Local variations in protein structure in the human eye lens: a Raman microspectroscopic study

Abstract

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.

Singlet Oxygen Production Correlates with His and Trp Destruction in Brunescent Cataract Water-Insoluble Proteins

“…Truscott and Augusteyn (1977) saw no loss of Trp in brunescent cataracts (144 nMol mg protein −" ), however, Kamei (1993) using magnetic circular dichroism found Trp values equivalent to 400 nMol mg protein −" , which decreased dramatically to 150 nMol mg −" protein in aged, dark brown lenses. Conversely, Smeets et al (1993) reported a marked increase in Trp content in the nucleus of the lens using Raman probe spectroscopy, which was even greater in aged human lenses and cataracts. Similar assays, however, showed a decrease in Trp in opaque compared to clear regions of cataract lenses (Duindem et al, 1998).…”

Raman Spectroscopy in Ophthalmology: From Experimental Tool to Applications In Vivo

“…The 'quest' is finding the cause of cataract, and for this, in vivo techniques could reveal the di#erent steps and changes in the lens. Raman spectroscopy is a unique tool in lens research since it provides structural information [80][81][82][83][84][85][86][87][88][89][90][91][92][93][94][95][96][97][98] on proteins, amino acids (like Try, Trp, Phe), and sulphur bonds (-SH, S-S). The maturation process of the proteins in the lens cortex and nucleus can be studied in the intact lens, thus preventing oxidation of sulphydryls and protein disruption.…”

“…Shun-Shin et al [95] studied Christmas tree cataracts in human donor eyes and revealed that cystine is the most likely candidate for the formation of the Christmas tree needles. Smeets et al [96] described an age-related increase in tryptophan (ratios integrated peak 760:1450 cm 1 ) in the human lens core as an increase in the -sheet signals in the Amide I and II bonds. Chiou et al [97] determined in pigeon eye lenses that the secondary structures of -and -crystallins are mainly antiparallel -pleated sheet in nature.…”

Development of cataract caused by diabetes mellitus: Raman study

“…Scanning electron microscopy of human lenses belonging to various age groups was employed for better understanding of tertiary and quaternary structure of protein fibers and membrane layers as well [2]. Raman microscopic study, also done on human lenses, concentrated on distribution of tyrosine and tryptophane amino acids in different sections of the lens [41]. What happens during cataract progression?…”