Ageing and changes in protein conformation in the human lens: a Raman microspectroscopic study.
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 beta-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 1425-1435 cm-1 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.
Siebinga
I
I
Department of Morphology, The Netherlands Ophthalmic Research Institute, Amsterdam.
Vrensen
G F
GF
Otto
K
K
Puppels
G J
GJ
De Mul
F F
FF
Greve
J
J
D000328:Adult; D000368:Aged; D000375:Aging / Q000378:metabolism*; D003459:Crystallins / Q000737:chemistry*; D006801:Humans; D007904:Lens Cortex, Crystalline / Q000737:chemistry; D007907:Lens Nucleus, Crystalline / Q000737:chemistry; D007908:Lens, Crystalline / Q000737:chemistry*; D008875:Middle Aged; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman
D003459:Crystallins