Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level

Abstract

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.

Imaging Lenticular Autofluorescence in Older Subjects

“…Two types of z-axis (axial) profiles have been previously reported for lenticular AF: two-peaked profiles 20 – 22 , 26 , 27 , 43 versus single-peaked profiles. 19 , 29 Our results using a retinal cSLO generated a single-peaked profile (Figs. 2 , 3 ).…”

“…The current work used a narrow spectrum 488-nm excitation light. It is likely that different fluorophores that accumulate in the lens with age have not only different excitation spectra 14 , 19 but also different axial distribution. 54 Thus, L-AF measurements using a wider spectrum may be expected to excite different fluorophores compared with those using a narrow spectrum.…”

“…9 – 11 The human lens is comprised of mostly water and protein 12 and photo-oxidation over time leads to structural changes in the proteins, 13 which subsequently results in accumulation of tissue-bound, advanced glycation end products (AGEs) in the lens. 14 – 19 Accrual of AGEs in the lens has been demonstrated to decrease light transmission through the lens. 20 – 24 In particular, it has been shown that such reduction in light transmittance preferentially affects the short wavelength portion of the visible spectrum.…”

Strategies for the Development of Metallic‐Nanoparticle‐Based Label‐Free Biosensors and Their Biomedical Applications

“…Ray and co‐workers described the detection of proteins by using aluminium nanostructures . Nearly 99.5 % of human protein comprises at least one tryptophan, which constitutes a significant part of the fluorescence of a protein in UV region . These native fluorescence intensities of a protein are very low for detection, so the MEF protocol has been followed to enhance the fluorescence intensity.…”

Glycation-mediated inter-protein cross-linking is promoted by chaperone–client complexes of α-crystallin: Implications for lens aging and presbyopia

“…Lens proteins, largely crystallins, have negligible turnover and therefore accumulate posttranslational modifications throughout life. Some of the major modifications are deamidation, racemization, glycation and oxidation (2)(3)(4)(5)(6)(7).…”