Mass spectrometric determination of early and advanced glycation in biology

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N^ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.     

Positive and negative electrospray ionisation tandem mass spectrometry as a tool for structural characterisation of acid released oligosaccharides from olive pulp glucuronoxylans

Xylo-oligosaccharides with degrees of polymerisation 5-13, formed by partial acid hydrolysis from an extract representative of olive pulp glucuronoxylans (GX), were analysed by electrospray ionisation mass spectrometry (ESI-MS), both in positive and negative modes. The positive spectrum showed the presence of xylo-oligosaccharides in the mass range between m/z 500 and 1500 corresponding to singly [M+Na](+) charged ions of neutral (Xyl(7-9)) and acidic xylo-oligosaccharides (Xyl(5-9)MeGlcA), and doubly [M+2Na](2+) charged ions of Xyl(9-13) and Xyl(7-11)MeGlcA. Ammonium adducts [M+NH(4)](+) were also observed for Xyl(5-9)MeGlcA. The negative spectra showed the contribution of ions in the mass range between m/z 600 and 1400, ascribed to the deprotonated molecules [M-H](-) of Xyl(3-9)MeGlcA. Tandem mass spectrometry (MS/MS) of the major ions observed in the MS spectra was performed. The MS/MS spectra of the [M+Na](+) adducts showed the loss of MeGlcA residues as the major fragmentation pathway and glycosidic fragment ions of Xyl(n) and Xyl(n)MeGlcA structures. The MS/MS spectra of the [M+NH(4)](+) adducts suggests the occurrence of isomers of Xyl(5-9)MeGlcA oligosaccharides with the MeGlcA residue at the reducing end and at the non-reducing end of the molecules, although other structural isomers can also occur. Both glycosidic bond and cross-ring cleavages in the MS/MS spectra of the [M-H](-) ion suggest the occurrence of Xyl(3-9)MeGlcA with the substituting group at the reducing end position of the xylose backbone, as the main fragmentation ions. The results obtained by ESI-MS/MS, both in positive and negative modes, of Xyl(7-13)- and Xyl(5-11)MeGlcA, allow to identify fragmentation patterns of the structural isomers with MeGlcA linked to the terminal xylosyl residues of the oligosaccharides. The occurrence of these higher molecular weight oligosaccharides with a low substitution pattern allows to infer a scatter and random distribution of MeGlcA along the xylan backbone of olive pulp.     

Molecular basis of maillard amide-advanced glycation end product (AGE) formation in vivo

The Maillard reaction in vivo entails alteration of proteins or free amino acids by non-enzymatic glycation or glycoxidation. The resulting modifications are called advanced glycation end products (AGEs) and play a prominent role in various pathologies, including normoglycemic uremia. Recently, we established a new class of lysine amide modifications in vitro. Now, human plasma levels of the novel amide-AGEs N(6)-acetyl lysine, N(6)-formyl lysine, N(6)-lactoyl lysine, and N(6)-glycerinyl lysine were determined by means of LC-MS/MS. They were significantly higher in uremic patients undergoing hemodialysis than in healthy subjects. Model reactions with N(1)-t-butoxycarbonyl-lysine under physiological conditions confirmed 1-deoxy-d-erythro-hexo-2,3-diulose as an immediate precursor. Because formation of N(6)-formyl lysine from glucose responded considerably to the presence of oxygen, glucosone was identified as another precursor. Comparison of the in vivo results with the model experiments enabled us to elucidate possible formation pathways linked to Maillard chemistry. The results strongly suggest a major participation of non-enzymatic Maillard mechanisms on amide-AGE formation pathways in vivo, which, in the case of N(6)-acetyl lysine, parallels enzymatic processes.     

Amides are novel protein modifications formed by physiological sugars

The Maillard reaction, or nonenzymatic browning, proceeds in vivo, and the resulting protein modifications (advanced glycation end products) have been associated with various pathologies. Despite intensive research only very few structures have been established in vivo. We report here for the first time N(6)-[2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl]lysine (GOLA) and N(6)-glycoloyllysine (GALA) as prototypes for novel amide protein modifications produced by reducing sugars. Their identity was confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with N(alpha)-t-butoxycarbonyl-lysine showed that glyoxal and glycolaldehyde are immediate precursors, and reaction pathways are directly linked to N(epsilon)-carboxymethyllysine via glyoxal-imine structures. GOLA, the amide cross-link, and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), the imidazolium cross-link, share a common intermediate. The ratio of GOLA to GOLD is greater when glyoxal levels are low at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose and lysine depends directly upon oxidation. With the advanced glycation end product inhibitors aminoguanidine and pyridoxamine we were able to dissect oxidative fragmentation of the Amadori product as a second mechanism of GOLA formation exactly coinciding with N(epsilon)-carboxymethyllysine synthesis. In contrast, the formation of GALA appears to depend solely upon glyoxal-imines. After enzymatic hydrolysis GOLA was found at 66 pmol/mg of brunescent lens protein. This suggests amide protein modifications as important markers of pathophysiological processes.     

Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging

Until now, the glycation reaction was considered to be a nonspecific reaction between reducing sugars and amino groups of random proteins. We were able to identify the intermediate filament vimentin as the major target for the AGE modification N(epsilon)-(carboxymethyl)lysine (CML) in primary human fibroblasts. This glycation of vimentin is neither based on a slow turnover of this protein nor on an extremely high intracellular expression level, but remarkably it is based on structural properties of this protein. Glycation of vimentin was predominantly detected at lysine residues located at the linker regions using nanoLC-ESI-MS/MS. This modification results in a rigorous redistribution of vimentin into a perinuclear aggregate, which is accompanied by the loss of contractile capacity of human skin fibroblasts. CML-induced rearrangement of vimentin was identified as an aggresome. This is the first evidence that CML-vimentin represents a damaged protein inside the aggresome, linking the glycation reaction directly to aggresome formation. Strikingly, we were able to prove that the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in human tissues such as skin into strong relationship with loss of organ contractile functions.     

Ultra performance liquid chromatography-mass spectrometric determination of the site specificity of modification of β-casein by glucose and methylglyoxal

Modification of protein by carbonyl compounds under in vitro physiological conditions is site-directed. There are few reports of the site specificity of glycation of proteins using heating conditions of relevance to food processing. The aim of this study was to determine the site specificity of modification of β-casein (βCN) by glucose and methylglyoxal (MGO). βCN (1.33 M, 3.2%) was heated with either glucose (1.345 M, 4.6%) or MGO (1 mM) at 95°C for up to 4 h. Tryptic digests were prepared and analysed by ultra performance liquid chromatography electrospray ionisation mass spectrometry (UPLC-ES/MS). The sites of formation of the Amadori product, N ^ε -(fructosyl)lysine (FL), and the advanced glycation end-products, N ^ε -(carboxymethyl)lysine (CML), MGO-derived dihydroxyimidazolidine (MG-DH) and MGO-derived hydroimidazolone (MG-HI), were located. FL and CML were detected at K107 and K176 residues in βCN/glucose incubations. Indigenous N ^ε -(lactulosyl)lysine was detected at K107 only. MG-DH and MG-HI were detected at R202 and possibly R183 residues in both βCN/glucose and βCN/MGO incubations. Glycation of βCN by glucose and MGO resulted in similar site specificity for MG-DH and MG-HI formation.     

Studies on the Reaction of Dipeptides with Glyoxal

The reaction of various dipeptides with glyoxal at 100°C, pH 5.0 was studied. Carbon dioxide, ammonia, amino acids and aldehydes were detected from the reaction solutions. Besides, a series of new pyrazinones—2-(3′-alkyl-2′-oxo-pyrazin-1′-yl)alkyl acid—were isolated, and their chemical structures were confirmed by UV, IR, MS and NMR spectra. These pyrazinones seem to play a role in the browning of the reaction.

In the reaction with glyoxal, acetaldehyde and glucose, reactivity of peptides was proved to be much higher than that of amino acids.

The reaction mechanism of dipeptides with glyoxal was also proposed.     

Glycation of amino groups in protein. Studies on the specificity of modification of RNase by glucose

Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.     

Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease

Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N(epsilon)-(carboxymethyl)lysine (CML). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degrees C for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CML formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CML is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.     

Gramicidin S: a peptide model for protein glycation and reversal of glycation using nucleophilic amines.

Nonenzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. Glycation of proteins proceeds through the stages of Schiff base formation, conversion to ketoamine product and advanced glycation end products. Gramicidin S, which has two ornithine residues, was used as a model system to study the various stages of glycation of proteins using electrospray ionization mass spectrometry. The proximity of two ornithine residues in the peptide favors the glycation reaction. Formation of advanced glycation end products and diglycation on ornithine residues in gramicidin S were observed. The formation of Schiff base adduct is reversible, whereas the Amadori rearrangement to the ketoamine product is irreversible. Nucleophilic amines and hydrazines can deglycate the Schiff base adduct of glucose with peptides and proteins. Hydroxylamine, isonicotinic acid hydrazide and aminoguanidine effectively removed glucose from the Schiff base adduct of gramicidin S. Hydroxylamine is more effective in deglycating the adduct compared with isonicotinic acid hydrazide and aminoguanidine. The observation that the hydrazines are effective in deglycating the Schiff base adduct even in the presence of high concentrations of glucose, may have a possible therapeutic application in preventing complications of diabetes mellitus. Hydrazines may be used to distinguish between the Schiff base and the ketoamine products formed at the initial stages of glycation.     

Adducts between nucleophilic amino acids and hexahydrophthalic anhydride, a structure inducing both types I and IV allergy.

Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC-UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N(alpha)-acetylated lysine and cysteine and the non-acetylated alpha-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine-HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine-HHPA adducts. The results will be useful for understanding the formation of HHPA-protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy.     

Rapid characterization and identification of steroidal alkaloids in Sarcococca coriacea using liquid chromatography coupled with electrospray ionization quadropole time-of-flight mass spectrometry

Rapid characterization of 23 pregnane-type steroidal alkaloids was studied using a positive ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. ESI-QqTOF-MS (positive ion mode) showed the presence of the protonated molecules [M+H](+) which through low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis showed the characteristic loss of dimethylamine moiety [M+H-45](+) followed by the sequential lossess of attached substituents. Steroidal alkaloids having tigloyl or senecioyl group at C-3 produced diagnostic fragment ions at m/z 100 and 83. Our study also demonstrates the influence of unsaturation, and number and nature of substitutents on product ion abundance and fragment ions. Moreover, the generalization of the fragmentation pattern was linked with the structural features in steroidal skeleton. This strategy was successfully applied in LC-ESI-QqTOF-MS/MS analysis of Sarcococca coriacea extract to investigate and characterize pregnane-type steroidal alkaloids in complex mixture.     

Formation of the molten globule-like state during prolonged glycation of human serum albumin

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reaction. We studied the impact of incubation of human serum albumin (HSA) with glucose, at various concentrations and incubation times, on the extent of HSA glycation and structural changes using circular dichroism (CD), fluorescence, and microviscometer techniques. The number of moles of glucose bound per mole of HSA (r), the number of reacted lysine and arginine residues, and the Amadori product formation during glycation were determined using 3-(dansylamino) phenyl boronic acid, fluorescamine, 9, 10 phenanthrenequinone, and p-nitroblue tetrazoliumchloride, respectively. The formation of advanced glycation end products (AGE) was detected using the autofluorescence characteristic of samples. We identified three stages of Maillard reaction for HSA upon incubation with the physiological level of glucose (0-630 mg/dl): the early, intermediate and late stages, which occurred after 7-14, 21, and >28 days of incubation, respectively. Structural information, Stokes radius, and 1-anilinonaphthalene-8-sulfonate (ANS) binding data indicated the formation of a molten globule-like state of HSA after 21 days of incubation with 35 mM (630 mg/dl) glucose. Thus, the extent of the Maillard reaction was influenced by the concentration of glucose and incubation time, such that longer exposure of HSA to glucose may have a more deleterious effect on its structure and especially on its half-life and turnover in the circulation. Our results suggest that in acute diabetes mellitus patients, HSA, after 21 days of glycation, passes through a molten globule-like state and may contribute to the pathogenesis of diabetes, and perhaps other diseases.     

Adducts between nucleophilic amino acids and hexahydrophthalic anhydride,a structure inducing both types I and IV allergy

Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC-UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N^α-acetylated lysine and cysteine and the non-acetylated α-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine–HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine–HHPA adducts. The results will be useful for understanding the formation of HHPA–protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy.     

Detection of dideoxyosone intermediates of glycation using a monoclonal antibody: characterization of major epitope structures

Glycation or the Maillard reaction in proteins forms advanced glycation end products (AGEs) that contribute to age- and diabetes-associated changes in tissues. Dideoxyosones, which are formed by the long-range carbonyl shift of the Amadori product, are newly discovered intermediates in the process of AGE formation in proteins. They react with o-phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate coupled to keyhole limpet hemocyanin. The antibody reacted strongly with ribose and fructose (+OPD)-modified RNase A and weakly with glucose and ascorbate (+OPD)-modified RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2-methyl group on the quinoxaline ring. We used high performance liquid chromatography to isolate and purify three antibody-reactive products from a reaction mixture of N alpha-hippuryl-L-lysine+ribose+OPD. The two most reactive products were identified as diastereoisomers of N1-benzoylglycyl-N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and the other less reactive product as N1-benzoylglycyl-N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine. Our study confirms that dideoxyosone intermediates form during glycation and offers a new tool for the study of this important pathway in diabetes and aging.     

Nonenzymatic glycation of type I collagen. The effects of aging on preferential glycation sites.

The present study was designed to investigate the effects of aging on preferential sites of glucose adduct formation on type I collagen chains. Two CNBr peptides, one from each type of chain in the type I tropocollagen molecule, were investigated in detail: alpha 1(I)CB3 and alpha 2CB3-5. Together these peptides comprise approximately 25% of the total tropocollagen molecule. The CNBr peptides were purified from rat tail tendon, obtained from animals aged 6, 18, and 36 months, by ion exchange chromatography, gel filtration, and high-performance liquid chromatography (HPLC). Sugar adducts were radiolabeled by reduction with NaB3H4. Glycated tryptic peptides were prepared from tryptic digests of alpha 2CB3-5 and alpha 1(I)CB3 by boronate affinity chromatography and HPLC. Peptides were identified by sequencing and by compositional analysis. Preferential sites of glycation were observed in both CB3 and alpha 2CB3-5. Of the 5 lysine residues in CB3, Lys-434 was the favored glycation site. Of the 18 lysine residues and 1 hydroxylysine residue in alpha 2CB3-5, 3 residues (Lys-453, Lys-479, and Lys-924) contained more than 80% of the glucose adducts on the peptide. Preferential glycation sites were highly conserved with aging. In collagen that had been glycated in vitro, the relative distribution of glucose adducts in old animals differed from that of young animals. In vitro experiments suggest that primary structure is the major determinant of preferential glycation sites but that higher order structure may influence the relative distribution of glucose adducts among these preferred sites.     

Effect of phosphate on the kinetics and specificity of glycation of protein

The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffers in order to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues in or near the active site. In contrast, in the cationic buffers, 3-(N-morpholino)propane-sulfonic acid and 3-(N-tris(hydroxymethyl)methyl-amino)-2-hydroxypropanesulfonic acid, the kinetics of glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNAse was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increased glycation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effects on the glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establish that buffering ions or ligands can exert significant effects on the kinetics and specificity of glycation of proteins.     

Chemical reactivity of the functional groups of insulin. Concentration-dependence studies.

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.     

Nonenzymatic glycation of DNA nucleosides with reducing sugars

Reducing sugars can react with the free amino groups of proteins to form a heterogeneous group of compounds known as advanced glycation endproducts (AGEs) or Maillard reaction products. The objective of this investigation was to monitor the nonenzymatic glycation of DNA nucleosides and to characterize the formation of nucleoside AGEs using capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), UV fluorescence spectroscopy, and mass spectrometry. Deoxyguanosine, deoxyadenosine, deoxythymidine, and deoxycytidine were used as the model nucleosides and were incubated over time with glucose, galactose, or glyceraldehyde. Under increasing concentrations and time, deoxyguanosine exhibited the highest rate of glycation with glyceraldehyde. Deoxyadenosine and deoxycytidine exhibited comparable reactivity with glyceraldehyde and no appreciable reactivity with galactose or glucose. No reactivity was observed between deoxythymidine and the sugars. A combination of CE, HPLC, UV fluorescence spectroscopy, and mass spectrometry provided a convenient method for characterizing nucleoside AGEs and for monitoring the physical factors that influence the formation of sugar adducts of DNA nucleosides.     

Reaction of ascorbate with lysine and protein under autoxidizing conditions: formation of N epsilon-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of glucose adducts to protein in vitro and has been detected in human tissue proteins and urine [Ahmed, M. U., Thorpe, S. R., & Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894; Dunn, J. A., Patrick, J. S., Thorpe, S. R., & Baynes, J. W. (1989) Biochemistry 28, 9464-9468]. In the present study we show that CML is also formed in reactions between ascorbate and lysine residues in model compounds and protein in vitro. The formation of CML from ascorbate and lysine proceeds spontaneously at physiological pH and temperature under air. Kinetic studies indicate that oxidation of ascorbic acid to dehydroascorbate is required. Threose and N epsilon-threuloselysine, the Amadori adduct of threose to lysine, were identified in the ascorbate reaction mixtures, suggesting that CML was formed by oxidative cleavage of N epsilon-threuloselysine. Support for this mechanism was obtained by identifying CML as a product of reaction between threose and lysine and by analysis of the relative rates of formation of threuloselysine and CML in reactions of ascorbate or threose with lysine. The detection of CML as a product of reaction of ascorbate and threose with lysine suggests that other sugars, in addition to glucose, may be sources of CML in proteins in vivo. The proposed mechanism for formation of CML from ascorbate is an example of autoxidative glycosylation of protein and suggests that CML may also be an indicator of autoxidative glycosylation of proteins in vivo.