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Near‐Infrared Fourier trans form (near‐IR FT) Raman spectros copy 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, in creased 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. More over our results for the in crease of tyrosine doubletratio 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 in creased ratio of tyro sine 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− (I880/I757) under went a precipitous de crease in the cortex and a rather gradual de crease 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 I880/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 be gins in the cortex. While no sulfhydryl (‐SH) signal was detected, there was a slow in crease of disulfide (‐S‐S‐) signal in the cortex of galactose‐fed lenses as compared to control lenses with out galactose. This suggested that a loss of lens glutathione occurred early and oxidation of cysteines in crystallins started in the first week, i.e., be fore 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 be gins in the cortex, and that the lens cortex suffers more structural alteration in crystallins than the nucleus.

A solution of y-crystallin became turbid upon heating at 65 "C for 30 minutes; however, addition of acrystallin suppressed this thermal aggregation. It was found the effective chaperone function could be achieved with the molar ratio of a/y greater than 1120. In terms of crystallin subunit, five molecular a-crystallin subunits could afford chaperone for one molecular y-crystallin. The gel til tration profile of the sample solution, containing a-and y-crystaJlins and preincubation at 65 0 C for 30 minutes, showed complex formation between a-and y-crystallins, indicating a-crystallin was bound to thermally denatured y-crystaIlin. A l-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence study showed that a-crystallin has more hydrophobic regions exposed after thermal incubation. In the presence of urea, both the a-crystallin chaperone activity and the ANS fluorescence intensity decreased. Accordingly, hydrophobic regions of a-crystallin play an indispensiblc role in its chaperone activity.

Raman vibrations of the fingerprint of aromatic amino acid residues were analyzed to study the changes of cataractous 'ens protein in the cortex and nucleus at various ages, Tryptophan content, analyzed by the quantification of I m /l l44 g ratio, shows the damage (modification) of tryptophan residue in the nucleus is caused primarily by the formation of cataracts, not by the aging process, Microenvironmental changes of tryptophan and tyrosine were analyzed by the intensity ratios of Il!7Jl m and 1 829/1m, respectively. The decrease of the ratio of 1 819 /1758' from 0.9 to 0.6 in the nucleus and from 0.7 to 0.6 for the cortex, reveal that more buried tryptophan residues become exposed in the cortex than in the nucleus during cataractogenesis, especially for non-senile cataractous lenses. The ratio of 1829/lm is around 1,0for both cortical and nuclear proteins at various ages, indicating some tyrosine residues have undergone a change in their hydrogen bonding environment. When compared to previous studies, we found that a normal (clear) lens bas a higher peak at 1617em:' than at 1604em", while a dense opaque or brunescent lens shows stronger intensity at 1604 em:' than at 1617 em", suggesting the ratio of 116l7/J1604 can be used to evaluate the human lens morphology.