Abstract
We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and (1)H, (13)C, and two-dimensional NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and fluorescence at 410 nm when excited at 343 nm. Analysis of the purified compound by reversed-phase HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues, and a five-carbon ring. Because this cross-link contains two lysine residues and a dihydropyridinium ring, we assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests were made using a high-performance liquid chromatograph equipped with a diode array detector. These measurements revealed a significant enhancement of K2P in cataract lens proteins (613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of WS protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of water-soluble (WS) protein). These data provide chemical evidence for increased protein cross-linking during aging and cataract development in vivo. This new cross-link may serve as a quantitatively more significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.
Highlights
In the past two decades, enormous progress has been made in our understanding of the Maillard reaction and its contribution to age-related changes and complications of diabetes
We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and 1H, 13C, and two-dimensional NMR
Novel Yellow Chromophore in Human Lens port the possibility that glycation plays an important role in lens proteins insolubilization and precipitation during aging and cataract development
Summary
We report the isolation and characterization of a novel lysine-lysine cross-link, 1-(5-amino-5-carboxypentyl)-4(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium (K2P), from enzymatic digests of aged and cataract human lens proteins. The age-dependent increase of this cross-link in normal human lenses and its significant enhancement in cataract lenses suppentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium; NOESY, nuclear Overhauser effect spectroscopy; RP-HPLC, reversed-phase high performance liquid chromatography; TOCSY, total correlation spectroscopy; Th, Thompsons (atomic mass unit); MOLD, methylglyoxal lysine dimer; GOLD, glyoxal lysine dimer. Novel Yellow Chromophore in Human Lens port the possibility that glycation plays an important role in lens proteins insolubilization and precipitation during aging and cataract development
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