Identification of glutathionyl-3-hydroxykynurenine glucoside as a novel fluorophore associated with aging of the human lens.
A novel fluorophore was isolated from human lenses using high performance liquid chromatography (HPLC). The new fluorophore was well separated from 3-hydroxykynurenine glucoside (3-OHKG) and its deaminated isoform, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, which are known UV filter compounds. The new compound exhibited UV absorbance maxima at 260 and 365 nm, was fluorescent (Ex(360 nm)/Em(500 nm)), and increased in concentration with age. Further analysis of the purified compound by microbore HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 676 Da. This mass corresponds to that of an adduct of GSH with a deaminated form of 3-OHKG. This adduct was synthesized using 3-OHKG and GSH as starting materials. The synthetic glutathionyl-3-hydroxykynurenine glucoside (GSH-3-OHKG) adduct had the same HPLC elution time, thin-layer chromatography R(F) value, UV absorbance maxima, fluorescence characteristics, and mass spectrum as the lens-derived fluorophore. Furthermore, the (1)H and (13)C NMR spectra of the synthetic adduct were entirely consistent with the proposed structure of GSH-3-OHKG. These data indicate that GSH-3-OHKG is present as a novel fluorophore in aged human lenses. The GSH-3-OHKG adduct was found to be less reactive with beta-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by molecular modeling.
Garner
B
B
Australian Cataract Research Foundation, University of Wollongong, Wollongong, New South Wales 2522, Australia.
Vazquez
S
S
Griffith
R
R
Lindner
R A
RA
Carver
J A
JA
Truscott
R J
RJ
D000375:Aging / Q000378:metabolism*; D015415:Biomarkers; D005960:Glucosides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007737:Kynurenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007908:Lens, Crystalline / Q000502:physiology*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D015415:Biomarkers; D005960:Glucosides; D020033:Protein Isoforms; C120691:glutathionyl-3-hydroxykynurenine glucoside; C029366:hydroxykynurenine glucoside; D007737:Kynurenine; D005978:Glutathione