Role of the Maillard Reaction in Aging of Tissue Proteins
1998; Elsevier BV; Volume: 273; Issue: 30 Linguagem: Inglês
10.1074/jbc.273.30.18714
ISSN1083-351X
AutoresElisabeth Brinkmann Frye, Thorsten P. Degenhardt, Suzanne R. Thorpe, John Baynes,
Tópico(s)Connexins and lens biology
ResumoDicarbonyl compounds such as glyoxal and methylglyoxal are reactive dicarbonyl intermediates in the nonenzymatic browning and cross-linking of proteins during the Maillard reaction. We describe here the quantification of glyoxal and methylglyoxal-derived imidazolium cross-links in tissue proteins. The imidazolium salt cross-links, glyoxal-lysine dimer (GOLD) and methylglyoxal-lysine dimer (MOLD), were measured by liquid chromatography/mass spectrometry and were present in lens protein at concentrations of 0.02–0.2 and 0.1–0.8 mmol/mol of lysine, respectively. The lens concentrations of GOLD and MOLD correlated significantly with one another and also increased with lens age. GOLD and MOLD were present at significantly higher concentrations than the fluorescent cross-links pentosidine and dityrosine, identifying them as major Maillard reaction cross-links in lens proteins. Like theN-carboxy-alkyllysinesNε-(carboxymethyl)lysine andNε-(carboxyethyl)lysine, these cross-links were also detected at lower concentrations in human skin collagen and increased with age in collagen. The presence of GOLD and MOLD in tissue proteins implicates methylglyoxal and glyoxal, either free or protein-bound, as important precursors of protein cross-links formed during Maillard reactions in vivo during aging and in disease. Dicarbonyl compounds such as glyoxal and methylglyoxal are reactive dicarbonyl intermediates in the nonenzymatic browning and cross-linking of proteins during the Maillard reaction. We describe here the quantification of glyoxal and methylglyoxal-derived imidazolium cross-links in tissue proteins. The imidazolium salt cross-links, glyoxal-lysine dimer (GOLD) and methylglyoxal-lysine dimer (MOLD), were measured by liquid chromatography/mass spectrometry and were present in lens protein at concentrations of 0.02–0.2 and 0.1–0.8 mmol/mol of lysine, respectively. The lens concentrations of GOLD and MOLD correlated significantly with one another and also increased with lens age. GOLD and MOLD were present at significantly higher concentrations than the fluorescent cross-links pentosidine and dityrosine, identifying them as major Maillard reaction cross-links in lens proteins. Like theN-carboxy-alkyllysinesNε-(carboxymethyl)lysine andNε-(carboxyethyl)lysine, these cross-links were also detected at lower concentrations in human skin collagen and increased with age in collagen. The presence of GOLD and MOLD in tissue proteins implicates methylglyoxal and glyoxal, either free or protein-bound, as important precursors of protein cross-links formed during Maillard reactions in vivo during aging and in disease. The Maillard reaction is a complex series of reactions between reducing sugars and amino groups on proteins, which lead to browning, fluorescence, and cross-linking of protein (1Hodge J.E. J. Agric. Food Chem. 1953; 1: 928-943Crossref Scopus (1462) Google Scholar, 2Ledl F. Schleicher E. Angew. Chem. Int. Ed. Engl. 1990; 29: 565-594Crossref Scopus (674) Google Scholar). Advanced glycation end products, formed during later stages of the Maillard reaction, accumulate in long lived tissue proteins, such as tissue collagens and lens crystallins, and may contribute to the development of complications in aging, diabetes, and atherosclerosis (3Vlassara H. J. Lab. Clin. Med. 1994; 124: 19-30PubMed Google Scholar, 4Thorpe S.R. Baynes J.W. Drugs Aging. 1996; 9: 69-77Crossref PubMed Scopus (266) Google Scholar). Glyoxal (GO), 1The abbreviations and trivial names used are: GO, glyoxal; CEL,Nε-[1-(1-carboxy)ethyl]lysine; CML,Nε-(carboxymethyl)lysine; ESI, electrospray ionization; RP-HPLC, reverse phase high performance liquid chromatography assay; GOLD, glyoxal-lysine dimer, 1,3-di-(Nε-lysino)imidazolium salt; HPLC, high performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; MGO, methylglyoxal; MOLD, methylglyoxal-lysine dimer, 1,3-di-(Nε-lysino)-4-methyl-imidazolium salt; PITC, phenylisothiocyanate; PTC, phenylthiocarbamoyl; RNase, bovine pancreatic ribonuclease A; PAGE, polyacrylamide gel electrophoresis; GC/MS, gas chromatography/mass spectrometry; SIM, selected ion monitoring; amu, atomic mass units. 1The abbreviations and trivial names used are: GO, glyoxal; CEL,Nε-[1-(1-carboxy)ethyl]lysine; CML,Nε-(carboxymethyl)lysine; ESI, electrospray ionization; RP-HPLC, reverse phase high performance liquid chromatography assay; GOLD, glyoxal-lysine dimer, 1,3-di-(Nε-lysino)imidazolium salt; HPLC, high performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; MGO, methylglyoxal; MOLD, methylglyoxal-lysine dimer, 1,3-di-(Nε-lysino)-4-methyl-imidazolium salt; PITC, phenylisothiocyanate; PTC, phenylthiocarbamoyl; RNase, bovine pancreatic ribonuclease A; PAGE, polyacrylamide gel electrophoresis; GC/MS, gas chromatography/mass spectrometry; SIM, selected ion monitoring; amu, atomic mass units. methylglyoxal (MGO), and deoxyglucosones belong to a series of dicarbonyl compounds, identified as intermediates in the Maillard reaction. GO is formed on autoxidation of glucose under physiological conditions (5Wells-Knecht K.J. Zyzak D.V. Litchfield L.E. Thorpe S.R. Baynes J.W. Biochemistry. 1995; 34: 3702-3709Crossref PubMed Scopus (548) Google Scholar) and also as a product of lipid peroxidation (6Loidl-Stahlhofen A. Hannemann K. Spiteller G. Chem. Phys. Lipids. 1995; 77: 113-117Crossref PubMed Scopus (21) Google Scholar). MGO is formed nonenzymatically by spontaneous decomposition of triose phosphate intermediates in glycolysis (7Richard J.P. Biochemistry. 1991; 30: 4581-4585Crossref PubMed Scopus (185) Google Scholar) and by amine-catalyzed sugar fragmentation reactions (8Hayashi T. Mase S. Namiki M. Agric. Biol. Chem. 1986; 50: 1959-1964Crossref Google Scholar,9Hayashi T. Namiki M. Agric. Biol. Chem. 1986; 30: 1965-1970Google Scholar). It is also a product of metabolism of acetone (10Reichard G.A. Skutches C.L. Hoeldtke R.D. Owen O.E. Diabetes. 1986; 35: 668-674Crossref PubMed Google Scholar) and threonine (11Ray M. Ray S. J. Biol. Chem. 1987; 262: 5974-5977Abstract Full Text PDF PubMed Google Scholar). Both GO and MGO are detoxified by the glutathione-dependent glyoxalase pathway, yielding hydroxyacetic acid and d-lactate, respectively (12Thornalley P.J. Mol. Aspects Med. 1994; 14: 287-371Crossref Scopus (451) Google Scholar, 13Thornalley P.J. Amino Acids ( Vienna ). 1994; 6: 15-23Crossref PubMed Scopus (62) Google Scholar, 14Thornalley P.J. McLellan A.C. Lo T.W.C. Benn J. Soenksen P.H. Clin. Sci. ( Lond. ). 1996; 91: 575-582Crossref PubMed Scopus (125) Google Scholar). MGO can also be detoxified by the NADPH-dependent enzyme aldose reductase, yielding 1,2-propanediol (15Vander Jagt D.L. Robinson B. Taylor K.K. Hunsaker L.A. J. Biol. Chem. 1992; 267: 4364-4369Abstract Full Text PDF PubMed Google Scholar). The concentration of MGO is elevated in the blood of diabetic patients in vivo(16Thornalley P.J. Hooper N.I. Jennings P.E. Florkowski C.M. Jones A.F. Lunec J. Barnett A.H. Diabetes Res. Clin. Pract. 1989; 7: 115-120Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 17McLellan A.C. Thornalley P.J. Benn J. Soenksen P.H. Clin. Sci. ( Lond. ). 1994; 87: 21-29Crossref PubMed Scopus (487) Google Scholar), and the metabolites of MGO detoxification, acetol and 1,2-propanediol, are also increased in blood during diabetic ketoacidosis (10Reichard G.A. Skutches C.L. Hoeldtke R.D. Owen O.E. Diabetes. 1986; 35: 668-674Crossref PubMed Google Scholar). GO and MGO are reactive toward amino, guanidino, and sulfhydryl functional groups in protein (18McLaughlin J.A. Pethig R. Szent-Gyoergyi A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 949-951Crossref PubMed Scopus (46) Google Scholar, 19Lo T.W.C. Westwood M.E. McLellan A.C. Selwood T. Thornalley P.J. J. Biol. Chem. 1994; 269: 32299-32305Abstract Full Text PDF PubMed Google Scholar), leading to browning, denaturation, and cross-linking of proteins. Besides unidentified brown and fluorescent products, the reaction of GO and MGO with lysine and arginine residues in protein yields well characterized compounds, such as the N-(carboxyalkyl)lysines,Nε-(carboxymethyl)lysine (CML) (20Ahmed M.U. Thorpe S.R. Baynes J.W. J. Biol. Chem. 1986; 261: 4889-4894Abstract Full Text PDF PubMed Google Scholar) andNε-[1-(1-carboxy)ethyl]lysine (CEL) (21Ahmed M.U. Brinkmann Frye E. Degenhardt T.P. Thorpe S.R. Baynes J.W. Biochem. J. 1997; 324: 565-570Crossref PubMed Scopus (525) Google Scholar), and imidazolones and dehydroimidazolones (19Lo T.W.C. Westwood M.E. McLellan A.C. Selwood T. Thornalley P.J. J. Biol. Chem. 1994; 269: 32299-32305Abstract Full Text PDF PubMed Google Scholar, 22Henle T. Walter A.W. Haessner A. Klostermeyer H. Z.Lebensm.-Unters. Forsch. 1994; 199: 55-58Crossref Scopus (105) Google Scholar, 23Konoshi Y. Hayase F. Kato H. Biosci. Biotechnol. Biochem. 1994; 58: 1953-1955Crossref Scopus (80) Google Scholar). Imidazolones have been detected in tissue proteins only by immunological methods, whereas CML and CEL have been measured by gas chromatography/mass spectometry (GC/MS) and increase in skin collagen and lens protein with age (21Ahmed M.U. Brinkmann Frye E. Degenhardt T.P. Thorpe S.R. Baynes J.W. Biochem. J. 1997; 324: 565-570Crossref PubMed Scopus (525) Google Scholar, 24Dyer D.G. Dunn J.A. Thorpe S.R. Bailie K.E. Lyons T.J. McCance D.R. Baynes J.W. J. Clin. Invest. 1993; 91: 2463-2469Crossref PubMed Scopus (631) Google Scholar, 25Dunn J.A. McCance D.R. Thorpe S.R. Lyons T.J. Baynes J.W. Biochemistry. 1991; 30: 1205-1210Crossref PubMed Scopus (284) Google Scholar, 26Dunn J.A. Patrick J.S. Thorpe S.R. Baynes J.W. Biochemistry. 1989; 28: 9454-9468Google Scholar). Two other products of the reaction of GO or MGO with lysine, glyoxal-lysine dimer (GOLD) and methylglyoxal-lysine dimer (MOLD) (Fig. 1), were originally characterized in reactions of GO or MGO, respectively, with hippuryllysine (27Wells-Knecht K.J. Brinkmann E. Thorpe S.R. Baynes J.W. J. Org. Chem. 1995; 60: 6246-6247Crossref Scopus (92) Google Scholar, 28Brinkmann E. Wells-Knecht K.J. Thorpe S.R. Baynes J.W. J. Chem. Soc. Perkin Trans. I. 1995; 1: 2817-2818Crossref Google Scholar). Nagaraj and colleagues (29Nagaraj R.H. Shipanova I.N. Faust F.M. J. Biol. Chem. 1996; 271: 19338-19345Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) recently detected and measured MOLD in human serum proteins by reverse phase high performance liquid chromatography assay (RP-HPLC) and showed that cross-linking of serum proteins by MOLD was increased in diabetes. In the present study, we describe the reaction of MGO with the model protein bovine pancreatic RNase and quantify the role of MGO in cross-linking of the protein. Using a liquid chromatography/mass spectrometry (LC/MS) assay with15N-labeled GOLD and MOLD internal standards, we also measure the levels of GOLD and MOLD in lens protein and show that the concentrations of both cross-links increase in concert with chronological age in lens protein and skin collagen. Quantitative analysis indicates that MOLD is the major chemically characterized cross-link formed in lens protein during the Maillard reaction. Unless otherwise indicated, all chemical reagents were of the highest quality obtainable from Sigma, including MGO (40% aqueous solution) and RNase A (type XII-A). (Carboxymethyl)trimethylammonium chloride hydrazide (Girard's Reagent T) was obtained from Aldrich. SupelClean LC-18 cartridges were obtained from Supelco (Bellefonte, PA), and C-18 preparative material was obtained from the Waters Corporation (Milford, MA).15 N 2-l-Lysine·HCl was obtained from Cambridge Isotope Laboratories Inc. (Andover, MA). For identification of products formed in reactions of MGO with the ε-amino groups of lysine, mixtures of 100 mmhippuryllysine and 100 or 200 mm MGO were incubated in 0.2m phosphate buffer at pH 7.4 and 37 °C. Aliquots were removed at various time points and analyzed by RP-HPLC. MOLD and GOLD were prepared by incubating 100 mm hippuryllysine with 200 mmMGO or GO in aqueous 200 mm formaldehyde. The reaction of MGO and hippuryllysine was conducted at 37 °C, and the reaction of GO and hippuryllysine was conducted at 65 °C. Additional MGO or GO (100 mm) and formaldehyde (100 mm) were added 8 times at hourly intervals and the reaction continued overnight. The pH dropped from 6 initially to 2.5 at the end of the reaction. The solutions were applied to a 0.75 × 10-cm column of C-18 preparative resin and eluted with a gradient of increasing concentrations of acetonitrile in water. Fractions were analyzed for absorbance at 228 nm, and those containing hippuryl-MOLD or -GOLD, detected by RP-HPLC, were pooled, dried, and finally cleaned by semi-preparative HPLC, using the analytical RP-HPLC system described below. To obtain free MOLD or GOLD, the hippuryl group was removed by hydrolysis in 6 n HCl for 4 h at 110 °C. For preparation of the heavy labeled cross-links15N4-MOLD and15N4-GOLD,15N2-l-Nα-formyllysine (100 mm), prepared from15N2-l-lysine as described by Hofmann et al. (30Hofmann K. Stutz E. Spühler G. Yajima H. Schwartz E.T. J. Am. Chem. Soc. 1960; 82: 3727-3732Crossref Scopus (48) Google Scholar), was incubated with MGO or GO (200 mm) and formaldehyde (200 mm) in deionized water. The reactions were conducted for 30 h as described above. Deformylated MOLD and GOLD were obtained by hydrolysis in 6n HCl for 2 h at 110 °C. The hydrolysate was dried by centrifugal evaporation (Speed-Vac, Savant Instruments, Holbrook, NY) and reconstituted in water, and brown products were removed by applying the product to a Supelclean-LC18 column (3 ml) in water. The yield determined by cation exchange chromatography was approximately 60% for 15N4-MOLD and 50% for15N4-GOLD. RNase (20 mg/ml, 1.5 mm) was incubated with MGO (15 or 30 mm MGO at molar ratios to lysine of 1:1 or 2:1, respectively) in 0.2m phosphate buffer, pH 7.4, at 37 °C. Aliquots were removed at various times and reduced with NaBH4 as described above. After quenching the reaction with acetic acid, the reduced protein was dialyzed against deionized water, concentrated by centrifugal evaporation, and hydrolyzed in 6 n HCl at 110 °C for 22 h. The hydrolyzed protein was dried and applied to a Supelclean LC-18 column (1 ml) to remove brown products. The dried protein, dissolved in 5 ml of deionized water, was applied to sulfopropyl-cation exchange gel (SP-Sephadex C-25; 1.2 ml). Neutral and acidic amino acids were eluted with 25 ml of 0.05 n HCl and basic amino acids, including MOLD, with 7 ml of 1 n HCl. For phenylisothiocyanate (PITC) derivatization, the sample was dried, dissolved in 30 μl of coupling buffer (water:ethanol:triethylamine, 2:2:1), and dried. PITC derivatization was conducted with 50 μl of a mixture of ethanol, water, triethylamine, and PITC (7:1:1:1) as described by Bidlingmeyer et al. (31Bidlingmeyer B.A. Cohen S.A. Tarvin T.L. J. Chromatogr. 1984; 336: 93-104Crossref PubMed Scopus (2105) Google Scholar). The derivatization mixture was evaporated by centrifugal evaporation, and the residue was dissolved in deionized water. Excess derivatization reagent was extracted with n-heptane, discarding the organic layer. The phenylthiocarbamoyl (PTC) amino acids were dissolved in solvent A for RP-HPLC. Histidine, which was unaltered during reaction of the protein with MGO and was recovered quantitatively during this procedure, was used as the internal standard for quantitation of Lys, Arg, and MOLD. Amino acid analysis was performed using a Pickering sodium cation exchange column (25 cm × 4.6 mm) and sodium buffers (Pickering Laboratories Inc., Mountain View, CA), as described by the manufacturers. Amino acids were quantified by post-column derivatization with o-phthalaldehyde and fluorescence detection (20Ahmed M.U. Thorpe S.R. Baynes J.W. J. Biol. Chem. 1986; 261: 4889-4894Abstract Full Text PDF PubMed Google Scholar, 32Watkins N.C. Thorpe S.R. Baynes J.W. J. Biol. Chem. 1985; 260: 10629-10636Abstract Full Text PDF PubMed Google Scholar). Hippuryl-amino acids were analyzed by RP-HPLC either on a 25 cm × 4.6 mm Zorbax SB C-18 HPLC column (MAC-MOD Analytical Inc., Chadds Ford, PA) or on a 15 cm × 3 mm-Waters C-18 Symmetry column (Waters Corporation) using a detection wavelength of 228 nm. The gradient consisted of solvent A (0.05% acetic acid, 0.05% formic acid, and 0.1% triethylamine in water) and solvent B (75% solvent A in acetonitrile). The gradient program for the Zorbax column was: 0–50 min, 0–5% solvent B; 50–160 min, 5–100% solvent B; 160–190 min, wash with 100% acetonitrile, at a flow rate of 1 ml/min; and for the Symmetry column: 0–30 min, 0–10% solvent B; 30–100 min, 10–100% solvent B; 100–105 min, wash with 100% acetonitrile, at a flow rate of 0.5 ml/min. PTC amino acids were analyzed on a 15 cm × 4.6 mm-inner diameter 218TP54 protein and peptide C-18 column (VYDAC/The Separations Group, Hesperia, CA) using a detection wavelength of 246 nm. The mobile phase consisted of solvent A (12.5 mm sodium phosphate, pH 6.2) and solvent B (30% solvent A and 70% acetonitrile). The gradient was programmed as follows: 0–2 min, 0% solvent B; 2–40 min, 0–50% solvent B; 40–50 min, 50–100% solvent B; this was followed by a 10-min washing step with 100% acetonitrile, flow rate 1.2 ml/min. MGO was measured using Girard's Reagent T in 0.5 m formic acid, pH 2.9, as described by Mitchel and Birnboim (33Mitchel R.E.J. Birnboim H.C. Anal. Biochem. 1977; 81: 47-56Crossref PubMed Scopus (64) Google Scholar), using absorbance at 292 nm for quantitation. SDS-PAGE was conducted under reducing conditions using a 4% stacking gel and a 15% separating gel as described by Laemmli (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Human lenses were obtained from the South Carolina Lions Eye Bank (Columbia, SC) and stored at −70 °C until used. Lenses were decapsulated, homogenized and dialyzed against deionized water, as described previously (26Dunn J.A. Patrick J.S. Thorpe S.R. Baynes J.W. Biochemistry. 1989; 28: 9454-9468Google Scholar, 35Patrick J.S. Thorpe S.R. Baynes J.W. J. Gerontol. 1990; 45: B18-B23Crossref PubMed Scopus (29) Google Scholar). Total lens protein was hydrolyzed in 6 n HCl at 110 °C for 24 h. The hydrolysate was dried and then applied to an SP-Sephadex cation exchange resin to recover basic amino acids, washed, and eluted, as described above. The eluent was dried by centrifugal evaporation and reconstituted in buffer A (sodium eluent, pH 3.15) for amino acid analysis. Fractions in the time interval including lysine, MOLD, and GOLD (15–25 min, 6 ml total volume) were collected. These pools were diluted with deionized water to 50 ml, acidified with 4 nHCl to pH 1–2, applied to a 2 ml-DOWEX-50W cation (sodium form), and desalted by washing with 70 ml of 0.5 n HCl; then the basic amino acids were eluted with 4 n HCl. The eluent was dried and derivatized for PTC amino acid analysis as described above. Human skin collagen was isolated by full thickness biopsy from the upper buttock, as described previously in detail (24Dyer D.G. Dunn J.A. Thorpe S.R. Bailie K.E. Lyons T.J. McCance D.R. Baynes J.W. J. Clin. Invest. 1993; 91: 2463-2469Crossref PubMed Scopus (631) Google Scholar). Briefly, the skin was scraped to remove adventitious tissue, extracted sequentially for 24 h with 1 m NaCl in 10 mm phosphate buffer, pH 7.4, and with 0.5 m acetic acid to remove soluble proteins, and then extracted with chloroform:methanol (2:1) to remove any residual lipid. The collagen was hydrolyzed and analyzed as described above for lens proteins. For LC/MS analysis about 2 mg of lens protein was mixed with 3 nmol of 15N4-MOLD and 2.5 nmol of15N4-GOLD or 4 mg of young (18 years) and old (85 years) pools of human skin collagen with 1.5 nmol of15N4-MOLD and 1.25 nmol of15N4-GOLD. The samples were hydrolyzed, processed, and derivatized with PITC, as described above. Gradient HPLC/MS analysis was performed using a 100 × 1-mm Hypersil ODS column (Keystone Scientific Inc., Bellefonte, PA) with a 2 × 1-mm guard column. The gradient consisted of solvent A (90% water, 10% methanol, and 0.3% glacial acetic acid) and solvent B (20% water, 80% methanol, and 0.3% glacial acetic acid). The gradient was: 100% solvent A for 5 min, then to 100% solvent B at 15 min, and hold at 100% solvent B for 20 min; the flow rate was 50 μl/min. The ion source was an Analytica of Branford Inc. electrospray model 103443 (Branford, CT), operating with the following settings: cylinder voltage, −2600 V; end cap voltage, −3700 V; capillary voltage, −4800 V; current, 4 × 10−8 A; source temperature, 275 °C; needlegas pressure, 38 p.s.i.; lens 1–6 voltages, 147.8, 25.6, 25.4, 2.7, 0, and −49.1 V. The mass spectrometer was a VG TRIO triple quadrupole mass analyzer (Beverly, MA) operating at: dwell time, 150 ms; delay time, 20 ms; and photomultiplier voltage, 600 V. The masses monitored were 417 amu for PTC-Lys+H+, 597 amu for (PTC)2-GOLD, 601 amu for (PTC)2-15N4-GOLD, 611 amu for (PTC)2-MOLD, and 615 amu for (PTC)2-15N4-MOLD. All assays were analyzed in a single batch to exclude interassay variation. Results of single analyses of each sample are shown. The intra-assay coefficients of variation for assay of GOLD and MOLD, measured at the mid-range of the samples, were 14 and 12%, respectively (n = 5). As reported previously, GOLD and MOLD were originally isolated from reactions of GO or MGO, respectively, with the model peptideNα-hippuryllysine (27Wells-Knecht K.J. Brinkmann E. Thorpe S.R. Baynes J.W. J. Org. Chem. 1995; 60: 6246-6247Crossref Scopus (92) Google Scholar, 28Brinkmann E. Wells-Knecht K.J. Thorpe S.R. Baynes J.W. J. Chem. Soc. Perkin Trans. I. 1995; 1: 2817-2818Crossref Google Scholar). RP-HPLC analysis of reactions of MGO with hippuryllysine (MGO:Lys, 2:1) yielded two major products (Fig. 2 A) subsequently identified asNα-hippuryl-CEL and (Nα-hippuryl)2-MOLD. Acid hydrolysis and amino acid analysis also yielded two major products, CEL and MOLD (Fig. 2 B). The identity of CEL was confirmed by its elution time on HPLC analysis and by GC/MS analysis of its trifluoroacetyl methyl ester derivative (M+ = 438 amu) (21Ahmed M.U. Brinkmann Frye E. Degenhardt T.P. Thorpe S.R. Baynes J.W. Biochem. J. 1997; 324: 565-570Crossref PubMed Scopus (525) Google Scholar). ESI mass spectrometry was used for identification of (Nα-hippuryl)2-MOLD and MOLD (M+ = 663 and 341 amu, respectively, before and after acid hydrolysis) (28Brinkmann E. Wells-Knecht K.J. Thorpe S.R. Baynes J.W. J. Chem. Soc. Perkin Trans. I. 1995; 1: 2817-2818Crossref Google Scholar). Yields were ∼12% CEL and ∼32% MOLD, based on original Nα-hippuryllysine. Similar results were obtained from reactions of GO withNα-hippuryllysine, yielding ∼33% CML and ∼32% GOLD (27Wells-Knecht K.J. Brinkmann E. Thorpe S.R. Baynes J.W. J. Org. Chem. 1995; 60: 6246-6247Crossref Scopus (92) Google Scholar) (data not shown). In numerous reactions of GO or MGO with Nα-hippuryllysine or proteins (RNase or albumin) at a variety of concentration ratios, both carboxyalkyllysines and imidazolium salt compounds were always formed together. Yields and relative yields varied with absolute concentrations and concentration ratios, as described for RNase in Fig. 3. Browning was more rapid and intense in reactions containing MGO, indicating a higher yield of melanoidins from MGO, compared with GO.Figure 3Detection of MOLD in MGO-modified RNase by RP-HPLC. RNase was incubated with MGO (MGO:Lys = 2:1) in 0.2m phosphate buffer, pH 7.4, for 24 h at 37 °C. Basic amino acids were isolated from the protein hydrolysate by chromatography on SP-Sephadex as described under “Experimental Procedures” and then derivatized with PITC for HPLC analysis. Theinset shows the enlarged view of the elution time frame of MOLD.View Large Image Figure ViewerDownload (PPT) Because MGO is present in higher concentrations in biological systems than GO (14Thornalley P.J. McLellan A.C. Lo T.W.C. Benn J. Soenksen P.H. Clin. Sci. ( Lond. ). 1996; 91: 575-582Crossref PubMed Scopus (125) Google Scholar), we concentrated on the reactions of MGO with protein. Reactions of MGO with the model protein RNase were studied under physiological conditions (pH 7.4, 37 °C). MGO was reacted with RNase, which has 10 lysine residues, at molar ratios of MGO:Lys, 1:1 and 2:1. As shown in Fig. 3, MOLD was readily detectable in MGO- modified RNase by RP-HPLC of the PTC derivative. The identity of the MOLD was confirmed by co-elution with an authentic standard and by its molecular mass of (PTC)2-MOLD (611 amu) measured by ESI-MS. The kinetics of the formation of MOLD described in Fig. 4 A were consistent with the kinetics of the disappearance of MGO from the reaction mixture (Fig. 4 B). The half-life of MGO in the presence of the protein was ∼4 h at both concentrations, whereas in phosphate buffer alone it disappeared with a half-life of ∼30 h (Fig. 4 B). The yield of MOLD increased 4–5-fold on doubling of the MGO concentration, an observation confirmed in two independent experiments, consistent with either a rate-limiting second order process or the requirement for 2 mol of MGO/mol of MOLD formed. In contrast, lysine and arginine decreased to similar extents at both MGO concentrations, yielding maximal modification of 5–6 of 10 lysine and 3 of 4 arginine residues in RNase (Fig. 4, C and D). The possibility that some free lysine and arginine were generated during acid hydrolysis cannot be excluded; however, the extent of the lack of reactivity of 3–4 lysine residues was confirmed by reaction with trinitrobenzenesulfonic acid (data not shown). Although ≥50% of lysine residues were modified in these reactions with formation of 20 or 95 mmol MOLD, only 1 or 3.6%, respectively, of the lysine loss could be accounted for as MOLD. CEL, measured by GC/MS (data not shown), accounted for an additional 1.3 or 1.6% of the total lysine modification (21Ahmed M.U. Brinkmann Frye E. Degenhardt T.P. Thorpe S.R. Baynes J.W. Biochem. J. 1997; 324: 565-570Crossref PubMed Scopus (525) Google Scholar). Thus, MOLD and CEL accounted for ≤5% of the modification of lysine residues in the protein. Browning, cross-linking, and formation of fluorescent products also occurred, indicating that other unidentified products were formed. As shown in Fig. 5, the time course of cross-linking of RNase, estimated by SDS-PAGE, was consistent with the rate of formation of MOLD (Fig. 4 A).Figure 5SDS-PAGE analysis of cross-linking of RNase during reaction with MGO. Lanes 1 and 9,molecular mass markers, cytochrome c (12,400 Da), carbonic anhydrase (29,000 Da), bovine serum albumin (66,000 Da), and alcohol dehydrogenase (150,000 Da); lane 2, native RNase;lanes 3–8, RNase, reacted with MGO (MGO:Lys, 1:1) at 0, 1, 2, 8, 48, and 96 h.View Large Image Figure ViewerDownload (PPT) MGO-modified RNase was fractionated into monomer, dimer, and polymer by chromatography on Sephadex G-75 (Fig. 6). Fractions were pooled, as indicated in the figure, and then analyzed for their MOLD content. As shown in the inset to Fig. 6, the MOLD content of RNase increased with the extent of polymerization of the protein. These data indicate that MOLD contributes to both inter- and intramolecular cross-linking of lysine residues in RNase; however, the yield of MOLD in the RNase dimer fraction (Fig. 6,inset) accounted for only about 5% of intermolecular cross-links, indicating that other cross-links were also formed. Human lens proteins were analyzed for their MOLD and GOLD content using the RP-HPLC analytical procedure described in Fig. 3. The lens proteins were analyzed as a function of age, because both CEL and CML accumulate in these proteins with age (21Ahmed M.U. Brinkmann Frye E. Degenhardt T.P. Thorpe S.R. Baynes J.W. Biochem. J. 1997; 324: 565-570Crossref PubMed Scopus (525) Google Scholar, 26Dunn J.A. Patrick J.S. Thorpe S.R. Baynes J.W. Biochemistry. 1989; 28: 9454-9468Google Scholar). As shown in Fig. 7, both MOLD and GOLD were detectable by RP-HPLC in the hydrolysate of lens protein. The peak identities were confirmed by co-elution with an authentic standard, but the reliability of the assay was questionable because of the low levels of MOLD and GOLD in lens proteins and their possible co-elution with other trace compounds in protein. To obtain more reliable results, we applied an LC/MS procedure to analyze lens protein hydrolysates that were derivatized with PITC. Heavy labeled MOLD and GOLD were prepared from 15N2-lysine for use as internal standards. Nonlabeled PTC2-MOLD and PTC2-GOLD were detected at the masses 611 and 597 amu, respectively, and their heavy labeled counterparts at 615 and 601 amu, respectively. Fig. 8 shows an RP-HPLC/SIM-ESI chromatogram of PITC-derivatized lens protein, confirming detection of both GOLD and MOLD in lens protein. The standard curves (inset) were prepared by adding increasing amounts of natural GOLD and MOLD to a fixed amount of internal standard.Figure 8Determination of MOLD and GOLD in human lens proteins by LC/MS. Heavy labeled standards were added to lens protein prior to acid hydrolysis. The protein was hydrolyzed, basic amino acids isolated, and amino acid derivatized with PITC, as described under “Experimental Procedures.” Using HPLC-ESI-MS, the masses recorded were 597 amu for GOLD, 601 amu for15N4-GOLD, 611 amu for MOLD, and 615 for15N4-MOLD. Standard curves were prepared by adding known amounts of GOLD and MOLD (597 and 611 amu) to constant amounts of internal standards (601 and 615 amu) (insets).View Large Image Figure ViewerDownload (PPT) Using internal standardization with heavy labeled MOLD and GOLD, these compounds were m
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