Application of solid-phase extraction for the concentration of chromophores, fluorophores, and photosensitizers from lens protein digests.

Solid-phase extraction was applied for the separation of protein digests obtained from aged human lenses, cataractous human lenses, calf lens proteins in vitro glycated with dehydroascorbic acid and native calf lens proteins. Four fractions were collected after stepwise elution with different solvents. The first fraction contained about 80% of the digested material possessing free amino groups. At the same time, the third and the fourth fractions were enriched in chromophores, fluorophores, and photosensitizing structures that originate mainly from advanced protein glycation. The comparison between the total digest and the fourth fraction based on their UV absorption at 330 nm, intensity of fluorescence (excitation/emission 350/450 nm), and production of singlet oxygen upon UVA irradiation argues that the solid-phase extraction was capable of concentrating the advanced glycation end-products about a hundredfold. Thus, this technique is a useful step for separation and concentration of fluorophores, chromophores, and photosensitizers from aged and glycated lens protein digests.

Influence of glutathione fructosylation on its properties.

Incubation of fructose and glutathione leads to the formation of N-2-deoxy-glucos-2-yl glutathione as the major glycation product, with characteristic positive ion at 470 Th in LC-MS spectra. Glutathione disulfide and fructose generate two compounds: N-2-deoxy-glucos-2-yl glutathione disulfide (m/z=775 Th) and bis di-N,N'-2-deoxy-glucos-2-yl glutathione disulfide (m/z=937 Th). N-2-deoxy-glucos-2-yl glutathione is 2.5-fold less effective than glutathione in reducing dehydroascorbic acid. Glutathione peroxidase and glutahione-S-transferase exhibit marginal activity toward N-2-deoxy-glucos-2-yl glutathione, while glyoxalase I shows 44.9% of the enzyme's specific activity. Glutathione reductase demonstrates 6.9% of the enzyme's specific activity with bis di-N,N'-2-deoxy-glucos-2-yl glutathione, while with mono-N-glucosyl glutathione disulfide retained 5 6.1% of the original activity. Glutathione reductase could not reduce N-2-deoxy-glucos-2-yl glutathione in mixed disulfide with gammaS-crystallin, but reduced glutathione in mixed disulfide with gammaS-crystallin by 90%. The presence of N-2-deoxy-glucos-2-yl glutathione in mixed disulfide with gammaS-crystallin makes this molecule more susceptible to unfolding than native gammaS-crystallin.

K2P--a novel cross-link from human lens protein.

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 LC-MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure as being 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl-amino)-3-hydroxy-2, 3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues and a five-carbon atom 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 revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of water-soluble [WS] protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross-link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.

Structure elucidation of a novel yellow chromophore from human lens protein.

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.

2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses.

Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the nonproteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nalpha-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nalpha-t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties.

Isolation and characterization of a new advanced glycation endproduct of dehydroascorbic acid and lysine.

Proteins are subject of posttranslational modification by sugars and their degradation products in vivo. The process is often referred as glycation. L-Dehydroascorbic acid (DHA), an oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. A new product of modification of lysine epsilon -amino group by DHA was discovered as a result of the interaction between Boc-Lys and dehydroascorbic acid. The chromatographic and spectral analyses revealed that the structure of the product was 1-(5-ammonio-5-carboxypentyl)-3-oxido-4-(hydroxymethyl)pyridinium. The same compound was isolated from DHA modified calf lens protein after hydrolysis and chromatographic separation. The study confirmed that L-erythrulose is an important intermediate of modification of proteins by DHA. The structure of the reported product and in vitro experiments suggested that L-erythrulose could further transform to L-threose, L-erythrose and glycolaldehyde under conditions similar to physiological. The present study revealed that the modification of epsilon -amino groups of lysine residues by DHA is a complex process and could involve a number of reactive carbonyl species.

Region-specific pathophysiological alterations occurring in calf lenses in vitro during hyperglycemia.

BACKGROUND: The early onset of cataract during diabetes may come about via a variety of pathogenic pathways, but an uncertainty about the significance of each of them exists. METHODS: Calf lenses cultured in a high glucose medium were investigated for regional variations in sorbitol accumulation, changes in lactate dehydrogenase activity, and formation of carbonyl groups in proteins. The results obtained were used to evaluate the contributions of various pathways to the alterations in the lens during hyperglycemia and to relate these findings to morphologically diverse lens substructures. RESULTS: The highest sorbitol accumulation was found in both the anterior and posterior cortex of lenses incubated in hyperglycemic medium. Lactate dehydrogenase activity was strongly affected by high sugar concentration, but the alterations in the equatorial part of lenses were more moderate relative to other substructures. After incubation with glucose, the concentration of Amadori products did not increase significantly compared to non-incubated and incubated controls. Nuclear proteins exhibited the highest level of oxidation. CONCLUSION: The process of sorbitol accumulation is more evident than glycation in the initial stage of hyperglycemia. Lens cortex is affected faster by elevated glucose, while the nucleus is more susceptible to prolonged effects of oxidation, glycation, and glycoxidation.