Search

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.

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.

The formation of cross-links in elastin and collagen is a basic phenomenon of maturation, and this process is associated with the formation of covalent bonds within these extracellular matrix protein structures. Various amino acids and their derivatives have been implicated and the cross-links can be intramolecular-linking within the same molecule, and intermolecular-covalent bonds between chains in different molecules. The basic mechanism of cross-linking is oxidative deamination of the ε-amino groups in lysine or hydroxylysine residues, catalyzed by lysyl oxidase. It is believed that oxidative deamination is the only step in cross-link formation that is catalyzed enzymatically. The aldehydes, allysine and/or hydroxyallysine, thus formed contribute to further chemical reactions to form cross-linking structures. In this review, I describe the chemistry and biology of cross-link formation and the function of cross-links in collagen and elastin.

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.

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.

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.

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.

The lysine residue of bovine serum albumin was deaminated to allysine (α-aminoadipic-δ-semialdehyde) during the incubation with glucose, 3-deoxyglucosone (3-DG), and methylglyoxal (MG) in the presence of Cu2+ at a physiological pH and temperature but not with glyoxal. Further, glucose, 3-DG, and MG oxidatively deaminated benzylamine to benzaldehyde in the presence of Cu2+. The formation of benzaldehyde was greatest with Cu2+, and was accelerated in the presence of oxygen. EDTA, catalase, and dimethyl sulfoxide (DMSO) significantly inhibited the oxidation. Analysis of plasma proteins revealed significantly higher levels of allysine in streptozotocin (STZ)-induced diabetic rats compared with normal controls. From these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes 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.

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 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.

Isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. The structures of these amino acids were determined to have 3,4,5- and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of 396 (C18H28N406) identified as 4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine and 2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine. We have named these pyridine cross-links desmopyridine (DESP) and isodesmopyridine (IDP), respectively. Structure analysis of these pyridine cross-links implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. The elastin incubated with ammonium chloride showed that DESP and IDP levels increased as the allysine content decreased. DESP and IDP were measured by high pressure liquid chromatography (HPLC) with UV detection and were found in a variety of bovine tissues. The DESP/desmosine (DES) and IDP/isodesmosine (IDE) ratios in aorta elastin were higher than in other tissues. DESP and IDP contents in human aorta elastin were found to be gradually increased with age. The concentration of IDP was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride (mean ± S.D.; 11.1 ± 0.9 nmol/mg elastin) when compared with normal rats (5.9 ± 1.5 nmol/mg elastin). Although DESP and IDP are present at only trace concentrations in the tissue elastin, these pyridine cross-links may be useful biomarkers for the aortic elastin damaged by ammonia.

Polyphenols in several oxidation systems gained amine oxidase-like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o- and p-phenolic compounds exhibited amine oxidase-like activity, whereas only the o-phenolic compounds showed the activity in the presence of 1,1-diphenyl-2-picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to alpha-amino-adipic semialdehyde residue, indicating lysyl oxidase-like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not beta-aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.

Lactose is a principal carbohydrate in nearly all species of mammalian milk. In order to examine the acceptor substrate specificity of lactose synthase in vivo, d-xylose as an acceptor substrate was injected into the jugular vein of a Holstein cow during lactation, then a milk sample obtained by milking. β1,β1-galactopyranosyl xylopyranoside, a nonreducing disaccharide, was separated from the bovine milk sample after elimination of reducing sugars, identified by fast-atom bombardment (FAB)-MS and 1H-NMR analysis. A mixture of β1,β1- and β1,4-galactopyranosyl xylopyranoside fractions was also obtained by thin layer chromatography from the milk sample and elucidated by electrospray ionization (ESI)-MS and 1H NMR analysis. Comparison of the integrated intensity of the products shows that the β1,β1 and β1,4 isomers are present in a ratio of 1.0 : 1.4, suggesting that d-xylose, transported from capillary blood across the plasma membrane of the mammary gland, was recognized by lactose synthase in its normal and reverse orientation owing to high symmetry of its structure. While the β1,4-isomer is known as a fragment of the linkage region between the protein and the polysaccharide chain of proteoglycans, the β1,β1-isomer has not been identified in vivo. Here, we demonstrate that galactosylation of d-xylose transported from the capillary blood can occur by lactose synthase catalysis in the mammary gland while the usual galactosylation of d-glucose proceeds. In addition, these results suggest that the possibility of endogenous occurrence of the β,β-trehalose type disaccharide in the mammary gland of lactating mammals may not be ruled out.

Removing and collecting heavy and rare metal ions from industrial effluents and waste aqueous solutions are important problems. Our previous studies showed that animal fibrous proteins (AFPs) such as hen eggshell membrane, chicken feather (CF), wool, and silk were stable and insoluble proteins and had an excellent ability to bind not only hard (Mn2+ and Fe3+) but also soft (Ag+, Au+, Pd2+, Pt2+, and Hg2+) acids from aqueous solutions. In this study, we synthesized some adsorbents for transition (Cr6+, Mn2+, Co2+, Ni2+, and Cu2+) and heavy (Cd2+) metal ions from AFPs (gelatin, CF, and wool) and plant polyphenols (lignin and tannin) by heating a mixture of AFPs and plant polyphenols under acidic conditions. In batch experiments, pH profile, time dependency, and isotherm analysis were performed to determine binding properties of adsorbents for transition and heavy metal ions. Column experiments were also performed to remove copper ion from aqueous solution. The results showed that the new adsorbents were effective for collecting and removing transition and heavy metal ions from aqueous solutions.

We examined the formation of quaternary pyridinium crosslinks of elastin formed by condensation of lysine and allysine residues using the model compounds propanal (allysine) and n-butylamine (lysine) under quasi-physiological conditions. The resulting pyridinium compounds were characterized and the structure compared with the known pyridinium crosslinks. Three pyridinium compounds were identified and the structures were identical with the skeleton of the crosslinking amino acids, desmosine (DES), isodesmosine (IDE), and pentasine. We concluded that a non-enzymatic pathway is available for the spontaneous generation of pyridinium crosslinks. To elucidate the intermediates and the mechanism of the formation of DES and IDE, we synthesized model intermediates from propanal and n-butylamine, and they were allowed to react in three kinds of solvents. Then, the products were analyzed by an ion-pair reverse-phase HPLC. The results of this model system indicated that DES and IDE can be formed by condensation of dehydromerodesmosine with dehydrolysinonorleucine and by condensation of allysine with dehydrolysinonorleucine, respectively. We also describe the mechanism of DES and IDE crosslinking.

Animal fibrous proteins (AFPs) such as egg-shell membrane (ESM), chicken feather (CF), wool, silk, or elastin are an intricate network of stable and water-insoluble fibers with high surface area and are abundant bioresources. Every AFP tested was found to accumulate gold-cyanide ion from aqueous solutions in high yield, depending on pH and some other parameters. Gold-cyanide ion is adsorbed by AFP at low pH range, with maximum binding observed at approx pH 2.0. Under the certain conditions, gold-cyanide ion was accumulated up to 8.6, 7.1, 9.8, 2.4, and 3.9% of dry weight on ESM, CF, wool, silk, and elastin, respectively. In the case of ESM, it was found that ESM removed gold-cyanide ion almost quantitatively and almost all the gold uptake by ESM was easily desorbed with 0.1 M NaOH. ESM can be used repeatedly for the process of gold adsorption-desorption. The gold-biosorptive capacity of ESM that was chemically modified with glutaraldehyde was higher than that of control. In column procedure, ESM packed on column removed gold-cyanide ion from the dilute aqueous solution to extremely low concentrations (nondetectable concentration of below 1 ppb).

食品のタンパク質のグリケーションの程度は,フルクトシルリジンやラクチュロシルリジンなどアマドリ化合物を含むタンパク質の酸加水分解により生成する,フロシンの定量によって行なうことができる.本実験では,フロシンを効率的に生成する加水分解条件およびカウンターイオンとしてSDSを用いた逆相系HPLCによるフロシンの分析法について検討した.その結果,フロシンの生成の最適条件の設定と共に,フロシンの定量を約15分で効率よく行なう方法を開発した.本法を用いて,乳および乳製品タンパク質のグリケーションの程度を調べた結果,スキムミルク,乳児用調整粉乳および原料に脱脂粉乳を用いた発酵乳において高い値を示した.

An ion-paired high-performance liquid chromatographic method for the determination of aldosine, which is derived from the aldol crosslink of both elastin and collagen, is described. Aldosine was determined as the pyridine derivative 6-(3-pyridyl) piperidine-2-carboxylic acid, which was synthesized by oxidative decarboxylation of aldosine using iron(III). This method does not require radioisotopes to label the aldol crosslink. In addition, pyridinium crosslinks such as desmosines can also be measured.

With the widespread consumption of milk, the complete characterization of the constituents of milk and milk products is important in terms of functionality and safety. In this study, a novel nonreducing carbohydrate was separated from powdered skim milk and was identified using electron spray ionization-mass spectrometry ( m / z 385.1[M + H + ]), 1 H, 13 C, 1 H 1 H-correlation spectroscopy, and heteronuclear single quantum-nuclear magnetic resonance spectra. The carbohydrate was identified as a lactose derivative of urea, N -carbamoyl- o -β-d-galactopyranosyl-(1–4)-d-glucopyranosylamine (lactose ureide, LU). For the HPLC analysis of LU in milk and milk products, benzoylated LU, hepta- o -benzoyl lactose ureide (melting point 137–139°C; m / z 1,113 [M + H + ]; wavelength of maximum absorption, λ max , 229nm; molar extinction coefficient, e, 8.1037 × 10 7 ), was used as a standard. The crude nonreducing carbohydrate fraction from raw milk, thermally processed milk, and milk products such as powdered milks were directly benzoylated and subjected to HPLC analysis using an octadecylsilyl column to determine the quantity of LU. The content of LU in 10% solutions of powdered skim milk and powdered infant formula (5.0±1.1 and 4.9±1.5mg/L, respectively) were almost 3-fold higher than that of UHT milk (1.6±0.5mg/L) and higher than that of low-temperature, long-time-processed (pasteurized at 65°C for 30min) milk (1.2±0.3mg/L) and the fresh raw milk sample (0.3±0.1mg/L). A time-course of the LU content in raw milk during heating at 110°C revealed that LU increased with time. From these results, it is likely that LU is formed by the Maillard-type reaction between the lactose and urea in milk and milk products. Because the concentration of LU in milk increased with the degree of processing heat treatment, it could serve as an indicator of the thermal deterioration of milk. Although it is known that the human intestine is unable to digest LU, the gastrointestinal bacteria in human subjects are able to digest and utilize urea nitrogen in formation of essential amino acids that are available to the host human. These findings suggest that LU in milk might have a functional role in human health.

核酸構成塩基であるシトシン(Cyt)および5-メチルシトシン(5-MeCyt)を,牛および豚の肺および胸腺の加水分解物から分離調製する方法について検討した.これらの塩基は活性炭カラムおよび大口径シリカゲルカラムにより分離した.それぞれCytおよび5-MeCytは,カウンターイオンとしてSDSを用いた逆相系分取HPLCにより精製し,結晶として得た.一方で,これらの塩基の逆相HPLCによる分析法を開発し,定量を行なった.その結果,乾物1g当りのCytおよび5-MeCytの含量は,それぞれ牛肺が2.11mgと2.09mg,牛胸腺が3.83mgと4.41mgであった.豚肺においてもほぼ同量含まれていた.

Two new fluorescent crosslinking amino acids named oxodesmosine and isooxodesmosine were isolated from bovine aorta elastin. These amino acids have unique structure, both N-substituted dihydrooxopyridine skeletons.

A high-performance liquid chromatographic (HPLC) procedure for the determination and preparation of desmosine (DES) and isodesmosine (IDE), the major cross-links of elastin, has been developed. Hydrolysates of elastin are treated with a SEP-PAK silica-gel small column and then separated on a silica-gel normal phase column using n-propanol-water-25% ammonia (9:3:0.06, v/v/v) followed by detection at 275 nm. The quantitative yields of DES and IDE are 7.98 +/- 0.47 and 6.44 +/- 0.51 (mean +/- S.D., n = 10) mg/g dry weight, respectively, in bovine ligamentum nuchae. Preparations of cross-links are carried out by means of a preparative column (240 mm x 10 mm), using the same solvent system.

A new polyfunctional cross-linking amino acid was isolated from the hydrolysate of bovine ligamentum nuchae elastin. This compound was a very hygroscopic, white amorphous solid with a faint yellow tinge, and was soluble in aqueous solvents but not in dry methanol. Its proposed structure was verified by ultraviolet spectroscopy, fast atom bombardment mass spectroscopy, and 1H- and 13C-nuclear magnetic resonace spectroscopy. The data indicated it to be a pentafunctional amino acid with a quaternary pyridinium structure similar to desmosine. The mass spectral analysis indicated a parent compound with a mass of 655 (C30H51N6O10). The proposed structure is one derived from the condensation of one lysine residue and four allysine residues. Based on the names of other cross-linking amino acids found in elastin, the trivial name of allodesmosine is given for this compound. Allodesmosine was also detected in hydrolysates of bovine lung, aorta and skin by high-performance liquid chromatography.

A new pentafunctional cross-linking amino acid, termed allo-desmosine, was isolated from bovine ligamentum nuchae elastin. This compound was a very hygroscopic, white amorphous solid with a faint yellow tinge, soluble in aqueous solvents but not dry methanol; it was characterized by UV, FAB mass and NMR spectroscopy. The compound was shown by UV and 1H-NMR to have a pyridinium ring structure similar to desmosine. Mass spectral analysis indicated a parent compound with a mass of 655. We postulated that it arose by condensation of a reduced aldol condensation product of allysine, allysine and lysine.

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.

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.

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.

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.

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.

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 Cu2+ 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 Cu2+, 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.

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.

Lactosamine (beta-D-Galp-(1-->4)-D-GlcN) was isolated from bovine milk sampled after intravenous infusion of glucosamine through the jugular vein of a lactating cow. The chemical structure was established by 2D NMR spectroscopy and electrospray ionisation mass spectrometry (ESIMS). Selected ion monitoring liquid chromatography-mass spectrometry (SIMLC-MS) of the perbenzoylated carbohydrate fraction showed the presence of the novel disaccharide in the milk sample after infusion, but not in the control bovine milk sample. The results showed the uptake of glucosamine in bovine mammary gland, and also indicated that a part of glucosamine was metabolised to the product lactosamine.

We characterized a model compound for the lysine tyrosylquinone (LTQ) cofactor of lysyl oxidase which is one of the mammalian copper-dependent amine oxidases. The model compound, 4-butylamino-5-methyl-o-quinone, was prepared from n-butylamine and 4-methylcatechol by the oxidation with sodium iodate and characterized by spectroscopic analyses. The absorption maximum at 494 nm is consistent with that of lysyl oxidase. The model compound was capable of deaminating benzylamine to benzaldehyde at 37 degrees C in buffered aqueous acetonitrile. The aldehyde production was markedly elevated in the presence of the Cu(II)-EDTA complex but inhibited by free Cu(II). The catalytic cycle was observed at pH 10 in the presence of Cu(II), and the pH activity profile showed a broad optimum at about pH 9.0. In the presence of beta-aminopropionitrile and upon deoxygenation with N2 aldelyde, production was decreased. The important features of the reaction were consistent with the enzymatic reaction.

Allysine is the most important precursor of physiologically essential cross-links formation in collagen and elastin and is formed by enzymatic oxidative deamination of lysine residues. Because it is a highly reactive aldehyde, many cross-linking amino acid residues may arise from its reaction with other allysine residues or lysine or even histidine residues. We purified and isolated an allysine bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxyphenylpentane (ACPP), from the reaction products of phenol and allysine residue of bovine ligamentum nuchae by acid hydrolysis in 6 M HCl. The structure of ACPP was verified by UV, fast atom bombardment-MS, 1H- and 13C-nuclear magnetic resonance spectroscopies. The optimal reaction condition for ACPP synthesis accompanied by hydrolysis of such proteins was investigated and an ion-paired high-performance liquid chromatographic method for determination of allysine as ACPP was also developed.

A trifunctional crosslinking amino acid named cyclopentenosine (CP) was isolated from the hydrolysate of bovine nuchal ligament elastin. CP and its derivatives were identified by spectroscopic methods. CP was found to consist of a 2-cyclopenten-1-one structure and its imine-enamine tautomers with enantiomers in H(2)O. A model reaction for the formation of the CP crosslink using model compounds for allysine (propanal) and lysine (n-butylamine) demonstrated that CP is composed of 2-cyclopenten-1-one and alpha, beta, gamma, delta-unsaturated aldehyde derived from three allysine residues. An ion-paired high-performance liquid chromatographic method for the determination of CP was developed. Among various bovine tissues the nuchal ligament had the highest concentration of CP. The age-related changes in the concentration of CP were examined in the aorta from rat (short-lived species) and bovine (long-lived species). The CP content was very low in the newborn rat but increased markedly with growth. After maturity, the CP content remained nearly the same or slightly decreased. In bovine aorta, the CP content scarcely changed from 7 months to 16 years.

The activity of lysyl oxidase was found in egg shell membrane (ESM) of hens. The activity was determined by measuring the enzymatic conversion of n-butylamine and Nalpha-acetyl-L-lysine to n-butyraldehyde and Nalpha-acetyl-L-allysine, respectively. ESM lysyl oxidase was significantly inhibited by beta-aminopropionitrile, chelating agents, and deoxygenation, consistent with the known properties of lysyl oxidase. Nevertheless, ESM lysyl oxidase was insoluble in urea solution, suggesting that it complexes with ESM. These findings support previous reports indicating the presence of lysine-derived cross-links in ESM and the necessity of lysyl oxidase located in the isthmus of the hen oviduct for the biosynthesis of ESM. Lysyl oxidase secreted around the egg white from the isthmus may initiate the cross-linking reaction of ESM protein, and remain as the constituent of ESM. Moreover, the H(2)O(2) released by lysyl oxidase in ESM was completely decomposed by coexisting catalase activity. ESM lysyl oxidase activity was greatly elevated in the presence of H(2)O(2), probably due to the O(2) produced by catalase. These findings indicate that lysyl oxidase is coupled with catalase in ESM. This coupling enzyme system was considered to be involved in the biosynthesis of ESM and to protect the embryo against H(2)O(2).

Removal of radioactive elements from the effluent and waste aqueous solutions is an important problem. In previous laboratory batch experiments, hen egg-shell membrane (ESM) was stable as an insoluble protein and was very capable of binding heavy metal ions from aqueous solution. Batch laboratory pH profile, time dependency, and capacity experiments were performed to determine the binding of uranium (U) and thorium (Th) to ESM. Batch pH profile experiments indicated that the optimum pH for binding these actinides was approx 6.0 (U) or 3.0 (Th). The adsorption isotherms were developed at pH 5.0 (U) or 3.0 (Th) at 25 degrees C, and the adsorption equilibrium data fitted both Langmuir and Freundlich models. The maximum uptakes by the Langmuir model were about 240 mg U/g and 60 mg Th/g dry weight ESM. In addition, their adsorption capacities increased as salt concentration increased. ESM could also accumulate uranium from dilute aqueous solution by adjusting to the optimum pH. These results showed that ESM was effective for removing actinides from solution and would be useful in filtration technology to remove actinides from aqueous solution.

Removal of radioactive elements from the effluent and waste aqueous solutions is an important problem. In previous laboratory batch experiments, hen egg-shell membrane (ESM) was stable as an insoluble protein and was very capable of binding heavy metal ions from aqueous solution. Batch laboratory pH profile, time dependency, and capacity experiments were performed to determine the binding of uranium (U) and thorium (Th) to ESM. Batch pH profile experiments indicated that the optimum pH for binding these actinides was approx 6.0 (U) or 3.0 (Th). The adsorption isotherms were developed at pH 5.0 (U) or 3.0 (Th) at 25°C, and the adsorption equilibrium data fitted both Langmuir and Freundlich models. The maximum uptakes by the Langmuir model were about 240 mg U/g and 60 mg Th/g dry weight ESM. In addition, their adsorption capacities increased as salt concentration increased. ESM could also accumulate uranium from dilute aqueous solution by adjusting to the optimum pH. These results showed that ESM was effective for removing actinides from solution and would be useful in filtration technology to remove actinides from aqueous solution.