II), 11 and 75 msec (γ-III), and 4·2 and 68 msec (γ-IV), ±10%. The components of the γ-II emission were assigned to the four tryptophans based on X-ray structural information. Quantum yields of the phosphorescence emission were in the ratio of 20:7:1 for γ-II, III and IV, and comparison of lifetimes and quantum yields suggests that tryptophan rigidity increases in the order γ-IV < III < II. Acrylamide quenching constants for the long-lived components of γ-II and III were roughly equal, while the short-lived tryptophans of γ-III were an order of magnitude more accessible than those of γ-II. The wide range of phosphorescence lifetimes and quenching constants allowed for discrimination of distinct contributions to the phosphorescence emission, and we suggest that room-temperature phosphorescence measurements will be an effective tool for studying conformational changes of lens crystallins.
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