An HPLC assay of hydroxyl radicals by the hydroxylation reaction of terephthalic acid

An HPLC assay for hydroxyl radicals is described. The hydroxyl radical was trapped by terephthalic acid (non-fluorescent), and 2-hydroxyl terephthalic acid (fluorescent) was quantitated by HPLC-fluorescence detection. At a terephthalic acid concentration of 4.25 mmol/L, the hydroxyl radical formed in the Fenton reaction was successfully assayed in the concentration range of hydrogen peroxide of 2.5-50 micro mol/L, where the concentration of Fe(II) was 50 micro mol/L. The fluorescence of 2-hydroxy terephthalate was stable at 24 h, and its detection limit by this method was 5 nmol/L (100 fmol).     

Potentiometric flow-injection determination of trace hydrogen peroxide based on its induced reaction in iron(III)-iron(II) potential buffer containing bromide and molybdenum(VI)

A rapid and highly sensitive potentiometric flow-injection method for the determination of trace hydrogen peroxide was developed by use of an Fe(III)-Fe(II) potential buffer solution containing bromide and Mo(VI). The analytical method was based on a linear relationship between a concentration of hydrogen peroxide and a largely transient potential change of an oxidation-reduction potential electrode due to bromine generated by the reaction of hydrogen peroxide with the potential buffer solution. The oxidation of bromide to bromine by hydrogen peroxide occurred very rapidly with the assistance of Mo(VI) when Fe(II) existed in the potential buffer solution. It was estimated by batchwise experiments that hydroxyl radical, OH., was generated by the reaction of hydrogen peroxide with Fe(II) as an intermediate, and subsequently oxidized bromide to bromine. In a flow system, analytical sensitivities to hydrogen peroxide obtained by the detection of the transient change of potential were enhanced about 75 fold compared with those obtained by using the potential change caused by the reaction of hydrogen peroxide with the potential buffer solution without bromide and Mo(VI). Sensitivities increased with decreasing concentration of the Fe(III)-Fe(II) buffer in the reagent solution. The detection limit (S/N = 3) of 4 x 10(-7) M (13.6 ppb) was achieved by using the 1 x 10(-4) M Fe(III)-Fe(II) buffer containing 0.4 M NaBr, 1.0 M H(2)SO(4) and 0.5% (NH(4))(6)Mo(7)O(24). Analytical throughput was approximately 40 h(-1) and the RSD (n = 6) was 0.6% for measurement of 4 x 10(-6) M hydrogen peroxide. The proposed method was applied to the determination of hydrogen peroxide in real rainwater samples, and was found to provide a good recovery for H(2)O(2) added to rainwater samples.     

Reaction of hydrogen peroxide with low molecular weight iron complexes

Fe(II) complexed with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) reacts with hydrogen peroxide in neutral aqueous solution at room temperature to yield reactive species which are not scavenged by t-butanol, under conditions where >90% of hydroxyl radical would be scavenged. Further, the ratio of the rate constants for the reaction of the reactive species with Fe(II)CDTA and H₂O₂ is 6.2, in contrast to a ratio of 200 which would result if the species were the hydroxyl radical. Thus, it is concluded that the reactive species produced is not the hydroxyl radical, but an iron-oxo species such as the ferryl ion. The reactive species is formed in an apparent first order reaction, when either hydrogen peroxide or Fe(II)CDTA is in kinetic excess. The bimolecular reaction rate constant is (1.26 ± 0.19) × 10³ M^-1 s^-1. In experiments where H₂O₂ was in kinetic excess, a chain decomposition of H₂O₂ was observed in which the initially produced iron-oxo intermediate exhibits hydroperoxidase activity.     

The atmospheric chemistry of hydrogen peroxide: a review

This paper reviews the atmospheric chemistry of hydrogen peroxide, taking into account the formation processes of both gas-phase and aqueous H2O2, and the reactions involving hydrogen peroxide in the gas phase and in atmospheric hydrometeors. Gas-phase hydrogen peroxide mainly forms upon dismutation of the hydroperoxyl radical, a product of the reactions between atmospheric hydrocarbons, hydroxyl radicals, nitric oxide, and oxygen. Aqueous hydrogen peroxide originates from the dissolution of the gaseous one, the reduction of molecular oxygen, a series of reactions involving dissolved ozone, and the irradiation of anthraquinones, aromatic carbonyls, and semiconductor oxides. The reactions involving aqueous H2O2 are very important in the context of the chemistry of the atmosphere. They include oxidation of S(IV) to S(VI), photolysis, the Fenton reaction in the presence of Fe(II), and possibly the formation of peroxynitrous acid. Within this framework, the correlation of hydrogen peroxide with other atmospheric components and the time trends of hydrogen peroxide in the atmosphere are easily accounted for.     

Modifications of boronic ester pro-chelators triggered by hydrogen peroxide tune reactivity to inhibit metal-promoted oxidative stress

Several new analogs of salicylaldehyde isonicotinoyl hydrazone (SIH) and salicylaldehyde benzoyl hydrazone (SBH) that contain an aryl boronic ester (BSIH, BSBH) or acid (BASIH) in place of an aryl hydroxide have been synthesized and characterized as masked metal ion chelators. These pro-chelators show negligible interaction with iron(III), although the boronic acid versions exhibit some interaction with copper(II), zinc(II) and nickel(II). Hydrogen peroxide oxidizes the aryl boronate to phenol, thus converting the pro-chelators to tridentate ligands with high affinity metal binding properties. An X-ray crystal structure of a bis-ligated iron(III) complex, [Fe(SBH(m-OMe)(3))(2)]NO(3), confirms the meridonal binding mode of these ligands. Modifications of the aroyl ring of the chelators tune their iron affinity, whereas modifications on the boron-containing ring of the pro-chelators attenuate their reaction rates with hydrogen peroxide. Thus, the methoxy derivative pro-chelator (p-OMe)BASIH reacts with hydrogen peroxide nearly 5 times faster than the chloro derivative (m-Cl)BASIH. Both the rate of pro-chelator to chelator conversion as well as the metal binding affinity of the chelator influence the overall ability of these molecules to inhibit hydroxyl radical formation catalyzed by iron or copper in the presence of hydrogen peroxide and ascorbic acid. This pro-chelator strategy has the potential to improve the efficacy of medicinal chelators for inhibiting metal-promoted oxidative stress.     

The "somersault" mechanism for the p-450 hydroxylation of hydrocarbons. The intervention of transient inverted metastable hydroperoxides

A series of model theoretical calculations are described that suggest a new mechanism for the oxidation step in enzymatic cytochrome P450 hydroxylation of saturated hydrocarbons. A new class of metastable metal hydroperoxides is described that involves the rearrangement of the ground-state metal hydroperoxide to its inverted isomeric form with a hydroxyl radical hydrogen bonded to the metal oxide (MO-OH --> MO....HO). The activation energy for this somersault motion of the FeO-OH group is 20.3 kcal/mol for the P450 model porphyrin iron(III) hydroperoxide [Por(SH)Fe(III)-OOH(-)] to produce the isomeric ferryl oxygen hydrogen bonded to an *OH radical [Por(SH)Fe(III)-O....HO(-)]. This isomeric metastable hydroperoxide, the proposed primary oxidant in the P450 hydroxylation reaction, is calculated to be 17.8 kcal/mol higher in energy than the ground-state iron(III) hydroperoxide Cpd 0. The first step of the proposed mechanism for isobutane oxidation is abstraction of a hydrogen atom from the C-H bond of isobutane by the hydrogen-bonded hydroxyl radical to produce a water molecule strongly hydrogen bonded to anionic Cpd II. The hydroxylation step involves a concerted but nonsynchronous transfer of a hydrogen atom from this newly formed, bound, water molecule to the ferryl oxygen with a concomitant rebound of the incipient *OH radical to the carbon radical of isobutane to produce the C-O bond of the final product, tert-butyl alcohol. The TS for the oxygen rebound step is 2 kcal/mol lower in energy than the hydrogen abstraction TS (DeltaE() = 19.5 kcal/mol). The overall proposed new mechanism is consistent with a lot of the ancillary experimental data for this enzymatic hydroxylation reaction.     

A novel detection approach based on chromophore-decolorizing with free radical and application to photometric determination of copper with acid chrome dark blue

A novel detection approach named chromophore-decolorizing with free radicals is developed for determination of trace heavy metal. The hydroxyl radicals (HO) generated from Fe(III) and hydrogen peroxide will oxidize the free chromophore into almost colorless products. The copper-acid chrome dark blue (ACDB) complexation was investigated at pH 5.07. In the presence of Fe(III) and hydrogen peroxide, the excess ACDB was decolorized in the Cu-ACDB reaction solution, and the final solution contained only one color compound, the Cu-ACDB complex. After oxidation of free hydroxyl radicals, the complexation becomes sensitive and selective and it has been used for the quantitation of trace amounts of Cu(II) dissolved in natural water. Beer's law is obeyed in the range from 0 to 0.500 μg mL⁻¹ Cu(II) and the limit of detection is only 6 μg L⁻¹ Cu(II). Besides, the Cu-ACDB complex formed was characterized.     

The use of high field/frequency EPR in studies of radical and metal sites in proteins and small inorganic models

Low temperature electron paramagnetic resonance (EPR) spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T have been used to study radicals and metal sites in proteins and small inorganic model complexes. We have studied radicals, Fe, Cu and Mn containing proteins. For S = 1/2 systems, the high frequency method can resolve the g-value anisotropy. It was used in mouse ribonucleotide reductase (RNR) to show the presence of a hydrogen bond to the tyrosyl radical oxygen. At 285 GHz the type 2 Cu(II) signal in the complex enzyme laccase is clearly resolved from the Hg(II) containing laccase peroxide adduct. For simple metal sites, the systems over S = 1/2 can be described by the spin Hamiltonian: H(S) = BgS + D[Sz2 - S(S + 1)/3 + E/D (Sx2 - Sy2)]. From the high frequency EPR the D-value can be determined directly by, (I) shifts of g(eff) for half-integer spin systems with large D-values as observed at 345 GHz on an Fe(II)-NO-EDTA complex, which is best described as S = 3/2 system with D = 11.5 cm(-1), E = 0.1 cm(-1) and gx = gy = gz = 2.0; (II) measuring the outermost signal, for systems with small D values, distant of (2S - 1) x absolute value(D) from the center of the spectrum as observed in S= 5/2 Fe(III)-EDTA. In Mn(II) substituted mouse RNR R2 protein the weakly interacting Mn(II) at X-band could be observed as decoupled Mn(II) at 285 GHz.     

Aliphatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting

Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.     

Ru(III)-EDTA catalyzed oxidation of ascorbic acid by hydrogen peroxide in aqueous solution

The kinetics of oxidation of ascorbic acid to dehydroascorbic acid by hydrogen peroxide catalyzed by ethylenediaminetetraacetatoruthenate(III) has been studied over the pH range 1.50 – 2.50, at 30°C and μ = 0.1 M KNO₃. The reaction has a first-order dependence on ascorbic acid and Ru(III)-EDTA concentrations, an inverse first-order dependence on hydrogen ion concentration, and is independent of hydrogen peroxide concentration in the pH range studied. A mechanism has been proposed in which ascorbate anion forms a kinetic intermediate with the catalyst in a pre-equilibrium step. Ruthenium(III) is reduced to ruthenium(II) in a rate-determining step and is reoxidized with hydrogen peroxide back to the Ru(III) complex in a fast step.     

Kinetics and mechanisms of decomposition reaction of hydrogen peroxide in presence of metal complexes

Hydrogen peroxide was discovered in 1818 and has been used in bleaching for over a century [ 1 ]. H₂O₂ on its own is a relatively weak oxidant under mild conditions: It can achieve some oxidations unaided, but for the majority of applications it requires activation in one way or another. Some activation methods, e.g., Fenton's reagent, are almost as old [ 2 ]. However, by far the bulk of useful chemistry has been discovered in the last 50 years, and many catalytic methods are much more recent. Although the decomposition of hydrogen peroxide is often employed as a standard reaction to determine the catalytic activity of metal complexes and metal oxides [ 3 , 4 ], it has recently been extensively used in intrinsically clean processes and in end‐of‐pipe treatment of effluent of chemical industries [ 5 , 6 ]. Furthermore, the adoption of H₂O₂ as an alternative of current industrial oxidation processes offer environmental advantages, some of which are (1) replacement of stoichiometric metal oxidants, (2) replacement of halogens, (3) replacement or reduction of solvent usage, and (4) avoidance of salt by‐products. On the other hand, wasteful decomposition of hydrogen peroxide due to trace transition metals in wash water in the fabric bleach industry, was also recognized [ 7 ]. The low intrinsic reactivity of H₂O₂ is actually an advantage, in that a method can be chosen which selectively activates it to perform a given oxidation. There are three main active oxidants derived from hydrogen peroxide, depending on the nature of the activator; they are (1) inorganic oxidant systems, (2) active oxygen species, and (3) per oxygen intermediates. Two general types of mechanisms have been postulated for the decomposition of hydrogen peroxide in the presence of transition metal complexes. The first is the radical mechanism (outer sphere), which was proposed by Haber and Weiss for the Fe(III)‐H₂O₂ system [ 8 ]. The key features of this mechanism were the discrete formation of hydroxyl and hydroperoxy radicals, which can form a redox cycle with the Fe(II)/Fe(III) couple. The second is the peroxide complex mechanism, which was proposed by Kremer and Stein [ 9 ]. The significant difference in the peroxide complex mechanism is the two‐electron oxidation of Fe(III) to Fe(V) with the resulting breaking of the peroxide oxygen‐oxygen bond. It is our intention in this article to briefly summarize the kinetics as well as the mechanisms of the decomposition of hydrogen peroxide, homogeneously and heterogeneously, in the presence of transition metal complexes. © 2000 John Wiley & Sons, Inc. Int J Chem Kinet 32: 643–666, 2000     

The kinetics and mechanism of oxidation of isopropanol with the hydrogen peroxide-vanadate ion-pyrazine-2-carboxylic acid system

The vanadate anion in the presence of pyrazine-2-carboxylic acid (PCA) was found to effectively catalyze the oxidation of isopropanol to acetone with hydrogen peroxide. The electronic spectra of solutions and the kinetics of oxidation were studied. The conclusion was drawn that the rate-determining stage of the reaction was the decomposition of the vanadium(V) diperoxo complex with PCA, and the particle that induced the oxidation of isopropanol was the hydroxyl radical. Supposedly, the HO^· radical detached a hydrogen atom from isopropanol, and the Me₂ C^· (OH) radical formed reacted with HOO^· to produce acetone and hydrogen peroxide. The electronic spectra of solutions in isopropanol and acetonitrile and the dependences of the initial rates of isopropanol oxidation without a solvent and cyclohexane oxidation in acetonitrile on the initial concentration of hydrogen peroxide were compared. The conclusion was drawn that hydroxyl radicals appeared in the oxidation of alkanes in acetonitrile in the decomposition of the vanadium diperoxo complex rather than the monoperoxo derivative, as was suggested by us earlier.     

Determination of hydrogen peroxide by iron(III) complex of thiacalix[4]arenetetrasulfonate on a modified ion-exchanger with peroxidase-like catalytic activity.

An iron(III) complex of thiacalix[4]arenetetrasulfonate on a modified anion-exchanger (Fe3+-TCAS(A-500)) has shown high peroxidase-like activity at pH 5 - 6 for the reaction of quinoid-dye formation between 3-methyl-2-benzothiazolinone hydrazone and N-(3-sulfopropyl)aniline in the presence of hydrogen peroxide. Utilizing the peroxidase-like activity of Fe3+-TCAS(A-500) for this reaction, a method using Fe3+-TCAS(A-500) was applied for the spectrophotometric determination of hydrogen peroxide. The calibration curve by the method using Fe3+-TCAS(A-500) was linear over the range from 1 to 10 microg of hydrogen peroxide in a 1 ml sample solution. The apparent molar absorptivity for hydrogen peroxide was 2.4 x 10(4) l mol(-1) cm(-1). which was about 80% of that by peroxidase under the same conditions. This determination method of hydrogen peroxide using Fe3+-TCAS(A-500) was applied for the determination of glucose in diluted normal and abnormal control serum I and II.     

Nanoscale Hydroxyl Radical Generation from Multiphoton Ionization of Tryptophan

Exposure of solutions containing both tryptophan and hydrogen peroxide to a pulsed (∼180 fs) laser beam at 750 nm induces luminescence characteristic of 5-hydroxytryptophan. The results indicate that 3-photon excitation of tryptophan results in photoionization within the focal volume of the laser beam. The resulting hydrated electron is scavenged by hydrogen peroxide to produce the hydroxyl radical. The latter subsequently reacts with tryptophan to form 5-hydroxytryptophan. The involvement of hydroxyl radicals is confirmed by the use of ethanol and nitrous oxide as scavengers and their effects on the fluorescence yield in this system. It is postulated that such multiphoton ionization of tryptophanyl residues in cellular proteins may contribute to the photodamage observed during imaging of cells and tissues using multiphoton microscopy.     

Determination of hydroxyl radical photoproduction rates in natural waters.

The photochemical formation rates of hydroxyl radicals (OH radicals) in river water and seawater were determined by a simple, rapid and sensitive benzene probe method, in which phenol formed by the reaction between benzene and photochemically-generated OH radicals was analyzed by on-line preconcentration HPLC. The OH radical formation rates from well-known OH radical sources, such as nitrate, nitrite and hydrogen peroxide, were in good agreement with those reported previously. River water samples containing high concentrations of nitrate and nitrite were found to show high OH radical formation rates. Ten to 80% of the OH radical formation in river water and seawater was due to the photolysis of nitrate and nitrite, but OH radical formation from hydrogen peroxide was negligible. The OH radical formation from unknown sources other than nitrate, nitrite and hydrogen peroxide was strongly correlated to the amount of fluorescent matter.     

Metallodesferals as a new class of DNA cleavers: Specificity, mechanism and targetting of DNA scission reactions

Interaction of metal complexes with nucleic acids is currently attracting wide attention due to their potential utility as drugs, regulators of gene expression and tools for molecular biology. Many metal complexes exhibit nucleolytic activity, the most important examples being Cu(II)-OP, Fe(H)-BLM, Fe(II)-EDTA, metalloporphyrins, Ru and Co complexes of 4,7-diphenyl-l,10-phenanthroline and more recently by Ni(II) complexes. Desferal, a well known siderophore and a highly effective drug in chelation therapy of iron overload diseases, forms a stable octahedral co-ordination Fe(III) complex Eerrioxamine B. We have been interested in the DNA damaging properties of metallodesferals and this paper describes the DNA cleaving ability of metallodesferals, metal-dependent base selectively in DNA scission reactions, mechanistic studies on DNA cleavage by CuDFO and targetting of DNA cutting by covalent MDFO conjugates. This paper reports the synthesis of Cu(II), Co (III) and Ni(II) complexes of a siderophore chelating drug desferal, the studies on cleavage of plasmid DNA, the sequence preference of cleavage reactions, and C1’ as the primary site of hydroxyl radical attack in the reactions. Oligonucleotides covalently linked with this molecular scissor can direct the cleavage of either single or double strand DNA’s, mediated by duplex or triple helix structures respectively. Such targetting of DNA cleavage reactions, mediated by oligonucleotide-Cu(II)/Co(III) desferal conjugates has demonstrated reasonable site specificity and efficiency     

Potentiometric flow injection analysis of concentrated hydrogen peroxide by using an Fe(II)-Fe(III) redox potential buffer solution

The flow injection analysis of hydrogen peroxide is proposed, using a redox electrode and an Fe(II)-Fe(III) potential buffer solution. Influencing factors, such as the concentrations of Fe(II)-Fe(III) and sulfuric acid in the potential buffer on sensitivity of the proposed method are examined. The analysis of high concentrations of hydrogen peroxide up to approximately 10 M was conducted successfully with relative standard deviation of 0.7%.     

Chemiluminescence determination of metformin based on hydroxyl radical reaction and molecularly imprinted polymer on-line enrichment

A novel and simple chemiluminescence (CL) method has been developed and validated for determination of metformin. This method is based on hydroxyl radical chemiluminescence—the hydroxyl radical generated by reaction of Cu(II) and hydrogen peroxide oxidizes rhodamine B (RhB) to produce weak CL which can be enhanced by metformin. At the same time, metformin molecularly imprinted polymer (MIP) was synthesized. After enrichment based on the selectivity of metformin-MIP, the CL method was successfully applied to the determination of metformin in human serum. The linear range was from 1.0×10⁻⁸ to 1.0×10⁻⁶ g mL⁻¹ and the detection limit was 4×10⁻⁹ g mL⁻¹. The relative standard deviation at 2.0×10⁻⁷ g mL⁻¹ by use of MIP was 3.67% (n=7).     

Hydroxyl radical detection with a salicylate probe using modified CUPRAC spectrophotometry and HPLC

Reactive oxygen species (ROS) may attack biological macromolecules giving rise to oxidative stress-originated diseases, so it is important to establish efficient methods to screen hydroxyl radical scavengers for antioxidant therapy. Since *OH is very short-lived, secondary products resulting from *OH attack to various probes are measured. As a low-cost measurement technique, we used a salicylate probe for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2 h. The CUPRAC (cupric ion reducing antioxidant capacity) assay absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of *OH scavengers, the difference being proportional to the scavenging ability of the tested compound. Attack by *OH radicals upon salicylate produced 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, and 2,5-dihydroxybenzoate as major products. HPLC separation combined with CUPRAC spectrophotometry was used to identify and quantify hydroxylated salicylate derivatives in the presence of synthetic water-soluble antioxidants and green tea infusion. The developed spectrophotometric method for *OH detection was validated with HPLC, i.e., the concentrations of dihydroxybenzoates produced by radical attack from the probe were determined by HPLC, and the sum of (concentrationxabsorptivity) products of these components approximately agreed with the experimentally found CUPRAC absorbances, confirming the validity of Beer's law for the selected system. Statistical comparison of the results found with the proposed methodology and HPLC was made with two-way ANOVA (analysis of variance) test. Under optimal conditions, about 53% of the probe (salicylate) was converted into dihydroxybenzoate isomers in the absence of *OH scavengers, and these isomers were more specific markers of hydroxyl radicals than the non-specific malondialdehyde end-product of the TBARS test. Thus, the more costly and less speedy HPLC method could advantageously be substituted with the proposed spectrophotometric assay of *OH detection, which was also of much higher yield than the TBARS colorimetric assay.     

Biochemical Studies on Hemoglobin Modified with Reactive Oxygen Species (ROS)

Hemoglobin is the iron-containing oxygen transporting metalloprotein in the red cells of blood in mammals and other animals. Hemoprotein-mediated oxidative stress is thought to play a major role in pathophysiology of cerebral hemorrhage, blast pressure injury, crush injury, myocardial ischemia reperfusion injury. Hemoglobin undergoes oxidation-reduction reactions that lead to both generation and consumption of highly reactive oxygen and nitrogen species. In the present study, hemoglobin molecule was treated with hydrogen peroxide and the modification so incurred was analyzed by UV spectra, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detection of carbonyl content. Our observations suggest that carbonyl content increases with increase in concentration of hydrogen peroxide. Production of hydroxyl radical was assessed by using benzoate degradation analysis. Our results was in tandem with the fact that hemoglobin on treatment with hydrogen peroxide rapidly generates free-radical species that can degrade benzoate to thiobarbituric acid reactive material which on reacting with thiobarbituric acid gives color. The increase in absorbance of ROS-modified hemoglobin at 532 nm shows the increase in benzoate degradation, which is a parameter of hydroxyl radical formation with increase in concentration of hydrogen peroxide. Modified hemoglobin was treated with catalase, mannitol, thiourea, glutathion, sodium benzoate and their effect were detected by spectroscopy and SDS-PAGE (12%). Substantial scavenging effect of aforementioned antioxidants reiterates the formation of hydroxyl radical. Catalase shows the maximum scavenging effect followed by thiourea and mannitol.