Pyridoxamine scavenges protein carbonyls and inhibits protein aggregation in oxidative stress-induced human HepG2 hepatocytes.

Introduction of carbonyl groups into amino acid residues is a hallmark for oxidative damage to proteins by reactive oxygen species (ROS). Protein carbonylation can have deleterious effects on cell function and viability, since it is generally unrepairable by cells and can lead to protein dysfunction and to the production of potentially harmful protein aggregates. Meanwhile, pyridoxamine (PM) is known to scavenge various toxic carbonyl species derived from either glucose or lipid degradation through nucleophilic addition. PM is also demonstrated to catalyze non-enzymatic transamination reactions between amino and α-keto acids. Here, we found that PM scavenges protein carbonyls in oxidized BSA with concomitant generation of pyridoxal and recovers oxidized lysozyme activity. Moreover, we demonstrated that the treatment of H2O2-exposed HepG2 hepatocytes with PM significantly reduced levels of cellular carbonylated proteins and aggregated proteins, and also improved cell survival rate. Our results suggest that PM may have potential efficacy in ameliorating ROS-mediated cellular dysfunction.

Fluorescent detection of alpha-aminoadipic and gamma-glutamic semialdehydes in oxidized proteins.

The oxidative modification of proteins is believed to play a critical role in the etiology and/or progression of several diseases. alpha-Aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) residues represent major oxidized amino acids generated in oxidized proteins. This paper describes a novel procedure for the specific and sensitive determination of AAS and GGS after their reductive amination with sodium cyanoborohydride and p-aminobenzoic acid, a fluorescence reagent, to their corresponding derivatives, followed by a high-performance liquid chromatography (HPLC) analysis. This fluorescent labeling of protein-associated aldehyde moieties is a simple and accurate technique that may be widely used to reveal increased levels of oxidatively modified proteins with reactive oxygen species during aging and disease.

Assessment of prion inactivation by fenton reaction using protein misfolding cyclic amplification and bioassay.

An abnormal isoform of the prion protein, associated with transmissible spongiform encephalopathies, retains infectivity even after undergoing routine sterilization processes. We found that a formulation of iron ions combined with hydrogen peroxide effectively reduced infectivity and the level of abnormal isoforms of the prion protein in scrapie-infected brain homogenates. Therefore, the Fenton reaction has potential for prion decontamination.

Proteomic analysis of wheat flour allergens.

Wheat can cause severe IgE-mediated systematic reactions, but knowledge on relevant wheat allergens at the molecular level is scanty. The aim of the present study was to achieve a more detailed and comprehensive characterization of the wheat allergens involved in food allergy to wheat using proteomic strategies, referred to as "allergenomics". Whole flour proteins were separated by two-dimensional gel electrophoresis with isoelectric focusing and lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, IgE-binding proteins were detected by immunoblotting with sera of patients with a food allergy to wheat. After tryptic digestion, the peptides of IgE-binding proteins were analyzed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. In this study, we identified four previously reported wheat allergens or their sequentially homologous proteins [serpin, alpha-amylase inhibitor, gamma-gliadin, and low molecular weight (LMW) glutenin] by a database search. As a result of the high resolution of two-dimensional gel electrophoresis, nine subunits of LMW glutenins were identified as the most predominant IgE-binding antigens. The two-dimensional allergen map can be beneficial in many ways. It could be used, for example, for precise diagnosis of wheat-allergic patients and assessment of wheat allergens in food. Additionally, we compared allergenomics to conventional biochemical methods and evaluated the usefulness of a proteomic strategy for identifying putative allergens to wheat allergy.

Prion inactivation by the Maillard reaction.

Since variant Creutzfeldt-Jakob disease (vCJD) has been suspected to be attributable to the infectious agents associated with bovine spongiform encephalopathy (BSE), it is important to prevent the transmission of pathogenic forms of prion protein (PrP(Sc)) through contaminated feeding materials such as meat and bone meal (MBM). Here, we demonstrate that the Maillard reaction employing a formulation of glucose in combination with sodium hydrogen carbonates effectively reduced the infectivity (approximately 5.9-log reduction) of a scrapie-infected hamster brain homogenate. In addition to a bioassay, a protein misfolding cyclic amplification (PMCA) technique, in which PrP(Sc) can be amplified in vitro, was used as a rapid test for assessing PrP(Sc) inactivation. The PMCA analysis also indicated that the PrP(Sc) level in the infected material significantly decreased following the Maillard reaction. Therefore, the Maillard reaction can be employed for the decontamination of large amounts of byproducts such as MBM.

Metal-catalyzed oxidation of protein-bound dopamine.

Dopamine (DA) is an unstable neurotransmitter that readily oxidizes to the DA quinone and forms reactive oxygen species, such as superoxide and hydrogen peroxide. The oxidized dopamine also forms thiol conjugates with sulfhydryl groups on cysteine, glutathione, and proteins. In the present study, we determined the redox potential of the protein-bound DA and established a novel mechanism for the oxidative modification of the protein, in which the DA-cysteine adduct generated in the DA-modified protein causes oxidative modification of the DA-bound protein in the presence of Cu2+. Exposure of a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, to DA resulted in a significant loss of sulfhydryl groups and the formation of the DA-cysteine adduct. When the DA-modified protein was incubated with Cu2+, we observed aggregation and degradation of the DA-bound protein and concomitant formation of a protein carbonyl, a marker of an oxidatively modified protein. Furthermore, we analyzed the carbonyl products generated during the Cu2+-catalyzed oxidation of the DA-modified protein and revealed the production of glutamic and aminoadipic semialdehydes, consisting of the protein carbonyls generated. The cysteinyl-DA residue generated in the DA-modified protein was suggested to represent a redox-active adduct, based on the observations that the cysteinyl-DA adduct, 5-S-cysteinyldopamine, produced by the reaction of cysteine with DA, gave rise to the oxidative modification of bovine serum albumin in the presence of Cu2+. These data suggest that the DA-modified protein may be involved in redox alteration under oxidative stress, whereby DA covalently binds to cysteine residues, generating the redox-active cysteinyl-DA adduct that causes the metal-catalyzed oxidation of protein.

New method for the quantitative determination of major protein carbonyls, alpha-aminoadipic and gamma-glutamic semialdehydes: investigation of the formation mechanism and chemical nature in vitro and in vivo.

Alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) are identified as the major carbonyl products in oxidized proteins. To elucidate the formation pathway of AAS and GGS in vivo, we developed and validated a new quantification method. AAS and GGS in proteins were derivatized by reductive amination with NaCNBH(3) and p-aminobenzoic acid, a fluorescent reagent, followed by acid hydrolysis. It is noteworthy that the fluorescent derivatives were completely stable during acid hydrolysis. The present method permitted the specific, accurate, and sensitive quantification of both semialdehydes by fluorometric high-performance liquid chromatography. Analysis of proteins oxidized by various oxidation systems revealed that AAS and GGS are notably generated by the reaction of proteins with (*)OH, which is produced by metal-catalyzed oxidation (MCO). Furthermore, exposure of transferrin and human plasma to ascorbic acid and H(2)O(2) significantly promoted the formation of AAS and GGS in vitro, suggesting that both semialdehydes can be generated by MCO in vivo. We also demonstrated their generation through oxidative stress induced by acute iron overload in vivo. In this paper, we describe this analytical technique for simple and precise measurement of AAS and GGS and discuss their formation mechanism in vivo.

Oxidative deamination of benzylamine and lysine residue in bovine serum albumin by green tea, black tea, and coffee.

Oxidative deamination by various polyphenolic compounds is presumed to be due to the oxidative conversion of polyphenols to the corresponding quinones through autoxidation. Here we examined the oxidative deamination by the polyphenol-rich beverages green tea, black tea, and coffee at a physiological pH and temperature. Green tea, black tea, and coffee extracts oxidatively deaminated benzylamine and the lysine residues of bovine serum albumin to benzaldehyde and alpha-aminoadipic delta-semialdehyde residues, respectively, in sodium phosphate buffer (pH 7.4) at 37 degrees C in both the presence and absence of Cu2+, indicating the occurrence of an amine (lysyl) oxidase-like reaction. We also examined the effects of pH and metal ions on the reaction. The possible biological effects of drinking polyphenol-rich beverages on human are also discussed.

Formation of alpha-aminoadipic and gamma-glutamic semialdehydes in proteins by the maillard reaction.

Recent research has demonstrated that nonenzymatic glycation (the Maillard reaction) lead to the formation of carbonyl groups and advanced glycation end products (AGEs) in proteins. Such oxidative modifications are a major contributing factor to diabetic complications and aging. alpha-Aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) have been identified as the major carbonyl products in oxidized proteins both in vitro and in vivo. AAS is an oxidative deamination product of lysine residue, while GGS originates from arginine and proline residues. To evaluate oxidative damage to proteins by the Maillard reaction, we developed a method of detecting AAS and GGS by high-performance liquid chromatography (HPLC). The aldehydic residues in proteins were derivatized by reductive amination with NaCNBH3 and p-aminobenzoic acid (ABA), a fluorescence regent. After acid hydrolysis of the ABA-derivatized protein, ABA-AAS and ABA-GGS were measured by fluorometric HPLC. Thus, AAS and GGS could be detected in various proteins such as human plasma protein using the present method. Accumulation of both aldehydic residues was observed in oxidized proteins by reactive oxygen species. Furthermore, AAS and GGS were markedly formed in the incubation of BSA with ascorbic acid. The formation of both aldehydic residues was also observed in the incubation of BSA with 100 mM glucose or 1.0 mM methylglyoxal in the absence and presence of 100 microM Fe3+ for 2 weeks. These results suggest that the Maillard reaction can contribute to the formation of AAS and GGS in vivo.

Removal of selenium and arsenic by animal biopolymers.

The animal biopolymers prepared from hen eggshell membrane and broiler chicken feathers, which are byproducts of the poultry-processing industry, were evaluated for the removal of the oxyanions selenium [Se(IV) and Se(VI)] and arsenic [As(III) and As(V)] from aqueous solutions. The biopolymers were found to be effective at removing Se(VI) from solution. Optimal Se(IV) and Se(VI) removal was achieved at pH 2.5-3.5. At an initial Se concentration of 100 mg/L (1.3 mM), the eggshell membrane removed approx 90% Se(VI) from the solution. Arsenic was removed less effectively than Se, but the chemical modification of biopolymer carboxyl groups dramatically enhanced the As(V) sorption capacity. Se(VI) and As(V) sorption isotherms were developed at optimal conditions and sorption equilibrium data fitted the Langmuir isotherm model. The maximum uptakes by the Langmuir model were about 37.0 mg/g and 20.7 mg/g of Se(VI) and 24.2 mg/g and 21.7 mg/g of As(V) for eggshell membrane and chicken feathers, respectively.

Oxidative deamination of benzylamine by glycoxidation.

In the present study, model reactions for the oxidative deamination by glycoxidation using benzylamine were undertaken to elucidate the detail of the reaction. Glucose, 3-deoxyglucosone (3-DG), and methylglyoxal (MG) oxidatively deaminated benzylamine to benzaldehyde in the presence of Cu(2+) at a physiological pH and temperature but not glyoxal. 3-DG and MG were more effective oxidants than glucose. We have determined the effects of metal ions, pH, oxygen, and radical scavengers on the oxidative deamination. The formation of benzaldehyde was greatest with Cu(2+), and was accelerated at a higher pH and in the presence of oxygen. EDTA, catalase, and dimethyl sulfoxide significantly inhibited the oxidation, suggesting the participation of reactive oxygen species. From these results, we propose a mechanism for the oxidative deamination by the Strecker-type reaction and the reactive oxygen species-mediated oxidation during glycoxidation.

Production of hydrogen peroxide by polyphenols and polyphenol-rich beverages under quasi-physiological conditions.

To investigate the ability of the production of H(2)O(2) by polyphenols, we incubated various phenolic compounds and natural polyphenols under a quasi-physiological pH and temperature (pH 7.4, 37 degrees C), and then measured the formation of H(2)O(2) by the ferrous ion oxidation-xylenol orange assay. Pyrocatechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, and polyphenols such as catechins yielded a significant amount of H(2)O(2). We also examined the effects of a metal chelator, pH, and O(2) on the H(2)O(2)-generating property, and the generation of H(2)O(2) by the polyphenol-rich beverages, green tea, black tea, and coffee, was determined. The features of the H(2)O(2)-generating property of green tea, black tea, and coffee were in good agreement with that of phenolic compounds, suggesting that polyphenols are responsible for the generation of H(2)O(2) in beverages. From the results, the possible significances of the H(2)O(2)-generating property of polyphenols for biological systems are discussed.

Oxidative deamination of lysine residue in plasma protein of diabetic rats. Novel mechanism via the Maillard reaction.

The levels of alpha-aminoadipic-delta-semialdehyde residue, the oxidative deamination product of lysine residue, in plasma protein from streptozotocin-induced diabetic rats were evaluated. alpha-Aminoadipic-delta-semialdehyde was converted to a bisphenol derivative by acid hydrolysis in the presence of phenol, and determined by high performance liquid chromatography. Analysis of plasma proteins revealed three times higher levels of alpha-aminoadipic-delta-semialdehyde in diabetic subjects compared with normal controls. Furthermore, we explored the oxidative deamination via the Maillard reaction and demonstrated that the lysine residue of bovine serum albumin is oxidatively deaminated during the incubation with various carbohydrates in the presence of Cu2+ at a physiological pH and temperature. This experiment showed that 3-deoxyglucosone and methylglyoxal are the most efficient oxidants of the lysine residue. When the reaction was initiated from glucose, a significant amount of alpha-aminoadipic-delta-semialdehyde was also formed in the presence of Cu2+. The reaction was significantly inhibited by deoxygenation, catalase, and a hydroxyl radical scavenger. The mechanism we propose for the oxidative deamination is the Strecker-type reaction and the reactive oxygen species-mediated oxidation. Based on these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residue via the Maillard reaction.

Oxidative deamination by hydrogen peroxide in the presence of metals.

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H2O2/Cu2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O2 on the oxidative deamination of benzylamine by H202. The formation of benzaldehyde was the greatest with Cu2+, and catalysis occurred with Co2+, VO2+, and Fe3+. The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O2/Cu2+ and hemoglobin/H2O2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H2O2/Cu2+ system is discussed.

Selective recovery of uranium and thorium ions from dilute aqueous solutions by animal biopolymers.

Selective actinide ion recovery from dilute, aqueous, multication waste streams is an important problem. The recovery of uranium (U) and thorium (Th) by various animal biopolymers was examined. Of four species of biopolymers tested, a high uptake of uranium and thorium was found in hen eggshell membrane (ESM) and silk proteins, with the maximum uranium and thorium recovery exceeding 98% and 79%, respectively. The uptake of U and Th was significantly affected by the pH of the solution. The optimum pH values were 6 and 3 for the uptake of U and Th, respectively. The effect of temperature differed with the metal. The uptake of U decreased with increasing temperature (30-50 degrees C), whereas the Th uptake increased with increasing temperature. Selective recovery of U and Th from dilute aqueous binary and multimetal solutions was also examined. ESM and silk proteins tested were effective and selective for removing each metal by controlling the pH and temperature of the solution. In multimetal systems, the order of sorption of ESM proteins was preferential: U > Cu > Cd > Mn > Pb > Th > Ni > Co > Zn at pH 6 and Th > U > Cu > Pb > Cd > Mn > Co > Ni = Zn at pH 3. These biopolymers appear to have potential for use in a commercial process for actinide recovery from actinide-containing wastewater.

Uptake and recovery of gold ions from electroplating wastes using eggshell membrane.

The animal byproduct, hen eggshell membrane (ESM), was evaluated for its ability to sorb gold ions (dicyanoaurate(I) and tetrachloroaurate(III)) from solutions and electroplating wastewater. The gold uptake was dependent on pH, temperature and co-ions present in the solutions, with pH 3.0 being the optimum value. The equilibrium data followed the Langmuir isotherm model with maximum capacities of 147 mg Au(I)/g dry weight and 618 mg Au(III)/g, respectively. Desorption of sorbed gold(I) with 0.1 mol/l NaOH resulted in no changes of the biosorbent gold uptake capacity through five consecutive sorption/desorption cycles. In column experiments, selective recovery of gold from electroplating wastewater containing various metal ions was noted. The affinity of metal sorption was in the order Au > Ag > Co > Cu > Pb > Ni > Zn.