Picosecond fluorescence decay in photolyzed lens protein alpha-crystallin.
Photolysis of calf lens protein alpha-crystallin in aqueous solutions has been monitored by observing changes in fluorescence decay following UV irradiation at 308 nm. The fluorescence decay was biexponential in dark controls and in photolyzed solutions. The recovered lifetime components in pH 7.4 phosphate buffer at 23 degrees C were 3.5 and 0.5 ns before irradiation and 2.7 and 0.5 ns after irradiation. As the UV dose increased, the relative weighting coefficient of the 2.7-ns decay component decreased, and that of the 0.5-ns component increased, resulting in an overall lifetime shortening. Similar results were obtained in 5 M guanidine hydrochloride solutions where lifetime components of 2.7 and 0.5 ns were observed. These observations were in contrast to the behavior of tryptophan monomer solutions which did not show any change in fluorescence decay kinetics upon UV photolysis but only a reduced fluorescence intensity. Steady-state fluorescence spectra and fluorescence quantum yields were also measured at 23 degrees C for unirradiated bovine alpha-crystallin and gave phi F = 0.11 +/- 0.01 in pH 7.4 buffer and phi F = 0.10 +/- 0.01 in 5 M guanidine hydrochloride solutions. The combined steady-state and fluorescence decay data were consistent with assignment of the long-lived fluorescence decay component in alpha-crystallin to emission from Trp-9, which is known to photolyze relatively rapidly. The short decay component was assigned to Trp-60, which photolyzed much more slowly. We thus provide an example of using steady-state photochemical data to assign fluorescence decay components in a multi-tryptophan protein.
Borkman
R F
RF
Institute for Molecular Science, Okazaki, Japan.
Douhal
A
A
Yoshihara
K
K
D003459:Crystallins / Q000737:chemistry / Q000528:radiation effects*; D005453:Fluorescence; D007700:Kinetics; D011487:Protein Conformation / Q000528:radiation effects; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry / Q000528:radiation effects; D014466:Ultraviolet Rays
D003459:Crystallins; D014364:Tryptophan