Raman spectroscopy of galactosemic rat lens crystallins: Correlation of microscopic changes of lens proteins at molecular levels with gross cataractous alteration

Near-Infrared Fourier transform (near-IR FT) Raman spectroscopy has been used to study the structural changes of lens proteins both in cortex and nucleus of galactosemic rat lenses. It was found that tyrosine doublet ratio of Raman bands, I₈₃₂/I₈₅₅, increased more rapidly in the cortex than in the nucleus during a 5-week period of galactose feeding, i.e., from 0.86 to 1.1 for the cortex and 0.88 to 0.92 for the nucleus. The ratio obtained for lens nucleus suggests that a lower ratio of less than 1 does not necessarily reflect the apparent transparent state of lens morphology. Moreover our results for the increase of tyrosine doublet ratio with the extent of cataract formation in galactosemic lens appear to indicate that there is more hydration in crystallins of the cortex than the nucleus since the increased ratio of tyrosine doublet has been shown to be due to the hydrogen-bond formation of hydroxyl groups in various tyrosines of proteins with water. The tryptophan band ratio at 880 cm^-1 and 757 cm^-1 (I₈₈₀/I₇₅₇) underwent a precipitous decrease in the cortex and a rather gradual decrease in the nucleus, suggesting buried tryptophan residues become more exposed in the cortex than in the nucleus during galactose-induced cataractogenesis. Based on the changes of the two ratios, I₈₃₂/I₈₅₅ and I ₈₈₀/I₇₅₇, the change of lens protein environment induced by galactosemic feeding appeared to take place in the cortex first, which was consistent with the observation that the development of an opaque lens begins in the cortex. While no sulfhydryl (-SH) signal was detected, there was a slow increase of disulfide (-S-S-) signal in the cortex of galactose-fed lenses as compared to control lenses without galactose. This suggested that a loss of lens glutathione occurred early and oxidation of cysteines in crystallins started in the first week, i.e., before the onset of cortical cataract. In contrast, for nucleus of galactosemic lenses the signal of the -SH group was detected and yet the -S-S- signal of crystallins could not be found. In this study, we have conclusively demonstrated using Raman vibrational band shift that galactosemic cataract begins in the cortex, and that the lens cortex suffers more structural alteration in crystallins than the nucleus.