Laser Raman spectroscopic studies of ocular lens and its isolated protein fractions.
The water-soluble proteins of the bovine lens were separated on a column of Sephadex G-200 into five fractions designated as alpha-, beta1-, beta2-, and gamma-crystallin. Laser Raman scattering studies on these isolated proteins (both in the lyophilized state and in solution) and insoluble albuminoid reveal that they contain predominantly antiparallel pleated sheet structure in the main chains and that sulfhydryl groups are highly localized in gamma-crystallin. This light-scattering technique was also applied to probe the homogeneity of protein structure in the intact lens. The analysis of the scattered light selectively collected from various parts of the lens indicated that these proteins also exist in an antiparallel beta structure throughout the entire lens. However, the central (nucleus) and outer (cortex) portions have somewhat different amino acid composition. Based on the relative intensities of the lines at 624 (phenylalanine) and 644 cm-1 (tyrosine), it is concluded that the nuclear part has the highest concentration of gamma-crystallin and that the content of alpha-crystallin increases significantly from the nucleus to the cortex. By examining the Raman spectra in the 2582 cm-1 and the amide I and III regions, we have demonstrated that the sulfhydryl groups and the beta conformation of the lens proteins are unaffected in the conversion of transparent to totally opaque lens by heat denaturation at 100 degrees. This means that the opacification of a lens does not necessarily involve the oxidation of sulfhydrul groups or conformation changes.
D000577:Amides; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000302:isolation & purification; D005260:Female; D006358:Hot Temperature; D007834:Lasers; D007908:Lens, Crystalline; D046911:Macromolecular Substances; D011487:Protein Conformation; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds
D000577:Amides; D000596:Amino Acids; D003459:Crystallins; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds