Encapsulation of myoglobin with a mesoporous silicate results in new capabilities

A metmyoglobin (Fe3+), an oxidized form of myoglobin (Fe2+), was confined in nanospaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a metmyoglobin (Fe3+)-FSM nanoconjugate. The spectral characteristics of metmyoglobin (Fe3+)- and myoglobin (Fe2+)-FSM show an absorption curve quite similar to that of native metmyoglobin, indicating that myoglobin retains its higher-order structure in the pores of FSM. The metmyoglobin (Fe3+)-FSM conjugate had not only a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfomic acid (ABTS) or guaiacol (a hydrogen donor) but also an advanced molecular recognition ability enabling it to distinguish between ABTS and guaiacol. Furthermore, the metmyoglobin (Fe3+)-FSM showed the peroxidase-like activity even in an organic media using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The simple immobilization of metmyoglobin (Fe3+) into FSM results in enhanced catalytic activity in organic media compared to that of native metmyoglobin (Fe3+).     

Hemin (Fe(3+))-- and heme (Fe(2+))--smectite conjugates as a model of hemoprotein based on spectrophotometry

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin-smectite conjugate. The hemin-smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as epsilon(398nm) = 11.6 x 10(4) M(-1) cm(-1), which is in good agreement with epsilon(398nm) = (12.2 +/- 3) x 10(4) M(-1) cm(-1) of monomeric hematin (1). Hemin (Fe(3+))-smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe(3+))-smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe(2+))-smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of alpha, beta, and Soret bands were located in only a 9--3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe(2+))-smectite conjugate was bound to O(2) to form O(2)-heme (Fe(2+))-smectite conjugate. The addition of carbon monoxide, CO, to O(2)-heme (Fe(2+))-smectite conjugate caused the formation of CO-heme (Fe(2+))-smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8--10 nm to shorter wavelength. Therefore, the transformation of O(2)-heme (Fe(2+))-smectite conjugate to CO-heme (Fe(2+))-smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the alpha, beta, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O(2)-heme (Fe(2+))-smectite conjugate and CO-heme (Fe(2+))-smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe(3+)) or heme (Fe(2+)), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.     

Thermodynamics of the two step formation of horseradish peroxidase compound I

The effects of temperature (20 to -38 degrees C), pressure (normal pressures to 1.2 kbar) and solvent (water, 60% DMSO and 50% methanol) on the reaction of hydrogen peroxide or ethyl peroxide with horseradish peroxidase were studied. The formation of compound I was followed at 403 nm in a stopped flow apparatus adapted for high pressure and low temperature work. As with the alkaline form (Job and Dunford 1978), the neutral form of the peroxidase binds peroxide substrates in two steps. It was the combined use of organic solvents and low temperatures which revealed saturation kinetics: (Formula: see text) compound I, where E = horseradish peroxidase and S peroxide substrate. In water and organic solvents at temperatures above -10 degrees C, K1 was too small and k2 too large to be measured, here K1 X k2 was obtained. k-2 was too small for measurement under all conditions. Whereas K1 was insensitive to the peroxide substrate and solvent composition, k2 was very sensitive. The thermodynamic parameters delta H, delta S and delta V for K1 and k2 were obtained under different experimental conditions and the data are interpreted within the available thermodynamic theories.     

Enzymes in Organic Synthesis VII—Enzymatic Activity of Hrp After Chemical Modification of the Carbohydrate Moiety

The carbohydrate moiety of horseradish peroxidase was conjugated with hexadecylamine or octylamine in a micellar medium. Recovery and purification of these conjugates was facilitated by the short length of the added spacers. The modification increased the liposolubility of the enzyme without detracting from its catalytic activity. For the hexadecylamine conjugate, the optimum reaction temperature was increased by 10d`C. In addition, activity in organic solvents, such as toluene or chloroform, remained high, even at 70d`C.     

水溶性有机溶剂对Fe3O4磁性纳米粒子过氧化物酶样活性的影响

采用化学共沉淀法合成10nm的Fe3O4磁性纳米粒子(MNPs)。以辣根过氧化物酶(HRP)为对照,研究了四氢呋喃、1,4-二氧六环、丙酮、N,N-二甲基酰胺、甲醇和二甲亚砜等6种水溶性有机溶剂对Fe3O4MNPs过氧化物酶样活性的影响。结果表明,在有机溶剂浓度(V/V)为30%～75%时,Fe3O4MNPs相对酶活力迅速下降至近于完全失活。在15%有机溶液中,Fe3O4MNPs的最适反应温度多为50oC,最适反应pH在3.6左右。经15%有机溶液处理后的水相反应酶活显示,Fe3O4MNPs表现出对有机溶剂较强的热稳定性和pH稳定性,且对75%有机溶液也具有良好的稳定性。以上多数性质均优于相同条件下的HRP组,表明Fe3O4MNPs是一种比HRP对水溶性有机溶剂更稳定的过氧化物酶。由于Fe3O4MNPs具有易制备、成本低、易于磁分离和可循环使用的特点,因此其具有替代HRP用于有机催化的应用潜力。     

Purification and characterization of chorion peroxidase from Aedes aegypti eggs

Previous study has shown that a peroxidase is present in the mature eggs of Aedes aegypti mosquitoes, and the enzyme is involved in the formation of a rigid and insoluble chorion by catalyzing chorion protein crosslinking through dityrosine formation. In this study, chorion peroxidase was solubilized from egg chorion by 1% SDS and 2 M urea and purified by various chromatographic techniques. The enzyme has a relative molecular mass of 63,000 as estimated by SDS-PAGE. Spectral analysis of the enzyme revealed the presence of the Soret band with a lambda(max) at 415 nm, indicating that chorion peroxidase is a hemoprotein. Treatment of the native enzyme with H2O2 in excess in the absence of reducing agents shifted the Soret band from 415 to 422 nm, and reduction of the native enzyme with sodium hydrosulfite under anaerobic conditions changed the Soret band from 415 to 446 nm. These results show that the chorion peroxidase behaves similarly to other peroxidases under oxidative and reductive conditions, respectively. Compared to other peroxidases, the chorion peroxidase, however, is extremely resistant to denaturing agents, such as SDS and organic solvents. For example, chorion peroxidase remained active for several weeks in 1% SDS, while horseradish peroxidase irreversibly lost all its activity in 2 h under the same conditions. Comparative analysis between mosquito chorion peroxidase and horseradish peroxidase showed that the specific activity of chorion peroxidase to tyrosine was at least 100 times greater than that of horseradish peroxidase to tyrosine. Chorion peroxidase is also capable of catalyzing polypeptide and chorion protein crosslinking through dityrosine formation during in vitro assays. Our data suggest that the characteristics of the chorion peroxidase in mosquitoes closely reflect its functions in chorion formation and hardening.     

Structural change and catalytic activity of horseradish peroxidase in oxidative polymerization of phenol

The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.     

Structural Change and Catalytic Activity of Horseradish Peroxidase in Oxidative Polymerization of Phenol

The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.     

Polyethylene glycol derivative-modified cholesterol oxidase soluble and active in benzene

Cholesterol oxidase from Nocardia sp. was modified with a synthetic copolymer of polyoxyethylene allylmethyldiether (PEG) and maleic acid anhydride (MA anhydride), poly(PEG-MA anhydride). The modified cholesterol oxidase, in which 64% of the amino groups in the protein molecule were coupled to poly(PEG-MA), was soluble in organic solvents and catalyzed the oxidation reaction of cholesterol in benzene to form 4-cholesten-3-one with the enzymic activity of 0.6 mumol/min/mg protein. Using the modified cholesterol oxidase together with polyethylene glycol-modified peroxidase, coupled reactions shown below took place in Cholesterol + O2----4-Cholesten-3-one + H2O2 H2O2 + o-Phenylenediamine----H2O + Oxidized o-Phenylenediamine transparent benzene solution, not in an emulsified system. The oxidation of cholesterol was directly determined in benzene by measuring the absorbance of oxidized o-phenylenediamine at 490 nm.     

Enzymatic enhancement of the catalytic rate of sulfhydryl oxidase

The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Moreover, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N-acetylcysteine could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/N-acetylcysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni ping-pong mechanism proposed previously for sulfhydryl oxidase.     

Alpha-bungarotoxin-horseradish peroxidase conjugate: preparation, properties and utilization for the histochemical detection of acetylcholine receptors.

A method is presented for the efficient conjugation of horseradish peroxidase to alpha-bungarotoxin. The 1:1 molar conjugate obtained is purified to completion by gel filtration on Sephadex G-100, followed by ion exchange chromatography on CM-Sephadex. The conjugate retains half of the activity of unmodified horseradish peroxidase and binds effectively to the nicotinic acetylcholine receptor of muscle. The conjugate is proven to be useful reagent for the histochemical staining of the receptor on muscle fibers for light and electron microscopy.     

A hydrophilic and hydrophobic organic solvent mixture enhances enzyme stability in organic media

Binary mixtures of hydrophilic and hydrophobic solvents were assessed for their ability to balance enzyme activity with the conservation of enzyme stability in organic media. Acetone, dioxane and dodecane were chosen as model organic solvents, and subtilisin Carlsberg and horseradish peroxidase (HRP) were chosen as model enzymes. Residual enzyme activities were measured to monitor enzyme stability, and the fluorescence intensity of HRP was monitored to investigate structural changes due to the presence of an organic solvent. Enzyme stability increased with the increasing hydrophobicity of the solvent mixture used, and a solvent mixture with a high log P value (~ >4) was capable of conserving enzyme stability. Enzyme stability in organic media can be conserved therefore with a mixture of hydrophilic and hydrophobic solvents: this approach might be used as a general and practical strategy for optimizing enzyme activity and stability for industrial applications.     

Tropolone as a substrate for horseradish peroxidase

Tropolone (2,4,6-cycloheptatrien-1-one), in the presence of hydrogen peroxide but not in its absence, can serve as a donor for the horseradish peroxidase catalysed reaction. The product formed is yellow and is characterized by a new peak at 418 nm. The relationship between the rate of oxidation of tropolone (ΔA at 418 nm/min) and various concentrations of horseradish peroxidase, tropolone and hydrogen peroxide is described. The yellow product obtained by the oxidation of tropolone by horseradish peroxidase in the presence of hydrogen peroxide was purified by chromatography on Sephadex G-10 and its spectral properties at different pHs are presented. The M, of the yellow product was estimated to be ca 500, suggesting that tropolone, in the presence of horseradish peroxidase and hydrogen peroxide is converted to a tetratropolone.     

Effect of dimethyl sulfoxide on the structure and the functional properties of horseradish peroxidase as observed by spectroscopy and cyclic voltammetry

Electrochemical biosensors have found wide application in food and clinical areas, as well as in environmental field. A large number of articles focused on horseradish peroxidase (HRP)-based biosensors have been published in the last decade, due to the capability of HRP to quantitatively detect the presence of hydrogen peroxide produced in a reaction. At present a large body of multi-enzymatic amperometric biosensors are realized by entrapping HRP together with other enzymes into a polymeric matrix; these systems represent a promising example of simple, low-cost electrochemical tools for the analysis of bioanalytes in solution, such as glucose, choline and cholesterol. Due to the fact that polymers used for HRP entrapping are soluble in organic solvents and that many solvents are strong denaturants of aquo-soluble proteins, in this paper we investigate (in particular, by circular dichroism and electron paramagnetic spectroscopies) the effect of dimethyl sulfoxide, one of the organic solvents employed for polymer solubilization, on the structure and the functionality of HRP, in order to determine the effect induced by the solvent concentration on the structure and activity of the hemoprotein. This is relevant for basic and applied biochemistry, HRP being largely employed in bioinorganic chemistry and sensor area.     

Poly(2-oxazoline)s terminated with 2,2′-imino diacetic acid form noncovalent polymer–enzyme conjugates that are highly active in organic solvents

A great limitation for the usability of free enzymes in organic solvents is their insolubility in these media. Some surfactants are capable of solubilizing enzymes in such media, but they are hard to remove. Covalent modification of enzymes with polymers has led to polymer–enzyme conjugates (PECs) that are soluble in organic solvents, but the process is quite elaborate. Poly(2-oxazoline)s (POx) with the end group 2,2′-imino diacetic acid were shown to form reversible, nano-sized noncovalent aggregates with enzymes. These PECs give clear solutions in organic solvents. The enzymes lysozyme, horseradish peroxidase (HRP), laccase, α-chymotrypsin (CT), catalase, and alcohol dehydrogenase could be solubilized in chloroform and toluene at concentrations of up to 2 mg protein/ml. Laccase, HRP, and CT were shown to survive the transfer into the organic medium and back to water in their active form. The distribution coefficient of the proteins between water and the organic solvent was shown to be dependent on the nature of the POx backbone. All three biocatalysts exhibit greatly enhanced activity in the respective organic solvent.     

Phytol-modified heme in mesoporous silica: conjugates as models of hemoproteins

A ferriprotoporphyrin, hemin (Fe(3+)), modified with 3,7,11,15-tetramethyl-2-hexadecen-1-ol, phytol, was adsorbed in nano-spaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material) forming a phytol-modified hemin (Fe(3+))-FSM nano-conjugate. The properties and the structure of the conjugate were studied by UV-visible light absorption, IR absorption spectroscopy, and a nitrogen adsorption isotherm. Although the hemin without phytol could not be adsorbed to the mesoporous silica, modification with phytol imparted preferential adsorption properties. The conjugate was not only stable but also had a peroxidase-like activity in a 0.1% hydrogen peroxide solution, while free hemin in the solution was easily destroyed. The hemin (Fe(3+)) in the FSM was reduced to heme (Fe(2+)) by hydrazine. The phytol-modified heme (Fe(2+))-FSM conjugate formed an O(2)-heme complex with a superoxide type structure, resembling oxyhemoglobin or oxymyoglobin, which has not been previously observed for free heme in solution. The addition of carbon monoxide or nitrogen monoxide to the phytol-modified heme (Fe(2+))-FSM conjugate caused the formation of CO- or NO-heme complex in the nano-spaces of the FSM. These properties are attributed not only to the Fe-complex but also to the cooperative functions of the heme with mesoporous silica, resembling properties of a natural heme-protein conjugate; hemoglobin or peroxidase. These results are an elegant example of biomimetic nano-technology.     

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.      

A model of peroxidase activity with inhibition by hydrogen peroxide

The rate of color formation in an activity assay consisting of phenol and hydrogen peroxide as substrates and 4-aminoantipyrine as chromogen is significantly influenced by hydrogen peroxide concentration due to its inhibitory effect on catalytic activity. A steady-state kinetic model describing the dependence of peroxidase activity on hydrogen peroxide concentration is presented. The model was tested for its application to soybean peroxidase (SBP) and horseradish peroxidase (HRP) reactions based on experimental data which were measured using simple spectrophotometric techniques. The model successfully describes the dependence of enzyme activity for SBP and HRP over a wide range of hydrogen peroxide concentrations. Model parameters may be used to compare the rate of substrate utilization for different peroxidases as well as their susceptibility to compound III formation. The model indicates that SBP tends to form more compound III and is catalytically slower than HRP during the oxidation of phenol.     

Enzymatic synthesis of a catechin conjugate of polyhedral oligomeric silsesquioxane and evaluation of its antioxidant activity

The antioxidant activity of catechin was amplified by conjugation with amine-terminated polyhedral oligomeric silsesquioxane (POSS) using horseradish peroxidase as catalyst. Compared to intact catechin, the scavenging activity of the POSS-catechin conjugate against superoxide anion was greatly improved. In addition, the conjugate strongly inhibited xanthine oxidase activity.     

A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.