Local variations in protein structure in the human eye lens: a Raman microspectroscopic study.
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/CH3 vibration peak. Tryptophan content was quantified 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 revealed an excess of tryptophan, tyrosine, phenylalanine, beta-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 alpha, beta and gamma crystallines. The age-related increase in tryptophan in the core may reflect the preferential breakdown by endo- and exopeptidases of alpha-crystallins damaged upon aging. The increase in beta-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.
Smeets
M H
MH
Department of Morphology, The Netherlands Ophthalmic Research Institute, Amsterdam.
Vrensen
G F
GF
Otto
K
K
Puppels
G J
GJ
Greve
J
J
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging; D003459:Crystallins / Q000032:analysis; D005136:Eye Proteins / Q000737:chemistry*; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry*; D008832:Microchemistry / Q000379:methods; D008875:Middle Aged; D010649:Phenylalanine / Q000032:analysis; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
D003459:Crystallins; D005136:Eye Proteins; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan