Fluorescence studies on tryptophan and sulfhydryl group changes of bovine lens crystallins in a photodynamic system.
Conformational changes in the three crystallins alpha-, beta-, and gamma- in a singlet-oxygen generating system were investigated by fluorescence studies of tryptophan and covalently-bound sulfhydryl probe 4-[(N-iodoacetoxy)N-methyl]amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD). Upon excitation at 295 nm, the tryptophan emission maxima of the crystallins were red-shifted by irradiation with visible light in the presence of the photosensitizer methylene blue. beta- crystallin showed the largest shift (4 nm) of the emission spectrum. Time course of the fluorescence changes by irradiation showed that the decrease in the tryptophan fluorescence yield occurs most rapidly for beta-crystallins, as compared to alpha- or gamma-crystallins. Fluorescence changes of IANBD-labeled crystallins show a 40% decrease in the fluorescence intensity of the sulfhydryl probe for beta-crystallin after one hour of irradiation. For alpha- and gamma-crystallin smaller decreases (7% and 15% respectively) were observed. Since all the sulfhydryl groups of beta-crystallin are known to be exposed on the surface of the protein (Andley et al, 1982, Biochemistry 21, 1853), these results suggest that the pronounced changes in conformation of beta-crystallin by singlet oxygen may be due to a rapid loss of the protein tertiary structure by oxidation of the sulfhydryl groups. These results have potential significance in understanding the age and cataract-related changes in the ocular lens in view of the fact that several key lens enzymes are associated with beta-crystallins in vivo.
D000042:Absorption; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000378:metabolism*; D005453:Fluorescence; D008027:Light; D008968:Molecular Conformation; D010777:Photochemistry; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013997:Time Factors; D014364:Tryptophan / Q000378:metabolism*
D003459:Crystallins; D013438:Sulfhydryl Compounds; D014364:Tryptophan