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 followin g UV irradiation at 308 nm. The fluorescence decay was biexponential i n dark controls and in photolyzed solutions. The recovered lifetime co mponents in pH 7.4 phosphate buffer at 23-degrees-C were 3.5 and 0.5 n s before irradiation and 2.7 and 0.5 ns after irradiation. As the UV d ose increased, the relative weighting coefficient of the 2.7-ns decay component decreased, and that of the 0.5-ns component increased, resul ting 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 t he behavior of tryptophan monomer solutions which did not show any cha nge in fluorescence decay kinetics upon UV photolysis but only a reduc ed fluorescence intensity. Steady-state fluorescence spectra and fluor escence quantum yields were also measured at 23-degrees-C for unirradi ated 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 solu tions. The combined steady-state and fluorescence decay data were cons istent with assignment of the long-lived fluorescence decay component in alpha-crystallin to emission from Trp-9, which is known to photolyz e relatively rapidly. The short decay component was assigned to Trp-60 , which photolyzed much more slowly. We thus provide an example of usi ng steady-state photochemical data to assign fluorescence decay compon ents in a multi-tryptophan protein.