Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study

Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (相似文献   

Factors that influence the deoxyribose oxidation assay for Fenton reaction products.

The mechanism of oxidation of deoxyribose to thiobarbituric acid-reactive products by Fenton systems consisting of H2O2 and either Fe2+ or Fe2+ (EDTA) has been studied. With Fe2+ (EDTA), dependences of product yield on reactant concentrations are consistent with a reaction involving OH.. With Fe2+ in 5-50 mM phosphate buffer, yields of oxidation products were much higher and increased with increasing deoxyribose concentration up to 30 mM. The product yield varied with H2O2 and Fe2+ concentrations in a way to suggest competition between deoxyribose and both reactants. Deoxyribose oxidation by Fe2+ and H2O2 was enhanced 1.5-fold by adding superoxide dismutase, even though superoxide generated by xanthine oxidase increased deoxyribose oxidation. These results are not as expected for a reaction involving free OH. or site localized OH. product on the deoxyribose. They can be accommodated by a mechanism of deoxyribose oxidation involving an iron(IV) species formed from H2O2 and Fe2+, but the overall conclusion is that the system is too complex for definitive identification of the Fenton oxidant.     

Iron and dioxygen chemistry is an important route to initiation of biological free radical oxidations: an electron paramagnetic resonance spin trapping study

Iron can be a detrimental catalyst in biological free radical oxidations. Because of the high physiological ratio of [O2]/[H2O2] (> or = 10(3)), we hypothesize that the Fenton reaction with pre-existing H2O2 is only a minor initiator of free radical oxidations and that the major initiators of biological free radical oxidations are the oxidizing species formed by the reaction of Fe2+ with dioxygen. We have employed electron paramagnetic resonance spin trapping to examine this hypothesis. Free radical oxidation of: 1) chemical (ethanol, dimethyl sulfoxide); 2) biochemical (glucose, glyceraldehyde); and 3) cellular (L1210 murine leukemia cells) targets were examined when subjected to an aerobic Fenton (Fe2+ + H2O2 + O2) or an aerobic (Fe2+ + O2) system. As anticipated, the Fenton reaction initiates radical formation in all the above targets. Without pre-existing H2O2, however, Fe2+ and O2 also induce substantial target radical formation. Under various experimental ratios of [O2]/[H2O2] (1-100 with [O2] approximately 250 microM), we compared the radical yield from the Fenton reaction vs. the radical yield from Fe2+ + O2 reactions. When [O2]/[H2O2] < 10, the Fenton reaction dominates target molecule radical formation; however, production of target-molecule radicals via the Fenton reaction is minor when [O2]/[H2O2] > or = 100. Interestingly, when L1210 cells are the oxidation targets, Fe2+ + O2 is observed to be responsible for formation of nearly all of the cell-derived radicals detected, no matter the ratio of [O2]/[H2O2]. Our data demonstrate that when [O2]/[H2O2] > or = 100, Fe2+ + O2 chemistry is an important route to initiation of detrimental biological free radical oxidations.     

Biosynthesis of heparin. O-sulfation of D-glucuronic acid units

Incubation of a microsomal fraction from murine mastocytoma, with UDP-[1-3H]GlcA, UDP-GlcNAc, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), yielded labeled, N-sulfated polysaccharides, in which most of the incorporated O-sulfate groups were located at C2 of L-iduronic acid and at C6 of D-glucosamine units. Analysis by anion-exchange high pressure liquid chromatography of disaccharides, generated by deaminative cleavage of these polysaccharides, revealed that, in addition, an appreciable portion of the -GlcNSO3-HexA-GlcNSO3- sequences in the intact polymers contained O-sulfated (at C2 or C3) D-glucuronic acid units. Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-cellulose, showed that glucuronosyl 2/3-O-sulfate accounted for approximately 12% of the total incorporated O-sulfate groups. With [35S]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, thus demonstrating that these components had in fact been formed by sulfation of glucuronic acid residues and not by "back epimerization" of sulfated iduronic acid units. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed O-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-O-sulfation of glucosamine residues.     

The phosphorylated and/or sulfated structure of the carbohydrate-protein-linkage region isolated from chondroitin sulfate in the hybrid proteoglycans of Engelbreth-Holm-Swarm mouse tumor.

The structure of the linkage region of chondroitin sulfate chains attached to the hybrid proteoglycans of the Engelbreth-Holm-Swarm mouse tumor was investigated. The peptidoglycan fraction which contains oversulfated chondroitin sulfate rich in the GlcA beta 1-3GalNAc-4,6-diO-sulfate unit and undersulfated heparan sulfate rich in GlcA beta 1-4GlcNAc and GlcA beta 1-4GlcN-2N-sulfate units was isolated after exhaustive protease digestion of the acetone powder of the tumor tissue, (GlcA, glucuronic acid; GalNAc, 2-deoxy-2-N-acetylamino-D-galactose). Glycosaminoglycans were released by beta-elimination using NaB3H4 and digested with chondroitinase ABC. The linkage region fraction was separated from heparan sulfate by gel filtration and fractionated by HPLC on an amine-bound silica column. Six radiolabeled compounds (L1-L6) were obtained and structurally analyzed by cochromatography with authentic hexasaccharide alditols recently isolated by us from the linkage region, and by digestion using chondroitinase ACII, alkaline phosphatase and beta-galactosidase in conjugation with HPLC. These compounds shared the conventional hexasaccharide backbone structure: delta GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol, (delta GlcA, delta 4.5-GlcA or D-gluco-4-enepyranosyluronic acid). L1 was not sulfated or phosphorylated. L2 and L4 were monosulfated at C-6 and C-4 of the GalNAc residue, respectively. Upon alkaline phosphatase digestion, L3, L5 and L6 were converted to L1, L2 and L4, respectively. Analysis of the periodate oxidation products indicated that the phosphate group in L3, L5 and L6 is located at C-2 of Xyl-ol. These results suggest that Xyl-2-O-phosphate is associated with both 4-O-sulfated and 6-O-sulfated GalNAc units and does not directly determine the sulfation pattern of chondroitin sulfate.     

Fenton chemistry. Amino acid oxidation

The oxidation of amino acids by Fenton reagent (H2O2 + Fe(II] leads mainly to the formation of NH+4, alpha-ketoacids, CO2, oximes, and aldehydes or carboxylic acids containing one less carbon atom. Oxidation is almost completely dependent on the presence of bicarbonate ion and is stimulated by iron chelators at levels which are substoichiometric with respect to the iron concentration but is inhibited at higher concentrations. The stimulatory effect of chelators is not due merely to solubilization of catalytically inactive polymeric forms of Fe(OH)3 nor to the conversion of Fe(II) to complexes incapable of scavenging hydroxyl radicals. The results suggest that an iron chelate and another as yet unidentified form of iron are both required for maximal rates of amino acid oxidation. The metal ion-catalyzed oxidation of amino acids is likely a "caged" process, since the oxidation is not inhibited by hydroxyl radical scavengers, and the relative rates of oxidation of various amino acids by the Fenton system as well as the distribution of products formed (especially products of aromatic amino acids) are significantly different from those reported for amino acid oxidation by ionizing radiation. Several iron-binding proteins, peptides, and hemoglobin degradation products can replace Fe(II) or Fe(III) in the bicarbonate-dependent oxidation of amino acids. In view of their ability to sequester metal ions and their susceptibility to oxidation by H2O2 in the presence of physiological concentrations of bicarbonate, amino acids may serve an important role in antioxidant defense against tissue damage.     

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems.

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H(2)O(2) (photooxidation), (2) ferrous ions/H(2)O(2) (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.     

The so-called Listeria innocua ferritin is a Dps protein. Iron incorporation, detoxification, and DNA protection properties

Listeria innocua Dps (DNA binding protein from starved cells) affords protection to DNA against oxidative damage and can accumulate about 500 iron atoms within its central cavity through a process facilitated by a ferroxidase center. The chemistry of iron binding and oxidation in Listeria Dps (LiDps, formerly described as a ferritin) using H(2)O(2) as oxidant was studied to further define the mechanism of iron deposition inside the protein and the role of LiDps in protecting DNA from oxidative damage. The relatively strong binding of 12 Fe(2+) to the apoprotein (K(D) approximately 0.023 microM) was demonstrated by isothermal titration calorimetry, fluorescence quenching, and pH stat experiments. Hydrogen peroxide was found to be a more efficient oxidant for the protein-bound Fe(2+) than O(2). Iron(II) oxidation by H(2)O(2) occurs with a stoichiometry of 2 Fe(2+)/H(2)O(2) in both the protein-based ferroxidation and subsequent mineralization reactions, indicating complete reduction of H(2)O(2) to H(2)O. Electron paramagnetic resonance (EPR) spin-trapping experiments demonstrated that LiDps attenuates the production of hydroxyl radical by Fenton chemistry. DNA cleavage assays showed that the protein, while not binding to DNA itself, protects it against the deleterious combination of Fe(2+) and H(2)O(2). The overall process of iron deposition and detoxification by LiDps is described by the following equations. For ferroxidation, Fe(2+) + Dps(Z)--> [(Fe(2+))-Dps](Z+1) + H(+) (Fe(2+) binding) and [(Fe(2+))-Dps](Z+1) + Fe(2+) + H(2)O(2) --> [(Fe(3+))(2)(O)(2)-Dps](Z+1) + 2H(+) (Fe(2+) oxidation/hydrolysis). For mineralization, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH((core)) + 4H(+) (Fe(2+) oxidation/hydrolysis). These reactions occur in place of undesirable odd-electron redox processes that produce hydroxyl radical.     

KfoA,the UDP-glucose-4-epimerase of Escherichia coli strain O5:K4:H4, shows preference for acetylated substrates

Applied Microbiology and Biotechnology - Capsule of Escherichia coli O5:K4:H4 is formed of a chondroitin-repeat disaccharide unit of glucuronic acid (GlcA)-N-acetylgalactosamine (GalNAc). This...     

Tannic acid inhibits in vitro iron-dependent free radical formation

The antioxidant activity of tannic acid (TA), a plant polyphenol claimed to possess antimutagenic and anticarcinogenic activities, was studied by monitoring (i) 2-deoxyribose degradation (a technique for OH detection), (ii) ascorbate oxidation, (iii) ascorbate radical formation (determined by EPR analysis) and (iv) oxygen uptake induced by the system, which comprised Fe(III) complexes (EDTA, nitrilotriacetic acid (NTA) or citrate as co-chelators), ascorbate and oxygen. TA removes Fe(III) from the co-chelators (in the case of EDTA, this removal is slower than with NTA or citrate), forming an iron-TA complex less capable of oxidizing ascorbate into ascorbate radical or mediating 2-deoxyribose degradation. The effectiveness of TA against 2-deoxyribose degradation, ascorbate oxidation and ascorbate radical formation was substantially higher in the presence of iron-NTA (or iron-citrate) than with iron-EDTA, which is consistent with the known formation constants of the iron complexes with the co-chelators. Oxygen uptake and 2-deoxyribose degradation induced by Fe(II) autoxidation were also inhibited by TA. These results indicate that TA inhibits OH formation induced by Fe(III)/ascorbate/O(2) mainly by arresting Fe(III)-induced ascorbate oxidation and Fe(II) autoxidation (which generates Fe(II) and H(2)O(2), respectively), thus limiting the production of Fenton reagents and OH formation. We also hypothesize that the Fe(II) complex with TA exhibits an OH trapping activity, which explains the effect of TA on the Fenton reaction.     

Oxodesmosine and isooxodesmosine, candidates of oxidative metabolic intermediates of pyridinium cross-links in elastin

We isolated two new dihydrooxopyridine cross-links, oxodesmosine (OXD) and isooxodesmosine (IOXD) from the acid hydrolysates of the bovine aortic elastin. OXD and IOXD were identified to have N-substituted 1,2-dihydro-2-oxopyridine and N-substituted 1,4-dihydro-4-oxopyridine skeletons, respectively, with three alpha-amino acid groups and mass of 495 (C23H37N5O7). These structures and distribution indicated that OXD and IOXD are oxidative metabolites generated from desmosine (DES) and isodesmosine (IDE), respectively, by reactive oxygen species (ROS). Effects of ROS derived from divalent metal (Fe2+, Cu2+)/H2O2 on DES, IDE, OXD, and IOXD in elastin were investigated. Changes in the contents of these cross-links in elastin were observed by using reverse-phase HPLC with UV detection. The time- and pH-dependent formation of OXD and reduction of DES and IDE in elastin by Cu2+/H2O2 and Fe2+/H2O2 were observed. OXD was found to be formed from DES by Fe2+/H2O2. No formation of IOXD was observed under the conditions of oxidation examined. By using a model compound of IDE, however, we found that 4-pyridone could be formed by Fe2+/H2O2. Elastin incubated in Cu2+/H2O2 was also solubilized dependent on solution pH and the concentration of H2O2. These results suggest that oxidative degradation of elastin with cross-links results in its weakening, followed by its solubilization. Pyridinium cross-links, such as DES and IDE, may be oxidatively metabolized by ROS, further changing to dihydrooxopyridine cross-links such as OXD and IOXD, respectively.     

Iron detoxification properties of Escherichia coli bacterioferritin. Attenuation of oxyradical chemistry

Bacterioferritin (EcBFR) of Escherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the "ferroxidase center." The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H(2)O(2) as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H(2)O(2) at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H(2)O(2) corresponds to [Fe(II)(2)-P](Z) + H(2)O(2) --> [Fe(III)(2)O-P](Z) + H(2)O, where [Fe(II)(2)-P](Z) represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)(2)O-P](Z) a micro-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2FeOOH((core)) + 4H(+), where two Fe(II) are again oxidized by one H(2)O(2). Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O(2) is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H(2)O(2) produced from O(2) is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H(2)O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H(2)O(2), consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H(2)O(2), thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.     

Generation of organic acids and monosaccharides by hydrolytic and oxidative transformation of food processing residues

Carbohydrate-rich biomass residues, i.e. sugar beet molasses, whey powder, wine yeast, potato peel sludge, spent hops, malt dust and apple marc, were tested as starting materials for the generation of marketable chemicals, e.g. aliphatic acids, sugar acids and mono-/disaccharides. Residues were oxidized or hydrolyzed under acidic or alkaline conditions applying conventional laboratory digestion methods and microwave assisted techniques. Yields and compositions of the oxidation products differed according to the oxidizing agent used. Main products of oxidation by 30% HNO(3) were acetic, glucaric, oxalic and glycolic acids. Applying H(2)O(2)/CuO in alkaline solution, the organic acid yields were remarkably lower with formic, acetic and threonic acids as main products. Gluconic acid was formed instead of glucaric acid throughout. Reaction of a 10% H(2)O(2) solution with sugar beet molasses generated formic and lactic acids mainly. Na(2)S(2)O(8) solutions were very inefficient at oxidizing the residues. Glucose, arabinose and galactose were formed during acidic hydrolysis of malt dust and apple marc. The glucose content reached 0.35 g per gram of residue. Important advantages of the microwave application were lower reaction times and reduced reagent demands.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

Generation of *OH initiated by interaction of Fe2+ and Cu+ with dioxygen; comparison with the Fenton chemistry

Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (*OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating *OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce *OH. The addition of Fe2+ and Cu+ (0-20 microM) to KH resulted in a concentration-dependent increase in *OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 microM) did not result in *OH formation, these ions mediated significant *OH production in the presence of a number of reducing agents. The *OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 microM, 5 microM or 10 microM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in *OH formation. For each Fe2+ concentration tested, the *OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting *OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the *OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate *OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.     

An electrochemical biosensor for the detection of tyrosine oxidation induced by Fenton reaction

A simple and sensitive electrochemical biosensor was used to detect tyrosine oxidation induced by hydroxyl radicals generated by Fenton reaction (Fe(2+)/H(2)O(2)). Poly(glu, tyr) (4:1) peptides were immobilized on indium tin oxide (ITO) electrode surface via layer-by-layer assembly technique, and Os(bpy)(3)(2+)-mediated tyrosine oxidation current was employed as the signal reporter of the biosensor. It was found that the electrochemical signal of the peptide decreased markedly after incubation with Fenton reagents. Interestingly, L-dopa, the oxidation product of tyrosine, was likely to form complexes with Fe(III), which could suppress the electro-oxidation of L-dopa and resulted in decrease of current response. Our results indicate that the peptide damage involved two steps and was a second-order reaction. X-ray photoelectron spectroscopy was used to quantitatively determine nitrogen elemental percentage on peptide-coated electrode surface, which eliminated the possibility that signal decrease was caused by peptide backbone cleavage. Moreover, the lowest concentration of Fenton reagents that could be detected was 10 μM Fe(2+) or H(2)O(2), similar to the level in vivo. We suggest that the biosensor can be used to detect protein damage induced by Fenton reaction.     

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.     

Efficient synthesis of an aldehyde functionalized hyaluronic acid and its application in the preparation of hyaluronan-lipid conjugates

An efficient method to synthesize hyaluronan oligosaccharide lipid conjugates is described. This strategy is based on the introduction of a double bond in the glucuronic acid of the hyaluronic acid (HA), by the biodegradation of HA with hyaluronate lyase, followed by the generation of a free aldehyde group at the nonreducing end of hyaluronic acid via ozonolysis and the subsequent reduction of the generated ozonide. The resulting aldehyde-functionalized HA is then coupled to dipalmitoyl phosphatidylethanolamine (DPPE) using reductive amination chemistry. This methodology can be extended to link molecules such as biotin, polymers, or proteins to HA for numerous applications in drug delivery and in the creation of biocompatible materials for tissue repair and engineering.     

Glucuronic acid directly linked to galacturonic acid in the rhamnogalacturonan backbone of beet pectins.

Sugar-beet pulp was de-esterified and submitted to 72 h hydrolysis by 0.1 M HCl at 80 degrees C. Oligomers containing a single glucuronic acid (GlcA) moiety in addition to n(>/= 2) repeats of the dimer -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1--> were isolated from the hydrolysate by ion-exchange and gel-permeation. Glycosyl linkage composition analysis and 1H NMR studies indicated that the GlcA was attached to O-3 of a galacturonic acid (GalA) residue, as shown for the two pentamers beta-D-GlcpA-(1-->3)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-L-Rhap and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-[beta-D-GlcpA-(1-->3)]-alpha-D-GalpA-(1-->2)-L-Rhap. Substitution by GlcA was estimated as occurring on one GalA residue out of 72 in the rhamnogalacturonan fraction of the backbone of beet pectins.     

Kinetics of hyaluronan hydrolysis in acidic solution at various pH values

Hyaluronic acid (HA) was hydrolyzed using varying temperatures (40, 60, and 80 degrees C) and acid concentrations (0.0010, 0.010, 0.10, 0.50, 1.0, and 2.0 M HCl). The degradation process was monitored by determination of weight average molecular weight ( M w) by size-exclusion chromatography with online multiangle laser light scattering, refractive index, and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples taken out continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave narrow molecular weight distributions with polydispersity indexes ( M w/ M n) of 1.3-1.7. Kinetic plots of 1/ M w versus time gave linear plots showing that acid hydrolysis of HA is a random process and that it follows a first order kinetics. For hydrolysis in HCl at 60 and 80 degrees C, it was shown that the kinetic rate constant ( k h) for the degradation depended linearly on the acid concentration. Further, the dependence of temperature on the hydrolysis in 0.1 M HCl was found to give a linear Arrhenius plot (ln k h vs 1/ T), with an activation energy ( E a) of 137 kJ/mol and Arrhenius constant ( A) of 7.86 x 10 (15) h (-1). (1)H NMR spectroscopy was used to characterize the product of extensive hydrolysis (48 h at 60 degrees C in 0.1 M HCl). No indication of de- N-acetylation of the N-acetyl glucosamine (GlcNAc) units or other byproducts were seen. Additionally, a low molecular weight HA was hydrolyzed in 0.1 M DCl for 4 h at 80 degrees C. It was shown that it was primarily the beta-(1-->4)-linkage between GlcNAc and glucuronic acid (GlcA) that was cleaved during hydrolysis at pH < p K a,GlcA. The dependence of the hydrolysis rate constant was further studied as a function of pH between -0.3 and 5. The degradation was found to be random (linear kinetic plots) over the entire pH range studied. Further, the kinetic rate constant was found to depend linearly on pH in the region -0.3 to 3. Above this pH (around the p K a of HA), the kinetic constant decreased more slowly, probably due to either a change in polymer conformation or due to an increased affinity for protons due to the polymer becoming charged as the GlcA units dissociated.