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• W2006297498 abstract "Methylglyoxal is a potent glycating agent under physiological conditions. Human serum albumin is modified by methylglyoxal in vivo. The glycation adducts formed and structural and functional changes induced by methylglyoxal modification have not been fully disclosed. Methylglyoxal reacted with human serum albumin under physiological conditions to form mainly the hydroimidazolone Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (92% of total modification) with a minor formation of argpyrimidine, Nϵ-(1-carboxyethyl)lysine, and methylglyoxal lysine dimer. When human serum albumin was modified minimally with methylglyoxal, tryptic peptide mapping indicated a hotspot of modification at Arg-410 located in drug-binding site II and the active site of albumin-associated esterase activity. Modification of Arg-410 by methylglyoxal was found in albumin glycated in vivo. Other sites of minor modification were: Arg-114, Arg-186, Arg-218, and Arg-428. Hydroimidazolone formation at Arg-410 inhibited ketoprofen binding and esterase activity; correspondingly, glycation in the presence of ketoprofen inhibited Arg-410 modification and loss of esterase activity. The pH dependence of esterase activity indicated a catalytic group with pKa = 7.9 ± 0.1, assigned to the catalytic base Tyr-411 with the conjugate base stabilized by interaction with the guanidinium group of Arg-410. Modification by methylglyoxal destabilized Tyr-411 and increased the pKa to 8.8 ± 0.1. Molecular dynamics and modeling studies indicated that hydroimidazolone formation caused structural distortion leading to disruption of arginine-directed hydrogen bonding and loss of electrostatic interactions. Methylglyoxal modification of critical arginine residues, therefore, whether experimental or physiological, is expected to disrupt protein-ligand interactions and inactivate enzyme activity by hydroimidazolone formation. Methylglyoxal is a potent glycating agent under physiological conditions. Human serum albumin is modified by methylglyoxal in vivo. The glycation adducts formed and structural and functional changes induced by methylglyoxal modification have not been fully disclosed. Methylglyoxal reacted with human serum albumin under physiological conditions to form mainly the hydroimidazolone Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (92% of total modification) with a minor formation of argpyrimidine, Nϵ-(1-carboxyethyl)lysine, and methylglyoxal lysine dimer. When human serum albumin was modified minimally with methylglyoxal, tryptic peptide mapping indicated a hotspot of modification at Arg-410 located in drug-binding site II and the active site of albumin-associated esterase activity. Modification of Arg-410 by methylglyoxal was found in albumin glycated in vivo. Other sites of minor modification were: Arg-114, Arg-186, Arg-218, and Arg-428. Hydroimidazolone formation at Arg-410 inhibited ketoprofen binding and esterase activity; correspondingly, glycation in the presence of ketoprofen inhibited Arg-410 modification and loss of esterase activity. The pH dependence of esterase activity indicated a catalytic group with pKa = 7.9 ± 0.1, assigned to the catalytic base Tyr-411 with the conjugate base stabilized by interaction with the guanidinium group of Arg-410. Modification by methylglyoxal destabilized Tyr-411 and increased the pKa to 8.8 ± 0.1. Molecular dynamics and modeling studies indicated that hydroimidazolone formation caused structural distortion leading to disruption of arginine-directed hydrogen bonding and loss of electrostatic interactions. Methylglyoxal modification of critical arginine residues, therefore, whether experimental or physiological, is expected to disrupt protein-ligand interactions and inactivate enzyme activity by hydroimidazolone formation. Methylglyoxal is an important glycating agent formed endogenously in physiological systems. It is formed by the spontaneous degradation of triosephosphates, oxidative metabolism of ketone bodies and catabolism of threonine (1Thornalley P.J. Mol. Aspects of Med. 1993; 14: 287-371Crossref PubMed Scopus (453) Google Scholar). Methylglyoxal reacts with proteins to form advanced glycation end products (AGEs). 1The abbreviations used are: AGEs, advanced glycation end products; HPLC, high performance liquid chromatography; HSA, human serum albumin; IPG, immobilized pH gradient; LC-MS/MS, liquid chromatography with triple quadrupole mass spectrometric detection; MGmin-HSA, human serum albumin minimally modified by methylglyoxal; MG-H1, Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine.1The abbreviations used are: AGEs, advanced glycation end products; HPLC, high performance liquid chromatography; HSA, human serum albumin; IPG, immobilized pH gradient; LC-MS/MS, liquid chromatography with triple quadrupole mass spectrometric detection; MGmin-HSA, human serum albumin minimally modified by methylglyoxal; MG-H1, Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine. Cellular and extracellular proteins show minimal extents of modification by methylglyoxal but significant concentrations of methylglyoxal-derived AGEs, approaching the concentrations of the early glycation adduct, fructosyl-lysine, in some cases (2Ahmed N. Thornalley P.J. Dawczynski J. Franke S. Strobel J. Stein G. Haik Jr., G.M. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5287-5292Crossref PubMed Scopus (216) Google Scholar, 3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar, 4Paul R.G. Avery N.C. Slatter D.A. Sims T.J. Bailey A.J. Biochem. J. 1998; 330: 1241-1248Crossref PubMed Scopus (49) Google Scholar). The major AGE quantitatively is the hydroimidazolone derived from arginine residues, Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1). There are two other related hydroimidazolone structural isomers, but MG-H1 dominates in proteins glycated under physiological conditions (2Ahmed N. Thornalley P.J. Dawczynski J. Franke S. Strobel J. Stein G. Haik Jr., G.M. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5287-5292Crossref PubMed Scopus (216) Google Scholar, 3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar, 5Henle T. Walter A. Hae βner R. Klostermeryer H. Z. Lebensm. Unters. Forsch. 1994; 199: 55-58Crossref Scopus (106) Google Scholar, 6Ahmed N. Argirov O.K. Minhas H.S. Cordeiro C.A. Thornalley P.J. Biochem. J. 2002; 364: 1-14Crossref PubMed Scopus (295) Google Scholar). Other minor methylglyoxal-derived AGEs are a tetrahydropyrimidine (Nδ-[5-(2,3,4-trihydroxybutyl)-5-hydro-4-imidazolon-2-yl]ornithine), argpyrimidine, Nϵ-carboxyethyllysine, and methylglyoxal-derived lysine dimer (1,3-di(Nϵ-lysino)-4-methyl-imidazolium salt) (reviewed in Ref. 6Ahmed N. Argirov O.K. Minhas H.S. Cordeiro C.A. Thornalley P.J. Biochem. J. 2002; 364: 1-14Crossref PubMed Scopus (295) Google Scholar).Glycation by methylglyoxal, unlike glycation by glucose, is mainly arginine-directed and produces a loss of positive charge by hydroimidazolone formation (7Ahmed N. Thornalley P.J. Biochem.J. 2002; 364: 15-24Crossref PubMed Scopus (142) Google Scholar). An important target of glycation in vivo is human serum albumin (HSA), the major protein of human blood plasma. The sites and functional effects of formation of the major glucose-derived glycation adduct fructosyl-lysine have been determined (8Garlick R.L. Mazer J.S. J. Biol. Chem. 1983; 258: 6142-6146Abstract Full Text PDF PubMed Google Scholar). In contrast, the sites of glycation of HSA by methylglyoxal have not, and neither have structural and functional changes induced by methylglyoxal modification been characterized. Related to this, however, glyoxal derivatives have been used to interrogate active sites and functional domains of proteins for critical arginine residues, exploiting the mainly arginine-directed modification by these agents (9Takahashi K. J. Biol. Chem. 1968; 243: 6171-6179Abstract Full Text PDF PubMed Google Scholar).In this report, we describe the sites of modification of HSA by methylglyoxal under physiological conditions and show that similar glycation occurs in vivo. The associated functional effects, inhibition of ligand binding and esterase activity, were characterized, and structural changes were predicted to explain the consequences of methylglyoxal modification for albumin functionality.EXPERIMENTAL PROCEDURESMaterials—HSA (fatty acid-free) was purchased from Sigma. Methylglyoxal, Nϵ-(1-deoxy-d-fructos-1-yl)lysine (referred to as fructosyl-lysine) and AGE authentic standards, including stable isotope-substituted analogues, were prepared as described (6Ahmed N. Argirov O.K. Minhas H.S. Cordeiro C.A. Thornalley P.J. Biochem. J. 2002; 364: 1-14Crossref PubMed Scopus (295) Google Scholar). High purity methylglyoxal was prepared and purified as described (10McLellan A.C. Thornalley P.J. Anal. Chim. Acta. 1992; 263: 137-142Crossref Scopus (81) Google Scholar).Preparation of Human Serum Albumin Glycated by Methylglyoxal— HSA glycated minimally by methylglyoxal-derived glycation adducts (MGmin-HSA) was prepared by incubation of methylglyoxal (500 μm) with HSA (6.6 mg/ml) in sodium phosphate buffer (100 mm, pH 7.4) at 37 °C for 24 h. A similar preparation was made of control protein of HSA incubated without methylglyoxal. Further preparations were made with and without methylglyoxal but preincubated for 30 min and throughout the glycation period with 120 μm ketoprofen (to block drug-binding site II). The glycated and control HSA preparations were dialyzed against 30 mm ammonium bicarbonate at 4 °C, lyophilized to dryness, and stored at -20 °C. Validation studies showed that lyophilization did not change the AGE content of these proteins significantly.Protein Glycation Adduct Determination by LC-MS/MS and Tryptic Digestion and Peptide Mapping—Protein glycation adduct residues were determined in exhaustive enzymatic digests (50 μg of protein equivalent) by LC-MS/MS and stable isotope-substituted standard internal standardization, as described (3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar). MGmin-HSA and HSA control (100 μg) were diluted to 20 μl with water. Aliquots of 40 mm HCl (25 μl), pepsin solution (2 mg/ml in 20 mm HCl; 5 μl), and thymol solution (2 mg/ml in 20 mm HCl; 5 μl) were added, and the sample was incubated at 37 °C for 24 h. The sample was then neutralized and buffered at pH 7.4 by the addition of 25 μlof0.5 m potassium phosphate buffer, pH 7.4, and 5 μl of 260 mm KOH. Pronase E solution (2 mg/ml in 10 mm potassium phosphate buffer, pH 7.4; 5 μl) was added, and the sample was incubated at 37 °C for 24 h under nitrogen. Aminopeptidase solution (2 mg/ml in 10 mm potassium phosphate buffer, pH 7.4; 5 μl) and prolidase solution (2 mg/ml in 10 mm potassium phosphate buffer, pH 7.4; 5 μl) were added, and the sample was incubated at 37 °C for 48 h under nitrogen (6Ahmed N. Argirov O.K. Minhas H.S. Cordeiro C.A. Thornalley P.J. Biochem. J. 2002; 364: 1-14Crossref PubMed Scopus (295) Google Scholar). This gave the final enzymatic hydrolysate (100 μl) for the AGE assay. AGE and amino acid recoveries were 87–100%; fructosyl-lysine recovery was 71% (3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar). Analytes released by self-digestion of proteases in assay blanks were subtracted from analyte estimates. The samples were assayed for glycation adducts by LC-MS/MS using a 2695 Separation module with a Quattro Ultima triple quadrupole mass spectrometric detector (Waters-Micromass, Manchester, UK). Interbatch coefficients were 2–10% (3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar).For peptide mapping, aqueous albumin samples (0.67 mg/ml) were reduced by the addition of dithiothreitol (4 mg/ml, 10 μl) and incubation at 37 °C in the dark for 30 min. Cysteinyl thiols were then blocked by the addition of iodoacetamide (9 mg/ml, 10 μl), and the sample was incubated at 37 °C in the dark for a further 30 min. Residual iodoacetamide was quenched by the addition of a further aliquot (10 μl) of dithiothreitol (4 mg/ml) and incubated at 37 °C in the dark for 30 min. l-1-Chloro-3-[4-tosylamido]-4-phenyl-2-butanone-treated trypsin (enzyme-to-substrate ratio, 1:50, w/w) in 1 mm CaCl2, 50 mm NH4HCO3, pH 8.5, and 37 °C, was added and incubated for 10 h. The digest was lyophilized and reconstituted in water. Tryptic digests of HSA derivatives were analyzed by LC-MS on the Waters-Micromass Quattro Ultima system in single mass analyzer mode. The column was a Symmetry™ ODS (2.1 × 100 mm; particle size, 100 Å) with a 10 × 2.1-mm guard column (Waters, Watford, UK). The flow rate was 0.2 ml/min. The eluent was 0.05% trifluoroacetic acid in water with a linear gradient of 0–10% methanol from 0 to 30 min. The eluate was monitored by absorbance in the range 200–400 nm and by positive ion electrospray mass spectrometry for parent ion masses in the range 100–2000 Da. Cumulative mass spectra were analyzed for tryptic peptide parent ions against a theoretical peptide map of an HSA tryptic digest using the Biolynx software and single ion response chromatograms generated from accumulated data. Peptide ion responses were normalized to that of the C-terminal (unmodified) peptide; coefficients of variance for normalized peptide responses were 1–8% (n = 3). Loss of specific peptides in MGmin-HSA was determined by comparing the mean of three normalized peptide responses in peptide digests modified and unmodified.Similar tryptic digests were prepared of plasma protein from human subjects. Plasma protein (0.33 mg) was washed with water on a 10-kDa cut-off ultrafiltration microspin filter, reduced, alkylated, and digested with trypsin as described above. Aliquots (30 ng of protein digest, 0.5 μl) were then analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry using a Bruker Daltonics Reflex IV mass spectrometer with a sample matrix containing α-cyano-4-hydroxycinnamic acid (5 mg/ml).Two-dimensional Electrophoresis and Circular Dichroism Spectra— Isoelectric focusing separation was done using precast immobilized pH gradient (IPG) gel strips (pH 5.3–6.5; strip length, 18 cm; Amersham Biosciences). The IPG strips were rehydrated overnight in trays loaded with the protein samples according to the manufacturer's instructions. Isoelectric focusing was performed using an Amersham Biosciences Ettan IPGphor isoelectric focusing cell unit at 20 °C with a maximum current of 50 μA/strip. The isoelectric focusing run involved a programmed voltage gradient of 500–8,000 V for a total 54 kVh. The IPG strips were equilibrated for 15 min in 20 mm dithiothreitol and for 5 min in 1 mm iodoacetamide solution. Following SDS-PAGE on a 1-mm-thick 4-20% gradient gel, albumin spots were stained with Coomassie Blue, destained for visualization, and then documented (11Turner M. Chantry D. Feldmann M. Biochem. Biophys. Res. Commun. 1988; 156: 830-839Crossref PubMed Scopus (17) Google Scholar).CD spectra of HSA and MGmin-HSA were recorded in 10 mm sodium phosphate buffer, pH 7.0, and room temperature, on a Jasco J720 spectropolarimeter. The instrument was flushed with nitrogen gas for a better performance below 200 nm. A rectangular 10-mm-path length cell and a cylindrical 0.2-mm-path length cell were employed in the regions of 360–230 and 260–185 nm, respectively. All of the spectra were solvent base line-corrected, and the concentration of the control HSA and MGmin-HSA was deduced assuming ϵ279 nm = 0.531 mg/ml (12Peters T. All about Albumin. Academic Press, New York1996: 25Google Scholar). The spectra were deconvoluted using a principle component regression analysis method to deduce the proportion of α-helix, β-sheet, and random coil in the secondary structure of the proteins (13Arvinte T. Bui T.T.T. Dahab A.A. Demeule B. Drake A.F. Elhag D. King P. Anal. Biochem. 2004; 332: 46-57Crossref PubMed Scopus (30) Google Scholar, 14Perzcel A. Park K. Fasman G.D. Anal. Biochem. 1992; 203: 83-93Crossref PubMed Scopus (420) Google Scholar).Drug Binding Studies and Esterase Activity Studies—The binding of ketoprofen to binding site II, domain 3A in HSA, and MGmin-HSA was studied. Ketoprofen (0.1–2 μm) and albumin derivative (10 μm) were incubated in 67 mm sodium phosphate buffer, pH 7.4, and 37 °C for 30 min. The unbound ligand fractions were separated by ultrafiltration centrifugation (20,000 × g at 4 °C for 5 min) using a 12-kDa cut-off membrane. Adsorption of ketoprofen on to the filtration membrane and apparatus were negligible. The concentration of unbound ligand was determined by HPLC. The HPLC system consisted of a Waters 717 plus autosampler (with samples maintained at 4 °C), Waters 600 quaternary pump, and Waters 481 Lambda Max absorbance detector. Columns for reversed phase HPLC were a 3.9 × 150-mm NOVAPAK™ ODS (4 μm) fitted with a 3.9 × 20-mm NOVAPAK™ ODS Sentry guard column. The mobile phase was 10 mm sodium phosphate buffer with 50% (v/v) methanol, pH 7.4. Ketoprofen was detected at 257 nm.Esterase activities of MGmin-HSA and HSA were determined with the synthetic substrate p-nitrophenylacetate following the hydrolysis to p-nitrophenol and acetate. The reaction was followed by monitoring the appearance of p-nitrophenol, detected spectrophotometrically at 400 nm. The reaction mixtures contained 50 μmp-nitrophenylacetate and 20 μm protein in 50 mm sodium phosphate buffer, pH 7.4, and 37 °C. The pH dependence of esterase activity was studied in the pH range 6.4–9.4 using phosphate buffer in the pH range 6.4–7.8 and pyrophosphate buffer in the pH range 8.4–9.4. Extinction coefficients for p-nitrophenol were determined at each pH value studied, and esterase activity was deduced from the initial rates of absorbance (dA400/dt)o.Molecular Dynamics, Arginine Residue Surface Exposure, and Surface Charge Density—The AMBER 7.0 force field (15Case D.A. Pearlman J.W. Caldwell J.W. Cheatham T.E. Wang III, J. Ross W.S. Simmerling C.L. Darden T.A. Merz K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Tsui V. Gohlke H. Radmer R.J. Duan Y. Pitera J. Massova I. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER 7 computer program. University of California, San Francisco2002Google Scholar) was used to simulate the effect of glycation on HSA. The crystal structure of native HSA (Protein Data Bank identification number 1ao6) at 2.5 Å (16Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1470) Google Scholar) was obtained from the Research Collaboratory for Structural Bioinformatics data base. Arg-410 was replaced with the hydroimidazolone MG-H1 residue. The native and modified protein structures were equilibrated using the Generalized Born solvation model (17Bashford D. Case D.A. Annu. Rev. Phys. Chem. 2000; 51: 129-152Crossref PubMed Scopus (963) Google Scholar) and then subjected to 100 ps of molecular dynamics using the AMBER force field until the total energy of each system reached equilibration. Final energy minimization of each protein was carried out within AMBER. Energy minimizations of modifications and molecular graphics were performed using DS Viewer Pro 5.0 (Accelrys, San Diego, CA). The electrostatic potential was calculated using the Poisson-Boltzmann (18Davis M.E. Madura J.D. Sines J. Luty B.A. Allison S.A. McCammon J.A. Methods Enzymol. 1991; 202: 473-497Crossref PubMed Scopus (62) Google Scholar) method as implemented in the University of Houston Brownian Dynamics (19Briggs J.M. Davis M.E. Desai B.H. Gilson M.K. Ilin A. Luty B.A. McCammon J.A. Madura J.D. Tan R.C. Wade R.C. Brownian Dynamics computer program (versions 4.1 and 5.1). University of Houston, Houston1989Google Scholar) program. The potential was displayed on the surface of the protein using Gopenmol (20Bergman D.L. Laaksonen L. Laaksonen A. J. Mol. Graph. Modelling. 1997; 15: 301-312Crossref PubMed Scopus (417) Google Scholar). The surface exposure of each of the Arg residues within the structure of HSA was calculated using the NACCESS software (21Hubbard S.J. Thornton J.M. NACCESS computer program. University College, London, UK1993Google Scholar) by rolling a probe of a defined radius (water molecule) over the surface of the protein to determine residue accessibility.RESULTSModification and Glycation Adduct Quantitation by LC-MS/MS—We and other investigators have previously prepared and studied the molecular characteristics of human and bovine serum albumins modified to minimal and high extents by methylglyoxal (22Westwood M.E. Thornalley P.J. J. Protein Chem. 1995; 14: 359-372Crossref PubMed Scopus (195) Google Scholar, 23Fan X. Subramaniam R. Weiss M.F. Monnier V.M. Arch. Biochem. Biophys. 2003; 409: 274-286Crossref PubMed Scopus (75) Google Scholar, 24Lee C. Yim M.B. Chock P.B. Yim H.S. Kang S.O. J. Biol. Chem. 1998; 273: 25272-25278Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We examined the presence of glycation adducts by quantifying the loss of lysine and arginine residues by chromophoric and fluorimetric lysine- and arginine-specific reagents and amino acid analysis with acid hydrolysis (22Westwood M.E. Thornalley P.J. J. Protein Chem. 1995; 14: 359-372Crossref PubMed Scopus (195) Google Scholar). This showed convincingly that arginine residues were lost preferentially by glycation with methylglyoxal. With recent advances in the application of LC-MS/MS to the detection of protein glycation adducts (3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar), we are now able to quantify methylglyoxal-derived glycation adducts in such modified proteins. Studies of the concentrations of methylglyoxal-derived glycation adducts in human plasma proteins and molecular mass measurements of human serum albumin in vivo indicated that HSA is also modified minimally by methylglyoxal-derived glycation adducts in vivo (3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar, 25Thornalley P.J. Argirova M. Ahmed N. Mann V.M. Argirov O.K. Dawnay A. Kidney Int. 2000; 58: 2228-2234Abstract Full Text Full Text PDF PubMed Google Scholar, 26Ahmed N. Thornalley P.J. Luthen R. Haussinger D. Sebekova K. Schinzel R. Voelker W. Heidland A. J. Hepatol. 2004; 41: 913-919Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Therefore, to quantify the glycation adducts formed and identify sites in HSA reactive toward methylglyoxal under physiological conditions in a physiologically relevant model glycated protein, we prepared HSA modified minimally by methylglyoxal (MGmin-HSA). The type and amount of glycation adducts in MGmin-HSA were assessed by quantitative glycation adduct screening with LC-MS/MS (Table I). Estimates of other glycation adducts, oxidation, and nitration adducts in HSA that were not changed significantly by glycation with methylglyoxal were (mmol/mol protein; mean ± S.D., n = 4): Nϵ-fructosyl-lysine, 10.0 ± 0.9; Nϵ-carboxymethyl-lysine, 5.7 ± 0.8; glyoxal-derived hydroimidazolone Nδ-(5-hydro-4-imidazolon-2-yl)ornithine, <0.5; 3-deoxyglucosone-derived hydroimidazolone Nδ-(5-hydro-5-(2,3,4-trihydroxybutyl)-4-imidazolon-2-yl)ornithine and related structural isomers, 7.8 ± 2.8; pentosidine, 0.073 ± 0.013; methionine sulfoxide, 124 ± 11; and 3-nitrotyrosine, 0.22 ± 0.05. The main methylglyoxal-derived glycation adduct in MGmin-HSA was the hydroimidazolone MG-H1, found at ∼2.5 molar equivalents and representing 91% of total methylglyoxal-derived glycation adducts. The LC-MS/MS detection of MG-H1 showed the expected partial resolution of two epimers of the hydroimidazolone (3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar) (Fig. 1, a and b). Minor methylglyoxal-derived adducts were: argpyrimidine, 7%; Nϵ-carboxyethyl-lysine, 1%; and methylglyoxal-derived lysine dimer, <1% of total methylglyoxal glycation adducts. The total methylglyoxal-derived adducts in MGmin-HSA, deduced by preparation of MGmin-HSA with radiolabeled [2-14C]methylglyoxal (27Westwood M.E. Argirov O.K. Abordo E.A. Thornalley P.J. Biochim. Biophys. Acta. 1997; 1356: 84-94Crossref PubMed Scopus (91) Google Scholar), was 2.45 ± 0.30 mol/mol protein, indicating that all of the methylglyoxal-derived adducts were detected. High resolution isoelectric focusing and SDS-PAGE of MGmin-HSA showed a small decrease in pI with respect to HSA (5.62 versus 5.76) and no evidence of significant intermolecular cross-linking (Fig. 2), consistent with loss of arginine residue positive charge by formation of MG-H1 residues.Table IGlycation adducts in human serum albumin modified minimally by methylglyoxalGlycation adductHSAMGmin-HSAmmol/molmmol/molMG-H141 ± 22493 ± 87Argpyrimidine0.08 ± 0.04200 ± 40Nϵ-Carboxyethyl-lysine4.0 ± 0.429.7 ± 1.8Methylglyoxal-derived lysine dimeraThis value was below the limit of detection (0.04)5 ± 1a This value was below the limit of detection (0.04) Open table in a new tab Fig. 2Two-dimensional gel electrophoresis of human serum albumin modified minimally by methylglyoxal.a, HSA (pI = 5.76) and b, MGmin-HSA (pI = 5.62). c, section of a matrix-assisted laser desorption ionization time-of-flight mass spectrum of a tryptic digest in protein of human plasma glycated in vivo. The arrow indicates the peak assigned to peptide FQNALLVRMG-H1YTK (predicted mass, 1406.9 Da). d, percentage surface exposure of arginine residues in HSA (see “Experimental Procedures”).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Location of the Hydroimidazolone MG-H1 by Peptide Mapping, Molecular Dynamics and Modeling, Surface Arginine Exposure, and Charge and Secondary Structure—The locations of MG-H1 residues in MGmin-HSA were identified by tryptic peptide mapping by cationic electrospray LC-MS. Tryptic peptide responses were normalized to the response of the C-terminal peptide. Loss of peptide (by methylglyoxal modification) was deduced by comparison of normalized peptide responses in MGmin-HSA to that of control HSA in triplicate. The normalized peptide ion response gave reproducible detection when singly charged peptide responses were quantified; doubly and triply charged peptide ions suffered variable ion quenching and were not used in deducing peptide detection responses. The summation of all peptide responses showing statistically significant decrease in MGmin-HSA, with respect to HSA, gave a total molar equivalent peptide loss of 2.06. This loss is assumed to be due to modification of peptides by MG-H1. The total modification by methylglyoxal in MGmin-HSA was ∼2.5 molar equivalents; the residual ∼0.5 molar equivalents of modified peptide difference between the total and that located in tryptic peptides is accounted for by hydrolysis of MG-H1 during the tryptic digestion (expect loss of 26% of the total modification or 0.6 molar equivalents) (6Ahmed N. Argirov O.K. Minhas H.S. Cordeiro C.A. Thornalley P.J. Biochem. J. 2002; 364: 1-14Crossref PubMed Scopus (295) Google Scholar). The individual peptides with decreased detection in MGmin-HSA are given in Table II. They represent modification of arginine residues (mol % modified) at Arg-114 (36%), Arg-186 (25%), Arg-218 (31%), Arg-410 (89%), and Arg-428 (25%). For the detection of T31213–218, there was significant interference from a dipeptide T67–68520–524. Peptide T31 contained a chromophoric tryptophan residue, the only tryptophan residue in HSA, whereas T67–68 did not. Hence, absorbance detection at 286 nm was used to quantify the loss of T31. The peptide containing the Arg-410 residue, T52, showed the most decreased response in MGmin-HSA and hence had the highest modification. Formation of MG-H1 residues in MGmin-HSA prevented trypsin cleavage at that site such that corresponding uncleaved tryptic dipeptides were detected. MG-H1-containing dipeptides in tryptic digests of MGmin-HSA were detected for Arg-186, Arg-218, Arg-410, and Arg-428 but not for Arg-114. Hence for the hotspot modification of Arg-410, the modified dipeptide T52–53 (FQNALLVRMG-H1YTK) was detected in digests of MGmin-HSA but not of HSA (Fig. 1, c and d). Tryptic digests of plasma protein glycated in vivo also contained the MG-H1-modified peptide detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (predicted FQNALLVRMG-H1YTK 1406.9; found peptide mass 1406.8 Da; Fig. 2c). This suggests that Arg-410 is a hotspot for albumin modification by methylglyoxal experimentally and may also be so physiologically too (Fig. 1, e and f). MG-H1 residues have been detected and quantified by LC-MS/MS in plasma protein in vivo where increases were found in diabetes and uremia (3Thornalley P.J. Battah S. Ahmed N. Karachalias N. Agalou S. Babaei-Jadidi R. Dawnay A. Biochem. J. 2003; 375: 581-592Crossref PubMed Scopus (557) Google Scholar, 28Ahmed N. Thornalley P.J. Howell S. Beisswenger P.J. Diabetes. 2003; 52: A441Google Scholar).Table IITryptic peptides containing arginine residues converted to MG-H1 residues in human serum albumin modified minimally by methy" @default.
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• W2006297498 title "Peptide Mapping Identifies Hotspot Site of Modification in Human Serum Albumin by Methylglyoxal Involved in Ligand Binding and Esterase Activity" @default.
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