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

Journal: Nature Scientific Reports

Authors: Anna Gakamsky, Rory R. Duncan, Nicola M. Howarth, Baljean Dhillon, Kim K. Buttenschön, Daniel J. Daly & Dmitry Gakamsky

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.


Exploring the possibility of early cataract diagnostics based on tryptophan fluorescence

Dmitry M. Gakamsky1, Bal Dhillon2,5, John Babraj3, Matthew Shelton4, S. Desmond Smith4,5

A novel route for early cataract diagnostics is investigated based on the excitation of tryptophan fluorescence (TF) at the red edge of its absorption band at 317 nm. This allows penetration through the cornea and aqueous humour to provide excitation of the ocular lens. The steepness of the red edge gives the potential of depth control of the lens excitation. Such wavelength selection targets the population of tryptophan residues, side chains of which are exposed to the polar aqueous environment. The TF emissions around 350 nm of a series of UV-irradiated as well as control lenses were observed. TF spectra of the UV cases were red-shifted and the intensity decreased with the radiation dose. In contrast, intensity of non-tryptophan emission with maximum at 435 nm exhibited an increase suggesting photochemical conversion of the tryptophan population to 435 nm emitting molecules. We demonstrate that the ratio of intensities at 435 nm to that around 350 nm can be used as a measure of early structural changes caused by UV irradiation in the lens by comparison with images from a conventional slit-lamp, which can only detect defects of optical wavelength size. Such diagnostics at a molecular level could aid research on cataract risk investigation and possible pharmacological research as well as assisting surgical lens replacement decisions.