Free Amino Groups Of Proteins Research Articles

Immunochemical detection of N2-[1-(1-carboxy)ethyl]guanosine, an advanced glycation end product formed by the reaction of DNA and reducing sugars or l-ascorbic acid in vitro

In the Maillard reaction, free amino groups of proteins and nucleic acids react with reducing sugars to form advanced glycation end products (AGE). A major product found in reaction mixtures of guanosine and glucose is N 2-[1-(1-carboxy)ethyl]guanosine (CEG), which, therefore, can be used as a marker of advanced glycation of DNA. An enzyme-linked immunosorbent assay (ELISA) was developed to detect and to semi-quantitate nonenzymatic glycosylation of DNA. A polyclonal antiserum was raised against CEG linked to keyhole limpet hemocyanin. A protocol for a competitive ELISA was developed, and the antiserum was tested for crossreactivity. Several unmodified nucleotides and N 2-modified guanosine derivatives showed no or negligible crossreactivity. Only very similar structures like N 2-(carboxymethyl)guanosine and N 2-(1-carboxy-3-hydroxypropyl)guanosine, which have been identified as reaction products of glucose or l-ascorbic acid and guanosine, display significant binding activity. The signal can be totally repressed by free CEG, yet protein-bound CEG is a stronger inhibitor. DNA incubated with d-glucose, dihydroxyacetone, l-ascorbic or l-dehydroascorbic acid shows a signal inhibition indicating the formation of CEG in vitro. The competitive ELISA procedure proved to be a sensitive method which can be used to detect glycation of DNA in vivo.

Diabetes, advanced glycation endproducts and vascular disease.

The high incidence of vascular complications in patients with diabetes mellitus remains incompletely understood. Several metabolic or endocrine abnormalities have been postulated as possible triggers for micro and macroangiopathies. This review article focuses on the consequences of hyperglycemia, leading to the formation of advanced glycation endproducts (AGE), on vascular function. Advanced glycation endproducts are the product of the binding of aldoses onto free amino groups of proteins or lipoproteins, which, after molecular rearrangement, result in a class of molecules of a brown color and specific fluorescence. Different cell membrane proteins have been shown to bind AGE and the best characterized receptor for AGE has been named RAGE. The AGE receptor is present on different cell types including endothelial cells, smooth muscle cells, lymphocytes and monocytes. Experimental studies have revealed that the binding of AGE to RAGE produces an activation of monocytes and endothelial cells. Activated endothelial cells produce interleukin and express vascular cell adhesion molecule and tissue factor. Advanced glycation endproducts, when infused into animals, induce an increase in vascular permeability. The blockade of RAGE by specific antibodies corrects the hypermeability observed in diabetic animals. The prevention of AGE formation by aminoguanidine treatment improves the microvascular lesions found in diabetic animals either in the retina or the glomerus. The infusion of recombinant RAGE in diabetic animals corrects hyperpermeability. The colocalization of RAGE and AGE at the microvascular site of the injury suggests that their interaction may play a significant role in the pathogenesis of diabetic vascular lesions.

Ageing promotes the increase of early glycation Amadori product as assessed by ϵ- N-(2-furoylmethyl)- l-lysine (furosine) levels in rodent skin collagen : The relationship to dietary restriction and glycoxidation

Glucose has been implicated in the aging process by its ability to react nonenzymatically with long-lived proteins like collagen to produce advanced glycosylated end-products (AGEs). In the initial phase of this reaction, referred to as glycation, glucose reacts with the free amino group of proteins resulting in Schiff base formation followed by rearrangement to an Amadori product. Since the Amadori product is transient due to its conversion to other products as well as its reversibility to the initial products, glycation as an age-related marker in collagen has questionable significance. In human studies, glycation of collagen has been found to increase modestly with age. In rodent studies, results are conflicting due to differences in methodology. Thus, it has been concluded that collagen glycation either does not vary or increases modestly with age. In the present study, a C8 HPLC column was used to measure Amadori product formation as the acid-hydrolyzed breakdown product furosine in the skin of rats and mice. Surprisingly, levels were found to increase at a rapid rate during aging of rodents. Impurity of the furosine peak from the use of crude acid-hydrolyzed skin samples was ruled-out because reductive properties and spectroscopic profiles matched those previously described for furosine. In the present study, glycemia was found important in furosine formation as shown by the glycation lowering effects of dietary restriction on collagen. Decreased collagen turnover probably plays a substantial role in explaining the age-related increase in furosine levels in rodent skin collagen.

Inhibitors of the Maillard Reaction

The Maillard reaction is a complex cascade of reactions initiated by glycation or the spontaneous reaction between the free amino groups of proteins and reducing sugars. The cascade reaction leads to the production of the so-called ‘advanced glycation end-products’ (AGEs), which include brown pigments, fluorescent compounds, free radicals and cross-linked protein products.

Free Radicals Generated during the Glycation Reaction of Amino Acids by Methylglyoxal: A MODEL STUDY OF PROTEIN-CROSS-LINKED FREE RADICALS

The formation of alpha-dicarbonyl compounds seems to be an important step for cross-linking proteins in the glycation or Maillard reaction. To elucidate the mechanism for the cross-linking reaction, we studied the reaction between a three-carbon alpha-dicarbonyl compound, methylglyoxal, and amino acids. Our results showed that this reaction generated yellow fluorescent products as formed in some glycated proteins. In addition, three types of free radical species were also produced, and their structures were determined by EPR spectroscopy. These free radicals are 1) the cross-linked radical cation, 2) the methylglyoxal radical anion as the counterion, and 3) the superoxide radical anion produced only in the presence of oxygen. The generation of the cross-linked radical cations and the methylglyoxal radical anions does not require metal ions or oxygens. These results indicate that dicarbonyl compounds cross-link free amino groups of protein by forming Schiff bases, which donate electrons directly to dicarbonyl compounds to form the cross-linked radical cations and the methylglyoxal radical anions. Oxygen can accept an electron from the radical anion to generate a superoxide radical anion, which can initiate damaging chain reactions. Time course studies suggest that the cross-linked radical cation is a precursor of yellow fluorescent glycation end products.

Plant Plasma Membrane Proteins

The ability of two biotinylating reagents, sulfosuccinimidobiotin and sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate, to label plasma membrane proteins was examined. These compounds form covalent bonds with the free amino groups of proteins and label the proteins with biotin. Biotinylated proteins can be detected with avidin-peroxidase staining. Protoplasts isolated from embryogenic Daucus carota suspension cells were labeled with biotin and the membranes were separated on linear sucrose gradients. The conditions used for labeling the protoplasts did not cause protoplast rupture or loss of viability. The distribution of the biotin label in these linear sucrose gradients was analyzed and compared to the distribution of vanadate-sensitive ATPase activity, a marker for the plasma membrane. Both the biotin label and the vanadate-sensitive ATPase activity were strongly localized in the gradient at peak density of 1.16 gram per cubic centimeter. When the protoplast surface was labeled, biotinylated polypeptides were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polypeptides of 153, 94, 51, 30, 20, 17, and 14 kilodaltons were shown to be plasma membrane in origin. When a crude membrane pellet was labeled, numerous biotinylated polypeptides were distributed throughout the gradient. Because the position of the biotin label in the gradient is strongly correlated with the distribution of vanadate-sensitive ATPase, it is concluded that these biotinylating reagents are effective and reliable labels for proteins of the plant plasma membrane. Furthermore, these labels permit the positive identification of plasma membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and can serve as convenient markers for solubilization and purification of these proteins.

Chemical analysis of the surface of microorganisms by X-ray photoelectron spectroscopy

Abstract A collection of microorganisms, including a microfungus and various yeasts and bacteria has been analyzed by X-ray photoelectron spectroscopy (XPS). A correlation is observed between the N/P atomic concentration ratio of the cell surface and the cell electrophoretic mobility measured at pH 4, indicating that the dehydrated surface analyzed by XPS is representative of the cell surface in contact with water. Deprotonation of phosphate groups plays a predominant role in the development of the cell negative charge, and carboxylic groups are not involved appreciably; a partial neutralization is allowed by protonation of free amino groups of proteins. These results advocate a broader use of XPS in order to understand physicochemical properties (electrostatic charge, hydrophobicity, ion binding) of the surface of cells, which are of prime importance in various processes occurring in nature and technology.

Polymerization of proteins caused by reaction with sugars and the formation of 3-deoxyglucosone under physiological conditions.

Lysozyme, ribonuclease A, ovalbumin and bovine serum albumin were reacted with reducing sugars under physiological conditions of 37°C and pH 7.4, and polymerization of proteins, changes in amino acid composition and carbonyl compounds formed during the reaction were investigated. Incubation of all the protein-sugar systems resulted in noticeable losses of arginine and lysine residues. Polymerization, as well as impairment of arginine residues of lysozyme, with fructose and aldopentose was higher than with aldohexoses. 3-Deoxyglucosone (3DG) was identified as one of the major carbonyl compounds generated in the reaction systems of proteins with glucose and fructose. The formation of 3DG in protein-fructose systems was 1.3-2 times that in protein-glucose systems. Glucose alone did not polymerize succinylated lysozyme, but fructose did and it impaired only arginine residues. These results indicate that 3DG was generated on the reaction between glucose and free amino groups in proteins, whereas it was generated from fructose alone with the modification of free amino groups. It is strongly suggested that 3DG was the cross-linker responsible for the polymerization of proteins and the attacker of arginine residues.

Genetic polymorphism of enzymes of alcohol metabolism and susceptibility to alcoholic liver disease

Differences in the pharmacokinetics of alcohol absorption and elimination are, in part, genetically determined. There are polymorphic variants of the two main enzymes responsible for ethanol oxidation in liver, alcohol dehydrogenase and aldehyde dehydrogenase. The frequency of occurrence of these variants, which have been shown to display strikingly different catalytic properties, differs among different racial populations. Since the activity of alcohol dehydrogenase in liver is a rate-limiting factor for ethanol metabolism in experimental animals, it is likely that the type and content of the polymorphic isoenzyme subunit encoded at ADH2, β-subunit, and at ADH3, the γ-subunit, are contributing factors to the genetic variability in ethanol elimination rate. The recent development of methods for genotyping individuals at these loci using white cell DNA will allow us to test this hypothesis as well as any relationship between ADH genotype and the susceptibility to alcoholism or alcohol-related pathology. A polymorphic variant of human liver mitochondrial aldehyde dehydrogenase, ADLH2, which has little or no acetaldehyde oxidizing activity has been identified. Individuals with the deficient ALDH2 phenotype do not have altered ethanol elimination rates but they do exhibit high blood acetaldehyde levels and dysphoric symptoms such as facial flushing, nausea and tachycardia, after drinking alcohol. Because acetaldehyde is so reactive, it binds to free amino groups of proteins including a 37 kilodalton hepatic protein-acetaldehyde adduct and may elicit an antibody response. We would predict that individuals who have low ALDH2 activity because of liver disease or because they have the inactive ALDH2 variant isoenzyme might form more protein-acetaldehyde adducts and elicit a greater immune response. These adducts may represent good biological markers of alcohol abuse and and may also play a role in liver injury due to chronic alcohol consumption.

Characterization of glycated proteins by 13C NMR spectroscopy. Identification of specific sites of protein modification by glucose.

13C NMR spectroscopy has been used to characterize Amadori (ketoamine) adducts formed by reaction of [2-13C]glucose with free amino groups of protein. The spectra of glycated proteins were acquired in phosphate buffer at pH 7.4 and were interpreted by reference to the spectra of model compounds, N alpha-formyl-N epsilon-fructose-lysine and glycated poly-L-lysine (GlcPLL). The anomeric carbon region of the spectrum (approximately 90-105 ppm) of glycated cytochrome c was superimposable on that of N alpha-formyl-N epsilon-fructose-lysine, and contained three peaks characteristic of the alpha- and beta-furanose and beta-pyranose anomers of Amadori adducts to peripheral lysine residues on protein (pK alpha approximately 10.5). The spectrum of GlcPLL yielded six anomeric carbon resonances; the second set of three was displaced about 2 ppm to lower shielding of the first and was assigned to the Amadori adduct at the alpha-amino terminus (pK alpha approximately 7.5). The spectrum of glycated RNase was similar to that of GlcPLL, but contained a third set of three signals attributable to modification of active site lysine 41 (pK alpha approximately 8.8). The assignments for RNase were confirmed by analysis of spectra taken at pH 4 and under denaturing conditions. The spectrum of glycated hemoglobin was comparable to that of GlcPLL, and distinct resonances could be assigned to Amadori adducts at amino-terminal valine and intrachain N epsilon-lysine residues. Chemical analyses were performed to measure the relative extent of alpha- and epsilon-amino group modification in the glycated macromolecules, and the results were compared with estimates based on integration of the NMR spectra.

Theoretical and Practical Application of Non‐Enzymatic Glycosylationin Diabetes

AbstractDuring heating or prolonged storage of food, a brown color results, in the presence of reducing sugars. This browning of foods (i.e. Maillard reaction) results from a direct non‐enzymatic reaction between free amino groups in proteins and reducing sugars, and results in a decrease in nutritional availability of lysine. Non‐enzymatic glycosylation occurs with proteins in vivo, is increased in diabetic subjects, and is dependent on concentrations of both proteins and sugars, turnover rates of proteins and accessibility of proteins to sugars. Functional changes have been demonstrated to occur in an increasing variety of proteins as a consequence of non‐enzymatic glycosylation. Thus, aberrant function has been suggested with serum albumin, neural tubulin, collagen, low density lipoproteins, fibrin, lens crystallins following glucosylation.

Nonenzymatic browning in vivo: possible process for aging of long-lived proteins.

The incubation of lens proteins with reducing sugars leads to the formation of fluorescent yellow pigments and cross-like similar to those reported in aging and cataractous human lenses. Called nonenzymatic browning or the Maillard reaction, this aging process also occurs in stored foods. Reducing sugars condense with the free amino group of proteins, then rearrange and dehydrate to form unsaturated pigments and cross-linked products. Although most proteins in living systems turn over with sufficient rapidity to avoid nonenzymatic browning, some, such as lens crystallins and skin collagen, are exceptionally long-lived and may be vulnerable.

Immuno-suppressive lymphocyte factors. I. Purification of inhibitor of DNA synthesis to homogeneity.

Inhibitor of DNA synthesis (IDS) is a T-lymphocyte factor, whose role in immunnoregulation might be to nonspecifically suppress the immune system especially in situations where very high, prolonged tolerogenic doses of antigens are present. We have purified IDS-contained supernates of stimulated lymphocytes to homogeneity, through isoelectric focusing and Sephadex gel chromatography. IDS has an isoelectric point of 2.73-2.75 and in its monomeric form has a mol wt of 20,000 but exists in the supernate usually as an aggregated tetrameric form. Di- and trimeric forms are also seen. All forms are biologically active. Purity was confirmed by SDS gel electrophoresis and the binding of dansyl chloride to terminal or free amino groups of proteins and peptides. We have, further confirmed that pure IDS is not cytotoxic and is probably a glycoprotein whose activity depends on an intact carbohydrate moiety.

Irreversible Binding of Chlorinated Ethylenes to Macromolecules

Rats have been exposed in a closed system to the chlorinated ethylenes vinyl chloride and trichloroethylene and to carbon tetrachloride as a reference compound. Data of uptake of the compounds, of urinary excretion of metabolites, and of exhalation after exposure show that the chlorinated ethylenes are metabolized much faster than carbon tetrachloride, probably due to their common ethylene structure. To eliminate differences in uptake, calculation of metabolites of the three compounds in tissues was based on the amount actually taken up by the animals. Vinyl chloride, trichloroethylene, and carbon tetrachloride showed irreversible binding of metabolites to tissue proteins, mainly of the liver. Irreversible protein binding of either of these compounds ranged within the same order of magnitude, if related to the amount of compound which had been taken up. Also, no differences in the relative portion of irreversibly bound metabolites were found after exposure to different atmospheric concentrations of the three compounds. As already shown for vinyl chloride, trichloroethylene is metabolized in vitro by rat liver microsomes in presence of NADPH-regenerating system to intermediates that irreversibly bind to proteins. Albumin (bovine and rabbit) was a preferred target for binding. In contrast to vinyl chloride, significant irreversible binding of trichloroethylene metabolites also occurred to non-SH-proteins (γ-globulin, concanavalin A) and to polylysine. Hence it should be inferred that, unlike vinyl chloride, trichloroethylene metabolites not only bind to sulfhydryl groups but also, to a lesser extent, to free amino groups of proteins.

Irreversible binding of chlorinated ethylenes to macromolecules.

Rats have been exposed in a closed system to the chlorinated ethylenes vinyl chloride and trichloroethylene and to carbon tetrachloride as a reference compound. Data of uptake of the compounds, of urinary excretion of metabolites, and of exhalation after exposure show that the chlorinated ethylenes are metabolized much faster than carbon tetrachloride, probably due to their common ethylene structure. To eliminate differences in uptake, calculation of metabolites of the three compounds in tissues was based on the amount actually taken up by the animals. Vinyl chloride, trichloroethylene, and carbon tetrachloride showed irreversible binding of metabolites to tissue proteins, mainly of the liver. Irreversible protein binding of either of these compounds ranged within the same order of magnitude, if related to the amount of compound which had been taken up. Also, no differences in the relative portion of irreversibly bound metabolites were found after exposure to different atmospheric concentrations of the three compounds. As already shown for vinyl chloride, trichloroethylene is metabolized in vitro by rat liver microsomes in presence of NADPH-regenerating system to intermediates that irreversibly bind to proteins. Albumin (bovine and rabbit) was a preferred target for binding. In contrast to vinyl chloride, significant irreversible binding of trichloroethylene metabolites also occurred to non-SH-proteins (gamma-globulin, concanavalin A) and to polylysine. Hence it should be inferred that, unlike vinyl chloride, trichloroethylene metabolites not only bind to sulfhydryl groups but also, to a lesser extent, to free amino groups of proteins.

Synthesis of p-isothiocyanatophenyl 3- O-(3,5-dideoxy-α- d- ribo-hexopyranosyl)-α- d-mannopyranoside

The synthesis of the title disaccharide derivative ( 1C), corresponding to the Salmonella O-factor 2 1, is described. Treatment of 2- O-benzyl-4- O- p-nitrobenzoyl-α-paratosyl bromide ( 5) with p-nitrophenyl 2- O-benzyl-4,6- O-benzylidene-α- d-mannoside in dichloromethane, in the presence of mercuric cyanide, gave the α- and β-linked disaccharide derivatives ( 6a and 6b) in yields of 34 and 5%, respectively. The disaccharide derivative 10 can react with free amino groups in proteins to produce artificial antigens useful in studies on Salmonella immunology.

16] Phthalylation of amino groups

This chapter presents a procedure for the phthalylation of amino groups. It has been found that phthalic anhydride reacts rapidly and specifically at pH 8.5 with the free amino groups of proteins and peptides, thereby converting them quantitatively to o -carboxybenzoyl derivatives. This reaction is also quantitatively reversible by the incubation of the phthalyl derivative at pH 3.5 and 50° for 48 hr. Spectral changes occur during the reaction of phthalic anhydride with free amino groups, allowing the conclusion that open o -carboxybenzoyl derivatives, and not cyclic imides, are formed exclusively under slightly alkaline pH reaction conditions. The important contribution to the absorption, at 270 nm, of the incorporated benzene rings provides a sensitive way of ascertaining the extent of the reaction. Two methods have been found useful for checking that the reaction has taken place quantitatively. The first method relies on the spectral changes associated with the reaction. The second method makes use of the reaction of dansyl chloride with free amino groups.

Influence of water activity on the enzymatic changes in freeze-dehydrated muscle. II. Reactions of carbohydrates (author's transl)

During storage of prerigor freeze-dried beef, glycogen is not broken down even at 97.5% r.h. (moisture content of the meat about 30%). However, the metabolites of glycogen -- glucose, fructose, and their phosphoric acid esters -- are changed during storage at r.h. greater than 25%, mainly by the effect of glycolytic enzymes. Also nonenzymic reactions of the Maillard type seem to occur. An accelerated breakdown of these carbohydrates with increasing water activity was found. Even though the sugar monophosphates are broken down, no increase in C3 metabolites was found. The reason for this could be that the energy-rich compounds glyceraldehyde phosphate and dihydroxyacetone phosphate form complexes with some proteins. It is suggested that also pyruvate reacts with free amino groups of proteins. The breakdown of carbohydrates increases in uncooked freeze-dried samples above 60% r.h. whereas nonenzymatic reactions of the Maillard type reach a maximum rate at nonenzymatic reactions of the Maillard type reach a maximum rate at 60% r.h. This also shows that the disappearance of carbohydrates during storage of freeze-dried prerigor beef is mainly due to enzymatic processes.

Implication of rifampicin-quinone in the irreversible binding of rifampicin to macromolecules.

1. When [3H]rifampicin is incubated with rat liver microsomes or rat liver homogenate, minor amounts are bound irreversibly to protein. This effect does not depend on the presence of NAD, NADH, NADP or NADPH. 2. Rifampicin is autoxidized at physiological pH. The product of autoxidation, rifampicin-quinone, if incubated with albumin, shows a much greater irreversible binding to the protein than the parent compound rifampicin. Hence it is concluded that rifampicin may bind irreversibly to proteins in a non-enzymic reaction after autoxidation to rifampicin-quinone. 3. Rifampicin-quinone also binds irreversibly to RNA and poly-L-lysine, if incubated with these compounds. This suggests that free amino groups of protein or RNA are involved in the binding. 4. 48 h after dosage of [3H]rifampicin (33 mg/kg) to rats, 29-2 +/- 4-1 (S.D.) pmol are bound irreversibly to 1 mg liver RNA, 15.8 +/- 8-1 pmol to 1 mg liver protein and 5-0 +/- 0-47 pmol to 1 mg protein in brain tissue. 5. Microsomal NADPH-cytochromcin-quinone to rifampicin. The KM of this reaction is 10(-4) M. Induction of the NADPH-cytochrome c reductase by pre-treatment of rats with 20 mg/kg rifampicin over 5 days results in a corresponding increase of increase of rifampicin-quinone reduction. 6. These results suggest that microsomal NADPH-cytochrome c reductase prevents accumulation of higher amounts of possibly toxic rifampicin-quinone by reduction to rifampicin.

Preparation of 6α- and 6β-carboxymethyl steroid conjugates and their use in radioimmunoassay for progesterone

A method was devised to utilize the Δ 4-3-ketone structure of steroids to obtain a functional group at the 6 position which could be conjugated to proteins. The bis ethylene ketal of progesterone was formed and the resulting Δ 5 double bond was subjected to epoxidation. The 5α,6α-epoxide formed was opened with allyl magnesium bromide after which the terminal carbon-carbon double bond was oxidized to provide a carboxyl group which could be coupled to the free amino groups of proteins. The 6α and 6β carboxymethyl progesterone derivatives formed by this method were coupled to bovine serum albumin (BSA) using tributyl amine and i -butyl chlorocarbonate. The 6α conjugate averaged 19 residues per molecule of BSA and the 6β, 24 residues per BSA. Antisera to the conjugates produced in rabbits showed very little cross-reaction with steroids which differed from progesterone at positions other than C-5 or C-6. There appeared to be very little difference between the specificities of the 6α and 6β antisera.