Phosphorescence measurements of calf gamma-II, III, and IV crystallins at 77 and 293 K.
Structural and dynamical features of bovine gamma-crystallin tryptophan residues were investigated by phosphorescence measurements at 77 and 293 K. The low temperature phosphorescence spectra and lifetimes of calf gamma-II, III, and IV crystallins did not reflect heterogeneity among the gamma-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 gamma-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 gamma-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 (gamma-II), 11 and 75 msec (gamma-III), and 4.2 and 68 msec (gamma-IV), +/- 10%. The components of the gamma-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 gamma-II, III and IV, and comparison of lifetimes and quantum yields suggests that tryptophan rigidity increases in the order gamma-IV less than III less than II. Acrylamide quenching constants for the long-lived components of gamma-II and III were roughly equal, while the short-lived tryptophans of gamma-III were an order of magnitude more accessible than those of gamma-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.
Berger
J W
JW
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Vanderkooi
J M
JM
Tallmadge
D H
DH
Borkman
R F
RF
D000818:Animals; D002417:Cattle; D003459:Crystallins; D008163:Luminescent Measurements; D011487:Protein Conformation; D013696:Temperature; D014364:Tryptophan
D003459:Crystallins; D014364:Tryptophan