Methylglyoxal Modification of Protein

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation ofN α-acetyl-l-arginine with MG at 37 °C, two nonfluorescent products and one fluorescent product were detected as the major products. The nonfluorescent products were identified as theN δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-l-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetrahydropyrimidine moiety (N δ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-l-ornithine).On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified asN δ-(5-methylimidazolon-2-yl)-l-ornithine (5-methylimidazolone), was found to be identical toN δ-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-l-ornithine (argpyrimidine): (i) the low and high resolution fast atom bombardment-mass spectrometry gave a molecular ion peak atm/z of 297 (M+H) and a molecular formula of C10H25O6N4, respectively, which coincided with argpyrimidine; (ii) the1H NMR spectrum of this product indMe2SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 °C for 14 days scarcely generated 5-methylimidazolone.On the other hand, as an immunochemical approach to the detection of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major fluorescent product, argpyrimidine, as the dominant epitope. The immunohistochemical analysis of the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by reperfusion. These results suggest that argpyrimidine may contribute to the progression of not only long term diabetic complications, such as nephropathy and atherosclerosis, but also the tissue injury caused by ischemia/reperfusion. Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation ofN α-acetyl-l-arginine with MG at 37 °C, two nonfluorescent products and one fluorescent product were detected as the major products. The nonfluorescent products were identified as theN δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-l-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetrahydropyrimidine moiety (N δ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-l-ornithine).On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified asN δ-(5-methylimidazolon-2-yl)-l-ornithine (5-methylimidazolone), was found to be identical toN δ-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-l-ornithine (argpyrimidine): (i) the low and high resolution fast atom bombardment-mass spectrometry gave a molecular ion peak atm/z of 297 (M+H) and a molecular formula of C10H25O6N4, respectively, which coincided with argpyrimidine; (ii) the1H NMR spectrum of this product indMe2SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 °C for 14 days scarcely generated 5-methylimidazolone. On the other hand, as an immunochemical approach to the detection of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major fluorescent product, argpyrimidine, as the dominant epitope. The immunohistochemical analysis of the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by reperfusion. These results suggest that argpyrimidine may contribute to the progression of not only long term diabetic complications, such as nephropathy and atherosclerosis, but also the tissue injury caused by ischemia/reperfusion. Nonenzymatic glycation (Maillard reaction) is a complex series of reactions between reducing sugars and amino groups of proteins, which leads to browning, fluorescence, and cross-linking of the proteins. The reaction is initiated with the reversible formation of a Schiff's base, which undergoes a rearrangement to form a relatively stable Amadori product. The Amadori product further undergoes a series of reactions through dicarbonyl intermediates to form advanced glycation end products (AGEs) 1The abbreviations used are: AGE, advanced glycation end product; MG, methylglyoxal; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; FAB-MS, fast atom bombardment-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; CEL, N ε-carboxyethyllysine; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.1The abbreviations used are: AGE, advanced glycation end product; MG, methylglyoxal; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; FAB-MS, fast atom bombardment-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; CEL, N ε-carboxyethyllysine; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.(1Brownlee M. Cerami A. Vlassara H. N. Engl. J. Med. 1988; 318: 1315-1321Crossref PubMed Scopus (2324) Google Scholar). It has been shown that the formation of AGEs in vivocontributes to the pathophysiologies associated with aging and the long term complications of diabetes (2Baynes J.W. Monnier V.M. Prog. Clin. Biol. Res. 1989; 304: 1-410Google Scholar).A number of aldehydes and ketones, in addition to sugars, are known to form AGEs. Methylglyoxal (MG), among them, has recently received considerable attention as a mediator to form AGEs. MG is known to be formed nonenzymically by amine-catalyzed sugar fragmentation reactions (3Hayashi T. Mase S. Namiki M. Agric. Biol. Chem. 1986; 50: 1959-1964Crossref Google Scholar, 4Hayashi T. Namiki M. Agric. Biol. Chem. 1986; 50: 1965-1970Google Scholar, 5Richard J.P. Biochemistry. 1991; 30: 4581-4585Crossref PubMed Scopus (186) Google Scholar) and by spontaneous decomposition of triose phosphate intermediates in glycolysis (5Richard J.P. Biochemistry. 1991; 30: 4581-4585Crossref PubMed Scopus (186) Google Scholar). It is also a product of the metabolism of acetol, an intermediate in the catabolism of both threonine (6Ray M. Ray S. J. Biol. Chem. 1987; 262: 5974-5977Abstract Full Text PDF PubMed Google Scholar) and the ketone body acetone (7Reichard G.A. Skutches C.L. Hoeldtke R.D. Owen O.E. Diabetes. 1986; 35: 668-674Crossref PubMed Google Scholar). A recent study on the formation of AGEs in endothelial cells cultured under hyperglycemic conditions indicated that MG was the major precursor of AGEs (8Shinohara M. Giardino I. Brownlee M. Diabetes. 1996; 45: 126ACrossref Google Scholar). Chaplen et al.(9Chaplen F.W.R. Fahl W.E. Cameron D.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5533-5538Crossref PubMed Scopus (155) Google Scholar) have shown that high levels of MG are indeed present in cultured Chinese hamster ovary cells. In addition, increased levels of MG are also found in blood from diabetic patients and in the lens of streptozotosin-induced diabetic rats (10Phillips S.A. Mirrlees D. Thonalley P.J. Biochem. Pharmacol. 1993; 46: 805-811Crossref PubMed Scopus (136) Google Scholar, 11McLellan A.C. Thornalley P.J. Benn J. Sonksen P.H. Clin. Sci. 1994; 87: 21-29Crossref PubMed Scopus (491) Google Scholar). Che et al.(12Che W. Asahi M. Takahashi M. Kaneto H.A. Okado A. Higashiyama S. Taniguchi N. J. Biol. Chem. 1997; 272: 18453-18459Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) have reported that MG induces gene expression of heparin-binding epidermal growth factor-like growth factor by provoking oxidative stress. It is also known that MG is the physiological substrate of the glyoxalase system, which catalyzes the conversion of MG tod-lactate via the intermediateS-d-lactoylgluthathione (13Thornalley P.J. Biochem. J. 1990; 269: 1-11Crossref PubMed Scopus (679) Google Scholar).MG irreversibly modifies proteins under physiological conditions (14Lo 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). The reactions proceed even at physiological concentrations of MG (14Lo 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) and form fluorescent products, characteristics of which resemble those occurring in proteins in aging and diabetes (15Vander Jagt D.L. Robinson B. Taylor K.T. Hunsaker L.A. J. Biol. Chem. 1992; 267: 4364-4369Abstract Full Text PDF PubMed Google Scholar). It has been reported that MG binds and modifies a number of proteins, including bovine serum albumin (15Vander Jagt D.L. Robinson B. Taylor K.T. Hunsaker L.A. J. Biol. 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MG modification of protein may be closely associated with cellular toxicity. It has been shown that MG selectively inhibits mitochondrial respiration and glycolysis in the cells (23Biswas S. Ray M. Misra S. Dutta D.P. Ray S. Biochem. J. 1997; 323: 343-348Crossref PubMed Scopus (99) Google Scholar). In relation to this, MG has been shown to inactivate membrane ATPases and glyceraldehyde-3-phosphate dehydrogenase, a key enzyme of the glycolytic pathway (24Halder J. Ray M. Ray S. Int. J. Cancer. 1993; 54: 443-449Crossref PubMed Scopus (60) Google Scholar). The high reactivity of MG with proteins and its relatively high concentration in the plasma (25Monnier V.M. Sell D.R. Miyata S. Nagaraj R.H. Finot P.A. Aeschbacher H.U. Hurrel R.F. Liardon R. The Maillard Reaction in Food Processing, Human Nutrition, and Physiology. Birkhaeuser Verlag, Basel1990: 393Google Scholar) suggest that MG represents a common intermediate in the formation of AGEs in vivo. This assumption may be supported by the fact that macrophages have a specific receptor for proteins modified by reaction with MG, as well as for glucose-derived AGEs (18Westwood M.E. MacLellan A.C. Selwood T. Thornalley P.J. J. Biol. Chem. 1994; 269: 32293-32298Abstract Full Text PDF PubMed Google Scholar). It has been reported that MG primarily reacts with arginine residues to formN δ-(5-methyl-4-imidazolon-2-yl)-l-ornithine (5-methylimidazolone) andN δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-l-ornithine (5-hydro-5-methylimidazolone) (26Westwood M.E. Thornalley P.J. J. Protein Chem. 1995; 14: 359-372Crossref PubMed Scopus (195) Google Scholar). Shipanova et al.(27Shipanova I.N. Glomb M.A. Nagaraj R.H. Arch. Biochem. Biophys. 1997; 344: 29-36Crossref PubMed Scopus (242) Google Scholar) have recently identified a novel MG-arginine adduct,N δ-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-l-ornithine (argpyrimidine), as a major fluorescent product, and the presence of this adduct in the human serum and cornea has also been demonstrated (28Shamsi F.A. Partal A. Sady C. Glomb M.A. Nagaraj R.H. J. Biol. Chem. 1998; 273: 6928-6936Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). MG also reacts with lysine residues to generate an MG-derived lysine-lysine cross-link (imidazolysine) (29Nagaraj R.H. Shipanova I.N. Faust F.M. J. Biol. Chem. 1996; 271: 19338-19345Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar) andN ε-carboxyethyllysine (CEL) (30Ahmed M.U. Thorpe S.R. Baynes J.W. J. Biol. Chem. 1986; 261: 4889-4894Abstract Full Text PDF PubMed Google Scholar), which accumulate with aging and its related diseases.Here, we report the chemical and immunochemical characterization of the MG modification of arginine. On the basis of chemical and spectroscopic evidence, we identify a novel MG-arginine adduct having the tetrahydropyrimidine structure and also demonstrate that the major fluorescent product, originally identified as 5methylimidazolone, is argpyrimidine. In addition, as an immunochemical approach to the detection of MG-arginine adducts in vivo, we raised the monoclonal antibodies and characterize their specificities. An attempt to detect antigenic materials in vivo was also made in the kidneys from diabetic patients and in the rat brain after ischemia/reperfusion.EXPERIMENTAL PROCEDURESMaterialsMG (40% aqueous solution), bovine serum albumin (BSA) (essentially fatty acid-free) andN α-acetyl-l-arginine were purchased from Sigma. MG was purified by distillation under reduced pressure, and the concentration of MG in stock solutions was determined by HPLC as 2-methylquinoxaline (31Corderio C. Freire A.P. Anal. Biochem. 1996; 234: 221-224Crossref PubMed Scopus (54) Google Scholar). An authentic sample of CEL was kindly provided by Dr. J. W. Baynes (University of South Carolina). 3-Deoxyglucosone was kindly provided by Dr. F. Hayase (Meiji University). Keyhole limpet hemocyanin was obtained from Pierce. Horseradish peroxidase-conjugated goat IgG fraction to mouse IgG was obtained from Organon Teknika Co. (Durham, NC).General ProcedureSeparation of MG-treatedN α-acetylarginine was carried out on a Jasco Gulliver HPLC with a Jasco MD-910 UV-visible photodiode array detector. Low and high resolution fast atom bombardment-mass spectrometry (FAB-MS) was measured with a JEOL JMS-700 (MStation) instrument. NMR spectra were recorded with a Bruker AMX600 (600 MHz) instrument. Ultraviolet absorption spectra were measured with a Hitachi U-Best-50 spectrophotometer, and fluorescence spectra were recorded with a Hitachi F-2000 spectrometer. Liquid chromatography-mass spectrometry (LC-MS) was measured with a Jasco PlatformII-LC instrument.Reaction of Nα-Acetyl-l-arginine with MGN α-Acetyl-l-arginine (100 mm) was incubated with MG (100 mm) in 100 mm sodium phosphate buffer, pH 7.4, at 37 °C for 14 days, with adjustment of the pH to 7.4 with sodium hydroxide solution (5 m) as required. The reaction mixture was then lyophilized to dryness and extracted with ethanol. After evaporation of the ethanol under vacuum, the residual solid product was purified by reversed-phase HPLC.HPLC AnalysisSeparation was carried out in a Develosil ODS-HG-5 column (4.6 × 250 mm, Nomura Chemicals Co., Seto, Japan) by applying a linear gradient of 10–50% methanol in 50 mm acetic acid from 15 to 30 min after the isocratic condition (0.1% methanol in 50 mm acetic acid) for 15 min at a flow rate of 0.8 ml/min. The eluent was monitored at 230 nm. For preparative HPLC, the reaction mixture was applied to a Develosil ODS-HG-5 column (8.0 × 250 mm), equilibrated in a solution of 15% methanol in 50 mmacetic acid, and eluted at a flow rate of 2.0 ml/min. The elution profiles were monitored by absorbance at 215 nm.Amino Acid AnalysisBSA (1 mg/ml) in 100 mm sodium phosphate buffer (pH 7.4) was treated with 100 mm MG at 37 °C for 24 h. After incubation, the reaction mixtures were treated with 10% trichloroacetic acid. Precipitated proteins were separated by centrifugation at 10,000 × g for 10 min. The pellets were collected and washed with 200 μl of diethyl ether. The resultant pellets were dried and subjected to acid hydrolysis with 6n HCl at 105 °C for 24 h. The hydrolysate was concentrated and dissolved with 50 mm sodium citrate buffer (pH 2.2). The amino acid analysis was performed with a JEOL JLC-500 amino acid analyzer equipped with a JEOL LC30-DK20 data analyzing system.Monoclonal AntibodiesFemale BALB/c mice were immunized three times with the MG-treated keyhole limpet hemocyanin. Spleen cells from the immunized mice were fused with P3/U1 murine myeloma cells and cultured in hypoxantine/aminopterin/thymidine selection medium. Culture supernatants of the hybridoma were screened using an enzyme-linked immunosorbent assay (ELISA), employing pairs of wells of microtiter plates on which were absorbed MG-treated BSA and native BSA as antigen (1 μg of protein/well). After incubation of 50 μl of hybridoma supernatants, and with intervening washes with phosphate-buffered saline, pH 7.8, containing 0.05% Tween 20 (PBS-Tween), the wells were incubated with alkaline phosphatase-conjugated goat antimouse IgG, followed by a substrate solution containing 1 mg/mlp-nitrophenyl phosphate. Hybridoma cells corresponding to supernatants that were positive on MG-modified BSA and negative on native BSA were then cloned by limiting dilution. After repeated screening, three clones were obtained. Among them, clones 3C and 6B showed the most distinctive recognition of MG-modified BSA.ELISADirect ELISAA coating antigen was prepared by incubating 1 mg of BSA with 10 mm aldehydic compounds in 1 ml of 50 mm sodium phosphate buffer, pH 7.2, for 24 h at 37 °C. A 100-μl aliquot of the antigen solution containing 0.4 mg of protein was added to each well of a 96-well microtiter plate and incubated overnight at 4 °C. The antigen solution was then removed, and the plate was washed with PBS-Tween. Each well was filled with 200 μl of 0.5% gelatin solution for 1 h at 37 °C. The primary antibody was then added to the wells at 100 μl/well of 1 μg/ml solution for 3 h at 37 °C. The plate was then washed once with PBS-Tween. After discarding the supernatants and washing three times with PBS-Tween, 100 μl of a 5 × 103 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase in PBS-Tween was added. After incubation for 1 h at 37 °C, the supernatant was discarded, and the plates were washed three times with PBS-Tween. Enzyme-linked antibody bound to the well was revealed by adding 100 μl/well 1,2-phenylenediamine (0.5 mg/ml) in 0.1 mcitrate/phosphate buffer (pH 5.0) containing 0.003% H2O2. The reaction was terminated by the addition of 50 μl of 2 m sulfuric acid, and the absorbance at 492 nm was read on a micro-ELISA plate reader.Competitive ELISAFor characterization of the antibody, a competitor was incubated with the antibody for 20 h at 4 °C to yield competitor/antibody mixtures containing antibody at 1 μg/ml and variable concentrations of the competitor. A 100-μl aliquot of the competitor/antibody mixture was added to each well and incubated for 1 h at 37 °C. After discarding the supernatants and washing three times with PBS-Tween, the second antibody was added, and the enzyme-linked antibody bound to the well was revealed as described previously. Results were expressed as the ratioB/Bo, where B = (absorbance in the presence of the competitor − background absorbance (no antibody)) and Bo = (absorbance in the absence of the competitor − background absorbance).Animal ExperimentsThe details of the operative method have been previously reported (32Urabe T. Hattori N. Nagamatsu H. Sawa Y. Mizuno Y. J. Neurochem. 1996; 67: 256-271Google Scholar). Animal experiments were carried out according to a protocol approved by the Animal Care Committee of Juntendo University. Adults male Wistar rats (SPF) weighing 250–300 g were used (Japan SLC, Shizuoka, Japan). In anesthetized rats, the right middle cerebral artery was occluded at its origin by the insertion of a thin nylon surgical thread via the external and internal carotid arteries according to the method of Longa et al. (33Longa E.Z. Weinstein P.R. Carlson S. Cummins R. Stroke. 1989; 20: 84-91Crossref PubMed Scopus (6580) Google Scholar) with slight modifications. After 3 h of right middle cerebral artery occlusion, the thread was removed to allow reperfusion. The body (rectal) temperature was maintained at 37 °C using a heating pad and lamp-heating. All rats exhibited neurogenic deficits characterized by severe left hemiparesis, more in the upper extremity, and right Horner's syndrome, and most of the rats died after 48 h due to severe brain edema. Five rats were perfused transcardially with 4% paraformaldehyde in PBS each at 6 and 24 h after reperfusion.ImmunohistochemistryHuman KidneysFormalin-fixed and paraffin-embedded renal tissues derived from autopsy samples of diabetic and nondiabetic patients were prepared. The immunohistochemical localization of argpyrimidine in the tissues was examined by a labeled streptavidin biotin method using Maxitags immunoenzyme universal kit (Shandon Lipshaw, Pittsburgh, PA). In brief, the tissue sections were deparaffinized with xylene and hydrated in a series of increasing water concentrations in ethanol. The slides were then placed in a chamber containing 0.3% H2O2 solution in methanol for 20 min at room temperature to inhibit the endogenous peroxidase activity, followed by the blocking step for 15 min. Subsequently, the slides were incubated with a 1:100 dilution of anti-argpyrimidine mouse monoclonal antibody (mAb6B) for 2 h at room temperature. To perform immunoabsorption experiments, the antibody was preincubated with and without free N-acetylargpyrimidine (final concentration, 25 μm) overnight at 4 °C before application to the tissue section as described above. After the reaction with primary antibody, the slides were washed with water and PBS and incubated with biotinylated anti-mouse IgG for 30 min at room temperature, followed by another wash. In the next step, the slides were incubated with peroxidase-conjugated streptavidin for 30 min at room temperature and then washed with water. The color reaction was carried out by incubating the slides with freshly prepared 3,3′-diaminobenzidine reagent. After stopping the reaction by washing the slides with water, the nuclei were counterstained with hematoxylin for 20 s.Rat Brain after Middle Cerebral Artery Occlusion Followed by ReperfusionThe brains were removed and then postfixed with 4% paraformaldehyde in PBS. The paraffin-embedded brains were cut coronary at a thickness of 5 mm, deparaffinized with xylene, and dehydrated with 100% ethanol. Immunostaining was performed by the avidin-biotin-peroxidase complex (ABC) method as previously reported (32Urabe T. Hattori N. Nagamatsu H. Sawa Y. Mizuno Y. J. Neurochem. 1996; 67: 256-271Google Scholar, 34Yoritaka A. Hattori N. Uchida K. Tanaka M. Stadtman E.R. Mizuno Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2696-2701Crossref PubMed Scopus (865) Google Scholar). After blocking endogenous peroxidase activities by treatment with 3% H2O2 in methanol, the sections were incubated with 10% normal goat serum in 0.01 m PBS containing 2% BSA for 10 min at room temperature to block nonspecific binding. After washing with PBS, the sections were incubated with the monoclonal antibody (mAb6B) overnight at 4 °C. After being rinsed in 0.01 m PBS, the sections were incubated with biotinylated anti-mouse IgG (1:100 dilution; Vectastain ABC kit, Vector Laboratories, Burlingame, CA) for 60 min at room temperature. They were then incubated with ABC (1:100 dilution; Vectastain ABC kit, Vector Laboratories) for 1 h. After rinsing, sections were finally incubated with 0.02% 3,3-diaminobenzidine and 0.03% H2O2 in distilled water for 7–10 min. To confirm the specificity of immunostaining, competition experiments were performed with the mAb6B that was preincubated for 2 h at 37 °C with an excess of argpyrimidine. Control sections were treated in the same manner with omission of the primary antibodies or with nonimmune rabbit or mouse IgG as a negative control.RESULTSCharacterization of Major Nonfluorescent and Fluorescent MG-Arginine AdductsThe selectivity of the binding of MG to arginine residues was assessed by changes in the amino acid composition of MG-treated proteins. When BSA (1 mg/ml) was incubated with 100 mm MG for 24 h at 37 °C, about 78% of the arginine and 27% of lysine residues were lost, and no significant change in other amino acids was observed. The data suggested that the arginine residues of proteins represented primary targets for reaction with MG. Hence, to determine the structure of the MG-arginine adduct generated in the protein, the reaction of MG withN α-acetylarginine was carried out. Upon incubation of N α-acetylarginine with MG for 14 days at 37 °C, two nonfluorescent products (a andb) and one fluorescent product (c) were detected (Fig. 1). After purification, they were characterized by 1H and 13C NMR, FAB-MS, and LC-MS along with N α-acetylarginine.Nonfluorescent ProductsThe LC-MS analysis of the peaks (product b) eluted at 22–24 min exhibited the same molecular weight of 361 (M+H) (data not shown). After purification, the product was characterized by FAB-MS and 1H and13C NMR. The FAB-MS on the glycerol matrix showed a molecular ion at m/z of 361 (M+H) (Fig.2). High resolution FAB-MS in the positive ion mode showed a molecular weight of 361.1729 (S.E., +1.6 ppm), that corresponded to the molecular formula of C14H25O7N4. The1H NMR spectrum exhibited the following signals: δH (D2O): 1.48 (g, 3H, s), 1.51 (g′,3H, s), 1.53 (f, 3H, s), 1.57 (f′,3H, s), 1.62 (d, 1H, m), 1.68 (d, 1H, m), 1.83 (c, 2H, m), 1.98 (a, 3H, s), 3.24 (e, 2H, t, Jd, e = 6.8 Hz), 3.75 (h, 1H, s), 3.94 (h′, 1H, s), 4.22 (b, 1H, m). The13C NMR spectrum showed the following signals; δC (D2O): 22.5 (C-a), 22.6 (C-f), 25.2 (C-d), 25.4 (C-g), 29.0 (C-c), 41.1 (C-e), 54.4 (C-b), 58.4 (C-l), 63.0 (C-l′), 72.5 (C-h′), 75.4 (C-h), 80.2 (C-m), 81.8 (C-m′), 151.6 (C-k), 174.8 (C-i), 177.1 (C-n′, COOH), 178.0 (C-j, COOH), 178.7 (C-n, COOH). Compared with the1H and 13C NMR spectra betweenN α-acetylarginine and the product, the following signals were assigned for the presence of nativeN α-acetylarginine structure in the product; δH 1.98 (a), 4.22 (b), 1.83 (c), 1.62 (d), 1.68 (d), 3.24 (e); δC 22.5 (C-a), 54.4 (C-b), 29.0 (C-c), 25.2 (C-d), 41.1 (C-e), 174.8 (C-i), 178.0 (C-j, COOH), 151.6 (C-k). The 1H and13C NMR spectra of the product showed the presence of one methine (h or h′). The 13C NMR spectrum showed the presence of two quaternary carbons linked with the guanidino group (C-l and C-m) and one carboxylic acid carbon (C-n or C-n′). It was suggested that the product had isomers, because C-m was an asymmetric carbon. Each signal of two methyl (f and g) and methine (h) showed two peaks by the effects of a chiral center. The methyl protons (f or f′) was correlated with C-h (or C-h′), C-l (or C-l′) and C-n (or C-n′) in the1H-detected multiple-bond heteronuclear multiple quantum coherence spectrum (Fig. 3). The two-dimensional spectra also showed the correlation of another methyl protons (g or g′) and C-m (or C-m′), and of methine (h or h′) and C-f (or C-f′), C-g (or C-g′), C-l (or C-l′), and C-m (or C-m′). Six signals of carbon (C-f, C-g, C-h, C-l, C-m, and C-n) seemed to have originated from two molecules of MG at the reaction with the guanidino group inN α-acetylarginine. Based on these characteristics, it was determined that the purified product was a novel tetrahydropyrimidine-type MG-arginine adduct,N δ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-l-ornithine (Fig. 3). The product was generated in the yield of 16.0% whenN α-acetylarginine was incubated with MG for 14 days at 37 °C. It is also notable that a similar tetrahydropyrimidine derivative was also isolated from the reaction mixture of N α-benzoylarginine with MG (data not shown).Figure 2FAB-MS spectrum of productb.View Large Image Figure ViewerDownload (PPT)Figure 3Assignment of spectroscopic data from1H-detected multiple-bond heteronuclear multiple quantum coherence spectrum experiments to structure elements of productb.View Large Image Figure ViewerDownload (PPT)All of the peaks (product a) eluted at 13–15 min in Fig.1 A showed an absorption maximum at 220 nm and a molecular weight of 271 upon LC-MS analysis, suggesting that they were a mix