Using confocal Raman microspectroscopy with laser light of 660 nm the secondary and tertiary conformation of crystallins was studied in human lenses of varying age (20-75 years). Differences in cortical and nuclear proteins in individual lenses and among lenses of different age and differences between small equatorial opacities and adjacent clear sites were analysed using a difference spectrum approach. Intensity calibration allows assessment of local variations in protein content.The main findings and conclusions are as follows. (1) Irrespective of the age of the lens nuclear proteins proved to contain more aromatic amino acids, i.e. tryptophan, tyrosine and phenylalanine. This change most probably reflects differences in crystallin composition between nucleus and cortex as described in biochemical studies. (2) Changes in the amide bands indicate a more pronounced p-sheet conformation of the nuclear proteins. Taking into account available biochemical evidence this most probably reflects a true post-translational change in the secondary conformation of the crystallins. (3) Cortical proteins in 'old' and 'young' lenses are largely identical indicating that ageing is not accompanied by gross alterations in the transcription/translation of the crystallin genes. (4) 'Old' and 'young' nuclear proteins deviate with respect to the amount of aromatic amino acids, being more abundant in 'young' nuclear proteins. (5) Proteins in small early opacities do not exhibit alterations in conformation. (6) A pronounced peak in the difference spectra in the region 142 5-1435 cm-* for nuclear proteins especially when compared with equatorial cortical proteins may be considered as evidence for the advanced photooxidation of tryptophan and/or deamidation of asparagine from superficial to deep regions of the lens.

Confocal Raman microspectroscopy was used to monitor local and age-related changes in protein conformation in human eye lenses. In clear human lenses of varying age (range 17-80 years) spectra were recorded along the visual axis, using laser light of 660 nm wavelength. The Raman vibrations in the 650-1750 cm -1 spectral region were analyzed. Difference spectra between central core and different positions along the visual axis were calculated after calibration for protein content using the I(1450) cm-1 CH2/CH 3 vibration peak. Tryptophan content was quantified using the peak at 760 cm-~ calibrated for protein. Changes in the 'exposed' vs. 'buried' position of tryptophan were analyzed using the peak heights at I(880) and 1(760) cm-L The difference spectra revealed an excess of tryptophan, tyrosine, phenylalanine,/3-sheet conformation and molecules or molecular groups responsible for a 1425 cm-1 peak in the core region in all lenses investigated. The excess peaks disappeared at about 0.6-0.9 mm below the surface. The tryptophan content increased from superficial to deep layers, levelling off between 0.4-0.8 mm below the surface. Upon aging, the tryptophan content increases in the core not in the cortex. No changes in the 'exposed' vs. 'buried' position of tryptophan were observed. Changes in tryptophan and tyrosine probably reflect the maturational shift from cortex to core in the relative content of a, /3 and ~/ crystallines. The age-related increase in tryptophan in the core may reflect the preferential breakdown by endo-and exopeptidases of a-crystallins damaged upon aging. The increase in /3-sheet conformation may indicate a post-translational shift in secondary conformation upon aging. These changes in protein conformation are largely completed in a small superficial zone, i.e., in the early life span of the crystallins.

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