LOCAL VARIATIONS IN PROTEIN-STRUCTURE IN THE HUMAN EYE LENS - A RAMANMICROSPECTROSCOPIC STUDY

Confocal Raman microspectroscopy was used to monitor local and age-rel ated changes in protein conformation in human eye lenses. In clear hum an lenses of varying age (range 17-80 years) spectra were recorded alo ng the visual axis, using laser light of 660 nm wavelength. The Raman vibrations in the 650-1750 cm-1 spectral region were analyzed. Differe nce spectra between central core and different positions along the vis ual axis were calculated after calibration for protein content using t he I(1450) cm-1 CH2/CH3 vibration peak. Tryptophan content was quantif ied using the peak at 760 cm-1 calibrated for protein. Changes in the 'exposed' vs. 'buried' position of tryptophan were analyzed using the peak heights at I(880) and I(760) cm-1. The difference spectra reveale d an excess of tryptophan, tyrosine, phenylalanine, beta-sheet conform ation and molecules or molecular groups responsible for a 1425 cm-1 pe ak in the core region in all lenses investigated. The excess peaks dis appeared 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. 'burie d' position of tryptophan were observed. Changes in tryptophan and tyr osine probably reflect the maturational shift from cortex to core in t he relative content of alpha, beta and gamma crystallines. The age-rel ated increase in tryptophan in the core may reflect the preferential b reakdown by endo- and exopeptidases of alpha-crystallins damaged upon aging. The increase in beta-sheet conformation may indicate a post-tra nslational shift in secondary conformation upon aging. These changes i n protein conformation are largely completed in a small superficial zo ne, i.e., in the early life span of the crystallins.