title: 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.
authors.affiliation: Edinburgh Instruments, 2 Bain Square, Livingston, EH54 7DQ, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Gakamsky
authors.affiliation: Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 6, UK.
authors.forename: Rory R
authors.initials: RR
authors.lastname: Duncan
authors.affiliation: Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 6, UK.
authors.forename: Nicola M
authors.initials: NM
authors.lastname: Howarth
authors.affiliation: Princess Alexandra Eye Pavilion, Edinburgh and Centre for Clinical Brain Sciences, School of Clinical Sciences, University of Edinburgh, UK.
authors.forename: Baljean
authors.initials: B
authors.lastname: Dhillon
authors.affiliation: Lein Applied Diagnostics, Reading Enterprise Centre, Whiteknights Rd, Reading RG6 6BU, UK.
authors.forename: Kim K
authors.initials: KK
authors.lastname: Buttenschön
authors.affiliation: Lein Applied Diagnostics, Reading Enterprise Centre, Whiteknights Rd, Reading RG6 6BU, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Daly
authors.affiliation: Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 6, UK.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Gakamsky
mesh_terms: D000818:Animals; D002386:Cataract / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000235:genetics* / Q000302:isolation & purification; D005453:Fluorescence; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism / Q000473:pathology; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013552:Swine; D014364:Tryptophan / Q000737:chemistry / Q000302:isolation & purification; D014466:Ultraviolet Rays
chemical_list: D003459:Crystallins; D014364:Tryptophan