Guanidino Group Of Arginine Research Articles

Development of selective class I protein arginine methyltransferase inhibitors through fragment-based drug design approach

Protein arginine methyltransferases (PRMTs) catalyze the methylation of the terminal nitrogen atoms of the guanidino group of arginine of protein substrates. The aberrant expression of these methyltransferases is linked to various diseases, making them promising therapeutic targets. Currently, PRMT inhibitors are at different stages of clinical development, which validated their significance as drug targets. Structural Genomics Consortium (SGC) has reported several small fragment inhibitors as Class I PRMT inhibitors, which can be the starting point for rational drug development. Herein, we report the successful application of a fragment-based approach toward the discovery of selective Class I PRMT inhibitors. Structure-based ligand optimization was performed by strategic incorporation of fragment hits on the drug-like quinazoline core and subsequent fragment growth in the desired orientation towards identified hydrophobic shelf. A clear SAR was established, and the lead compounds 55 and 56 displayed potent inhibition of Class I PRMTs with IC50 values of 92 nM and 37 nM against PRMT4. We report the systematic development of potent Class I PRMT inhibitors with good potency and about 100-fold selectivity when tested against a panel of 31 human DNA, RNA, and protein lysine and arginine methyltransferases. These improved small molecules will provide new options for the development of novel potent and selective PRMT4 inhibitors.

Determination of proteinaceous free amino acids by gas chromatography

A method was developed for determination of proteinaceous free amino acids by gas chromatography-mass spectrometry (GC-MS). The guanidino group of arginine in amino acid samples was modified with 1,2-cyclohexanedione at room temperature under basic conditions, and all amino acids were directly derivatized with isobutyl chloroformate. The amino acid derivatives formed were analyzed by GC-MS. The method developed was applied successfully for the determination of amino acids in the Japanese alcoholic beverage, sake.

Exceptionally versatile - arginine in bacterial post-translational protein modifications.

Post-translational modifications (PTM) are the evolutionary solution to challenge and extend the boundaries of genetically predetermined proteomic diversity. As PTMs are highly dynamic, they also hold an enormous regulatory potential. It is therefore not surprising that out of the 20 proteinogenic amino acids, 15 can be post-translationally modified. Even the relatively inert guanidino group of arginine is subject to a multitude of mostly enzyme mediated chemical changes. The resulting alterations can have a major influence on protein function. In this review, we will discuss how bacteria control their cellular processes and develop pathogenicity based on post-translational protein-arginine modifications.

Regulation of arginine biosynthesis, catabolism and transport in Escherichia coli.

Already very early, the study of microbial arginine biosynthesis and its regulation contributed significantly to the development of new ideas and concepts. Hence, the term "repression" was proposed by Vogel (The chemical basis of heredity, The John Hopkins Press, Baltimore, 1957) (in opposition to induction) to describe the relative decrease in acetylornithinase production in Escherichia coli cells upon arginine supplementation, whereas the term "regulon" was coined by Maas and Clark (J Mol Biol 8:365-370, 1964) for the ensemble of arginine biosynthetic genes dispersed over the E. coli chromosome but all subjected to regulation by the trans-acting argR gene product. Since then, unraveling of the molecular mechanisms controlling arginine biosynthesis, catabolism, and transport in and out the cell, have revealed moonlighting activities of enzymes and transcriptional regulators that generate unexpected interconnections between at first sight totally unrelated cellular processes, and have continued to replenish scientific knowledge and stimulated creative thinking. Furthermore, arginine is much more than just a common amino acid for protein synthesis. It may also be used as sole source of nitrogen by E. coli and a source of nitrogen, carbon and energy by many other bacteria. It is a substrate for the synthesis of polyamines, and important for the extreme acid resistance of E. coli. Furthermore, the guanidino group of arginine is well suited to engage in multiple interactions involving hydrogen bonds and ionic interactions with proteins and nucleic acids. Here, we combine major historical discoveries with current state of the art knowledge on arginine biosynthesis, catabolism and transport, and especially the regulation of these processes in E. coli, with reference to other microorganisms.

New class of early-stage enterovirus inhibitors with a novel mechanism of action

4-dimethylamino benzoic acid (compound 12, synonym: 4EDMAB) was identified as an in vitro inhibitor of Coxsackie virus B3 (CVB3) replication in CPE-based assays (EC50 of 9.1 ± 1.5 μM). Next, the activity of twenty-three analogues was assessed, their structure-activity relationship was deduced and a more potent analogue was identified (EC50 of 2.6 ± 0.5 μM). The antiviral activity of 4EDMAB was further confirmed by quantifying viral RNA yield. Time-of-drug-addition assay revealed that 4EDMAB exerts its antiviral activity at the early stages of virus replication. Six compound-resistant viruses were selected and genotyped and all the mutations appeared to be in the capsid protein VP1. Reverse engineering showed that single mutants Y75C, A88V, A98V, D133N and R219K were respectively 15-, 2-, 4-, 17- and 76-fold resistant to 4EDMAB. The compound protected both wild type (WT) CVB3 and the five resistant mutants from heat inactivation. The plaque size produced by the A88V, D133N and R219K mutants was smaller than that of WT and these mutants were also more heat-sensitive than WT in the absence of the compound. These findings suggest that these three mutations increase virion capsid flexibility and compensate for the stabilizing effects of 4EDMAB. Molecular modelling suggests that the compound binds to a small cavity in VP1, which is different from the hydrophobic pocket in the canyon where typical capsid binders (such as pleconaril) bind. Modelling studies also suggest a direct ionic interaction between the negatively charged carboxylic group of 4EDMAB and the positively charged guanidino group of arginine 219. Moreover, the in vitro combination of 4EDMAB and pleconaril resulted in synergistic antiviral effect. In conclusion, 4EDMAB is a novel early-stage inhibitor, which targets VP1 with a mechanism that is different from that of known capsid binders.

Study of an Unusual Advanced Glycation End-Product (AGE) Derived from Glyoxal Using Mass Spectrometry

Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.

Special edition of Amino Acids of selected papers from the eleventh international symposium on the Maillard reaction, September 16–20, 2012

We introduce this special edition of Amino Acids with selected papers from the eleventh international symposium on the Maillard reaction held in Nancy, France, September 16–20, 2012. Glycation of proteins occurs by a complex series of sequential and parallel reactions called collectively the Maillard reaction—named after the leading pioneer of glycation research, Louis Camille Maillard (1978–1936). Glycation researchers gathered at the symposium in 2012 organised by the International Maillard Reaction Society to commemorate the centennial of Maillard reaction research and review achievements and current standing of glycation research. In this special edition there are selected papers from this meeting. Subjects of study include: the involvement of advanced glycation endproducts (AGEs) in complications of diabetes, association of AGEs with insulin secretion, enzymatic metabolism of early glycation adducts, absorption into the body of early and advanced glycation endproducts (AGEs) of dietary origin, association of AGEs in dietary protein with chronic disease, the study of the effect of AGEs on bacterial growth and inhibition of AGE formation by natural products. The Centennial Maillard Symposium began with a presentation by Nobel laureate Jean-Marie Lehn (University of Strasbourg, France) on ‘‘Towards a chemistry of complex matter’’. The Maillard reaction exemplifies how a relatively simple chemical reaction of a reducing sugar or sugar derivative with an amino group becomes highly complex through multiple parallel and sequential reactions. In recent decades there has also been increasing awareness of dicarbonyl compounds and metabolites reacting with guanidino groups in the Maillard reaction. This expands the locus of glycation adduct formation from N-terminal and lysine side chain amino groups in amino acids and proteins to guanidino groups of arginine and arginine residues in proteins. Indeed, arginine-derived AGEs are now considered to be quantitatively major AGEs in physiological systems. In 100 years the Maillard reaction has seen applications in chemistry, food, nutrition, health and disease, microbiology and clinical diagnostics and repeated, on-going development of applications in pharmacology and therapeutics. We celebrate the achievements of Louis Camille Maillard in glycation research and suggest that, if he was here today, he would marvel at the advances and development that led from his earlier studies.

Gas-Phase Conjugation to Arginine Residues in Polypeptide Ions via N-Hydroxysuccinimide Ester-Based Reagent Ions

Gas-phase conjugation to unprotonated arginine side-chains via N-hydroxysuccinimide (NHS) esters is demonstrated through both charge reduction and charge inversion ion/ion reactions. The unprotonated guanidino group of arginine can serve as a strong nucleophile, resulting in the facile displacement of NHS from NHS esters with concomitant covalent modification of the arginine residue. This reactivity is analogous to that observed with unprotonated primary amines such as the N-terminus or ε-amino group of lysine. In solution, however, the arginine residues tend to be protonated at pH values low enough to prevent hydrolysis of NHS esters, which would render them relatively unreactive with NHS esters. This work demonstrates novel means for gas-phase conjugation to arginine side chains in polypeptide ions.

Formation of Arginine Modifications in a Model System of Nα-tert-Butoxycarbonyl (Boc)-Arginine with Methylglyoxal

The present study deals with the mechanistic reaction pathway of the α-dicarbonyl compound methylglyoxal with the guanidino group of arginine. Eight products were formed from the reaction of methylglyoxal with N(α)-tert-butoxycarbonyl (Boc)-arginine under physiological conditions (pH 7.4 and 37 °C). Isolation and purification of substances were achieved using cation-exchange chromatography and preparative high-performance liquid chromatography (HPLC). Structures were verified by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry. 2-Amino-5-(2-amino-4-hydro-4-methyl-5-imidazolinone-1-yl)pentanoic acid (3) was determined as the key intermediate precursor within the total reaction scheme. Kinetic studies identified N(δ)-(5-methyl-4-oxo-5-hydroimidazolinone-2-yl)-L-ornithine and N(7)-carboxyethylarginine as thermodynamically more stable products from compound 3. Further mechanistic investigations revealed an acidic hydrogen at C-8 of compound 3 to trigger aldol condensations. This reactivity of compound 3 allowed for the addition of another molecule of methylglyoxal to form products, such as N(δ)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-l-ornithine and argpyrimidine.

Conversion of arginine to ornithine for improving the fragmentation pattern of peptides labeled with the N-terminal tris(2,4,6-trimethoxyphenyl)phosphonium group in tandem mass spectrometry

We propose a method of converting arginine to ornithine residues by controlled hydrazinolysis, in order to facilitate the sequencing of peptides by tandem mass spectrometry (MS/MS). Whereas the presence of C-terminal arginine residue occurring in a tryptic peptide is generally desirable in MS and MS/MS analyses, that of arginine in the middle of a peptide often adversely affects the appearance of fragment peaks. This problem still arises in peptides, which are acylated with a reagent containing the tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) group at the N-termini. Using four model peptides, angiotensin III (RVYIHPF), a mucin-related peptide (APDTRPAPG), α-bag cell peptide (1–9) (APRLRFYSL), and [des-Pro2] bradykinin (RPGFSPFR), we optimized the protocol of hydrazinolysis to remove the guanidino group of arginine. Owing to this derivatization, we obtained much simpler MS/MS spectra composing mainly a-type ions characteristic of peptides tagged with the TMPP group. Formylation of the δ-amino group of ornithine further enhanced the efficacy of the derivatization, which would be applicable to the sequencing of non-tryptic peptides.

The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry

Qualitative and quantitative analysis of post-translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well-characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono- and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI-MS). Despite minor matrix-dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono- and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI-MS.

Determination ofNɛ‐(Carboxymethyl)lysine in Foods and Related Systems

The sensitive and specific determination of advanced glycation end products (AGEs) is of considerable interest because these compounds have been associated with pro-oxidative and proinflammatory effects in vivo. AGEs form when carbonyl compounds, such as glucose and its oxidation products, glyoxal and methylglyoxal, react with the epsilon-amino group of lysine and the guanidino group of arginine to give structures including N epsilon-(carboxymethyl)lysine (CML), N epsilon-(carboxyethyl)lysine, and hydroimidazolones. CML is frequently used as a marker for AGEs in general. It exists in both the free or peptide-bound forms. Analysis of CML involves its extraction from the food (including protein hydrolysis to release any peptide-bound adduct) and determination by immunochemical or instrumental means. Various factors must be considered at each step of the analysis. Extraction, hydrolysis, and sample clean-up are all less straight forward for food samples, compared to plasma and tissue. The immunochemical and instrumental methods all have their advantages and disadvantages, and no perfect method exists. Currently, different procedures are being used in different laboratories, and there is an urgent need to compare, improve, and validate methods.

Biotinylation of Peptides/Proteins Using Biocytin Hydrazide

AbstractBiocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In this study, however, biocytin hydrazide was found to be able to directly biotinylate peptides and proteins. This phenomenon may cause false identification of non‐glycopeptides/non‐glycoproteins as glycopeptides/glycoproteins. Here, we report a systematic investigation of the reaction of peptides/proteins with biocytin hydrazide. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry is used to analyze the biotinylation reaction between peptides/proteins and biocytin hydrazide. Peptides/proteins were reacted with biocytin hydrazide in diverse solvent systems with different biocytin hydrazide concentrations for up to 96 h at temperatures ranging from 4 °C to 65 °C. Singly biotinylated or multiply biotinylated peptides/proteins are observed. The efficiency of the biotinylation reaction increases with higher temperature, higher biocytin hydrazide concentration, or longer reaction time. The influence of buffer pH on the biotinylation reaction of peptides/proteins is less pronounced. The biotinylation efficiency is optimum at neutral pH. Data suggests that the peptides are biotinylated as efficiently as proteins. The observation that peptides/proteins condense only with biocytin hydrazide, 2‐iminobiotin hydrazide, adipic dihydrazide and phenyl hydrazine but not with biocytin HCl and 2‐iminobiotin, indicates that the biotinylation reaction of peptides/proteins occurs with the hydrazide moiety but not with biotin moiety of the biotinylated reagent. The postsource decay data of biotinylated P14R indicates that biocytin hydrazide condenses with the guanidino group of arginine's side chain of P14R, indicating that besides N‐terminal and lysine residue of peptides/proteins, arginine residue is capable of reacting with biocytin hydrazide.

Structure of the Whole Cytosolic Region of ATP-Dependent Protease FtsH

An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA+ ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA+ domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA+ domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.

A Polar Pathway for the S4 Arginines

Voltage-gated ion channels detect changes in transmembrane voltage by means of a specialized voltage-sensing domain through a mechanism that involves movement of positively charged arginines in the S4 helix across the membrane electric field. The precise details of this movement--and whether the S4 arginines reside in a polar protein environment or are exposed to the membrane lipid--have been the subject of some controversy. Tombola et al. found that substitution of the first S4 arginine (R1) of the Shaker potassium channel with smaller amino acids lacking the arginine guanidino group (alanine, cysteine, serine, or valine) enabled passage of an inward current, which they called the "omega current." Pharmacological blockade of the central potassium-conducting pore or closure of the pore through P-type inactivation did not affect the omega current, which was only apparent under conditions in which the S4 segment was in its resting conformation. The current, which could be carried by various cations, was weakly selective for Cs + over K + over Li + and could be carried by guanidinium ions, which suggests that it used the pathway through which arginine sidechains normally move. Mutation of a glutamate residue in the S2 segment that is believed to interact electrostatically with the S4 arginines increased the omega current, whereas extracellular Mg 2+ blocked it. The authors conclude that R1 likely faces into a polar protein environment within the voltage-sensing domain and propose a model in which the S4 segment ratchets through conformations in which one arginine after another occupies (and thereby blocks ion permeation through) the narrowest portion of a hydrophilic pore. F. Tombola, M. M. Pathak, E. Y. Isacoff, Voltage-sensing arginines in a potassium channel permeate and occlude cation-selective pores. Neuron 45 , 379-388 (2005). [PubMed]

Characterization of Bis(levuglandinyl) Urea Derivatives as Products of the Reaction between Prostaglandin H2 and Arginine

Levuglandins are gamma-keto aldehydes formed by rearrangement of prostaglandin (PG) H(2) in aqueous solution. Levuglandins are highly reactive with primary amines. We had previously characterized adducts formed after reaction of levuglandin E(2) (LGE(2)) or PGH(2) with lysine. In this study, we assessed whether reaction of PGH(2) with arginine yielded covalent adducts. Using N(alpha)-acetylarginine and both PGH(2) and synthetic LGE(2), we discovered a novel series of levuglandinyl adducts derived from reaction of two levuglandin moieties with the guanidino group of arginine. Subsequent spontaneous hydrolysis of the adducted amino acid yields bis(levuglandinyl) urea and the corresponding ornithine residue. Using liquid chromatography tandem mass spectrometry, we characterized the molecular structure of these novel adducts and demonstrated their formation after coincubation of PGH(2) with synthetic peptides and proteins. The soluble characteristic of these molecules provides a potential strategy for development of biological markers of lipid modification of proteins following cyclooxygenase activity or lipid peroxidation.

Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences.

The reaction of ninhydrin with primary amino groups to form the purple dye now called Ruhemann's purple (RP) was discovered by Siegfried Ruhemann in 1910. In addition, imines such as pipecolic acid and proline, the guanidino group of arginine, the amide groups of asparagine, the indole ring of tryptophan, the sulfhydryl group of cysteine, amino groups of cytosine and guanine, and cyanide ions also react with ninhydrin to form various chromophores of analytical interest. Since its discovery, extensive efforts have been made to apply manual and automated ninhydrin reactions as well as ninhydrin spray reagents to the detection, isolation, and analysis of numerous compounds of interest across a broad spectrum of disciplines. These include agricultural, biochemical, clinical, environmental, food, forensic, histochemical, microbiological, medical, nutritional, plant, and protein sciences. This reaction is unique among chromogenic reactions in that at pH 5.5 it results in the formation of the same soluble chromophore by all primary amines which react, be they amines, amino acids, peptides, proteins, and even ammonia. Because the chromophore is not chemically bound to the protein or other insoluble material, it is not lost when the insoluble substrate is removed by centrifugation or filtration after the reaction is completed. The visible color of the chromophore is distinctive and is generally not affected by the yellow colors present in many food, plant, and tissue extracts. Adaptations of the classical ninhydrin reaction to specialized needs in analytical chemistry and biochemistry include the use of acid, alkaline, and fluorogenic ninhydrin reagents. To cross-fertilize information among several disciplines wherein an interest in the ninhydrin reaction has developed, and to enhance its utility, this review attempts to integrate and correlate the widely scattered literature on ninhydrin reactions of a variety of structurally different compounds. Specifically covered are the following aspects: historical perspective, chemistry and mechanisms, applications, and research needs. A better understanding of these multifaceted ninhydrin reactions provide a scientific basis for further improvements of this important analytical technique.

Surface Modification Chemistry Based on the Electrostatic Adsorption of Poly-l-arginine onto Alkanethiol Modified Gold Surfaces

The reaction between the arginine's guanidino group and α-dicarbonyl functionalities was used to develop a novel surface modification chemistry. The first modification step consists of the electrostatic adsorption of a poly-l-arginine layer onto ionizable alkanethiol modified gold surfaces. The strongly basic character of the guanidino group of the arginine residues (pKa > 12) guarantees the robust attachment of the polypeptide to negatively charged gold surfaces until very high pH. By varying the pH of the solution from which poly-l-arginine is electrostatically adsorbed, it is possible to control the amount deposited. The availability of the surface guanidino groups of the poly-l-arginine layer for further derivatization with α-dicarbonyl reaction probes, yielding stable heterocyclic condensation adducts, is demonstrated. In addition, the reaction with the heterobifunctional reagent p-azidophenyl glyoxal (APG) provides a surface terminated with a photosensitive aryl azide group which was employed for the photochemical immobilization of proteins to the surface. The application of this surface modification chemistry to immobilize antibodies is demonstrated.

Isolation and characterization of Glyoxal–Arginine modifications

Glyoxal is generated in foods and in vivo by autoxidation of unsaturated fatty acids and by the Maillard reaction of reducing sugars and amines. Due to its high reactivity, the α-dicarbonyl easily reacts with protein-bound lysine and arginine residues. The quantitatively most important modification of the ε-amino group of lysine is Nε-carboxymethyllysine [1]. Reaction with glyoxal also leads to cross-linking of proteins by formation of amides (glyoxal–amide cross-link [2]), imidazole (MODIC [3]) and imidazolium structures (GOLD [4]).Concerning the reaction of glyoxal with the guanidino group of arginine, inconsistent results have been published in the literature. We could now verify 1-(4-amino-4-carboxybutyl)-2-imino-4,5-dihydroxy-4,5-dihydroimidazolidine (dihydroxyimidazolidine) as the single primary product in incubations of Nα-t-BOC-arginine with glyoxal under physiological conditions [5]. The structure was identified as a cis/trans-isomer mixture of ca. 1:4. In a much slower reaction, dihydroxyimidazolidine is degraded to N7-carboxymethylarginine (CMA), representing a novel stable endproduct. Both structures were unequivocally established by 1H-, 13C-, HMBC-NMR and high-resolution mass spectra. Other structures could not be found under these reaction conditions. Synthesis of 1-(4-amino-4-carboxybutyl)-2-imino-5-oxo-imidazolidine (imidazolinone), which has been published earlier as the only modification so far , could not be verified [6]. However, both arginine modifications, dihydroxyimidazolidine and CMA can be transformed to the imidazolinone under acid conditions. Therefore, the detection of the imidazolinone in incubations under physiological conditions must be evaluated as an artifact of the acid sample workup procedures applied.The mechanistic relationship of all three structures was clarified in incubations at various temperatures, pH values and in the presence of o-phenylenediamine and aminoguanidine. The latter experiment using α-dicarbonyl trapping reagents proved that the reaction to the dihydroxyimidazolidine can be reversed to regenerate arginine and a glyoxal derivative. As trapping reagents are commonly used to measure ‘free’ glyoxal in foods and in vivo samples, the published data has to be evaluated very critically. In addition, it was possible to elucidate the mechanism leading from the dihydroxyimidazolidine to CMA and to exclude any participation of the imidazolinone.

A novel monoclonal antibody against methylglyoxal–arginine adduct

Methylglyoxal (MG), a precursor of advanced glycation end products, reacts rapidly with arginine residue in proteins. Three products, one fluorescent and two non-fluorescent adducts, have been so far identified. The fluorescent MG–arginine adduct, putatively identified as 5-methylimidazolone, has been shown to be identical to argpyrimidine. The non-fluorescent products were identified as the 5-hydro-5-methylimidazol-4-one and a novel MG adduct, which we named tetrahydropyrimidine (THP). THP was found to be formed from the reaction of guanidino group of arginine with two-MG molecules under physiological conditions. In the present study, to detect the THP adduct in vivo, we developed a monoclonal antibody (mAb5A3) directed to protein-bound THP. During preparation of the monoclonal antibody, hybridomas were selected by comparing the reactivities of the culture supernatant to THP using a competitive enzyme-linked immunosorbent assay. It was found that mAb5A3 specifically recognized THP as the dominant epitope. The presence of THP adduct was attested by immunohistochemical analysis of human atherosclerotic lesion, in which macrophage-derived foam cells were positively stained by the antibody. Our present results suggest that accumulation of THP occurs in vivo and may contribute to the progression of vascular diseases.