Mechanisms of photochemically produced turbidity in lens protein solutions.
Calf alpha- and gamma-crystallin were photolyzed in 1-2 mg ml-1 aqueous solutions, using both laser and conventional UV radiation in the 297-320 nm wavelength region. Gamma-crystallin solutions became highly turbid upon UV irradiation, while alpha-crystallin developed no turbidity when irradiated under identical conditions. The photolyzed solutions were analyzed by SDS-PAGE. These gels revealed loss of normal 20 kDa polypeptide, and formation of higher molecular weight peptides, in both alpha- and gamma-crystallin, presumably as a result of photocross-linking reactions and/or protein insolubilization. Thus, although both crystallins underwent photocross-linking, significant turbidity production only occurred in gamma-crystallin. Some possible explanations for these differences are proposed, with one possibility being that most photocross-links in alpha-crystallin occur between subunits of the 1000-kDa oligomer, while in gamma-crystallin the cross-links occur between 20-kDa monomer units. Hence, cross-linking in alpha-crystallin does not affect the average size of particles in solution (or the turbidity), while cross-linking in gamma-crystallin results in a significant increase in average particle size with concomitant increase in turbidity. Another possible explanation is that UV-irradiated gamma-crystallin becomes insoluble (due to charge changes resulting in non-covalent aggregation) while alpha-crystallin does not. Other differences in the photochemical behavior of alpha- vs. gamma-crystallin were noted--gamma-crystallin photolysis rate was about 50% greater than alpha-crystallin. Alpha-crystallin photolysis yielded strong NFK-like fluorescence, while gamma-crystallin did not. One similarity was that photolyzed alpha- and gamma-crystallin lost amino acids His and Trp at about the same rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
D Y
DY
School of Chemistry, Georgia Institute of Technology, Atlanta 30332-0400.
Borkman
R F
RF
Wang
R H
RH
Dillon
J
J
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D009391:Nephelometry and Turbidimetry; D010782:Photolysis; D011489:Protein Denaturation / Q000528:radiation effects; D014466:Ultraviolet Rays
D000596:Amino Acids; D003459:Crystallins