Structure Elucidation of a Novel Yellow Chromophore from Human Lens Protein

• Published in: The Journal of biological chemistry 2004-10, Vol.279 (44), p.45441-45449
• Main Authors: Cheng, Rongzhu, Feng, Qi, Argirov, Ognyan K, Ortwerth, Beryl J
• Format: Article
• Language: English
• Subjects: Aged; Aging - metabolism; Cataract - etiology; Cataract - metabolism; Chromatography, High Pressure Liquid; Crystallins - chemistry; Humans; Mass Spectrometry; Nuclear Magnetic Resonance, Biomolecular
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M405664200

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 ϵ-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.