Peroxidase Activity Of Hemoglobin Research Articles

Estimate the Correlation Between Some Antioxidant Biomarkers and Pseudo Peroxidase Activity of Hemoglobin in Patients With Chronic Kidney Disease

Background: Hemoglobin, being a hemoprotein, may operate as a peroxidase due to the hemolysis process and the release of heme into the bloodstream. Objective: Evaluation of the oxidative biomarkers and peroxidase activity of hemoglobin in patients with the final stage of renal failure. Materials and Methods: The study encompassed a total of 200 blood samples, with 140 samples collected from patients diagnosed with Chronic Kidney Disease (CKD) and 60 samples obtained from individuals who were considered healthy control subjects. Results: Levels of various serum parameters, including total protein, urea, creatinine, albumin, free amino, disulfide, ischemia-modified albumin (IMA), peroxidase activity, and specific activity, exhibited a significant increase in patients with Chronic Kidney Disease (CKD) when compared to the control group consisting of healthy individuals. Conversely, CKD patients demonstrated a notable decrease in levels of glomerular filtration rate (GFR), hemoglobin, native thiols, and total thiols compared to the control group. Conclusion: Oxidative biomarkers may present useful information about oxidative stress and help to diagnose CKD patients in their advanced stages of the disease.

(Invited) A Biosensor Platform for on-Site Virus Visualization

Introduction The detection of small amounts of specific targets such as toxins, pathogens, and viruses has become increasingly important since the SARS-CoV-2 pandemic. Particularly, the on-site virus detection by naked eye or camera, which allows direct and rapid visualization of the virus on a surface directly and rapidly, is one of the most appropriate ways to control and prevent from the infection. Currently available technologies for the virus detection such as quantitative PCR, ELISA, and lateral flow immunoassay are useful, but they require complicated sample extractions and sometimes bound/free (B/F) separation procedures, which makes the detection time-consuming and inconvenient. One realistic example of the on-site visualization is a forensic luminol blood test based on chemiluminescence because complicated detection procedures must be eliminated for the on-site visualization. The forensic test is based on the peroxidase activity of hemoglobin for the chemiluminescence emission upon the spraying of hydrogen peroxide solution. Therefore, we focused on generation of hydrogen peroxide in the proximal of virus and the associated emission of chemiluminescence, which enables us to the on-site visualization.To realize the localized hydrogen peroxide generation and successive chemiluminescence emission, we designed sequential enzymatic reactions using oxidase and peroxidase to be occurred only on the viral surface. In detail, we prepared antibody-enzyme complex (AEC) composed of oxidase and anti-virus antibody as a donor, and peroxidase-based molecules with virus-recognition ability as an acceptor. In the presence of virus, the donor and the acceptor bind to the virus, and a hydrogen peroxide relay from the donor and the acceptor on the viral surface will lead to the detection and visualization only after mixing of reagents (Fig.). Here, glucose oxidase (GOx) was used as a model donor enzyme because the substrate, glucose, is safe and highly stable, and conveniently prepared the AEC by SpyCatcher/SpyTag system, a protein coupling module for biosensing1). As the acceptor, bispecific aptamer targeting virus and peroxidase-based molecules was used. Then, we validated the utility of the platform by applying it to the detection and visualization of inactivated SARS-CoV-2. Methods The recombinantly produced SpyTag (ST)-fused anti-SARS-CoV-2 surface protein single-chain variable fragment (scFv-ST) and SpyCatcher (SC)-fused GOx (SC-GOx) were equivalently mixed and incubated at 4℃ to prepare the homogenous AEC. The bivalent AEC can be prepared because the GOx forms homo-dimer. The enzymatic activity and affinity were evaluated. Then, we constructed the detection systems using different peroxidase-based molecules as acceptors, and applied them to inactivated SARS-CoV-2 detection and visualization using a conventional smartphone camera. Results & Discussion SDS-PAGE analysis revealed that the AEC was prepared by only mixing of scFv-ST and SC-GOx via the SpyCatcher/SpyTag reaction. The AEC showed high binding ability, and K D values of scFv-ST and the AEC were 32 nM and 19 nM, respectively. This affinity enhancement was caused by the bivalency of the AEC. The AEC showed 72% of oxidase activity compared to the original GOx, suggested that the AEC retained sufficient activity for the signal generation as the donor.As the acceptor, myoglobin (Mb) and a bispecific aptamer composed of Mb aptamer2) and SARS-CoV-2 surface protein binding aptamer were used. The peroxidase activity of Mb is relatively weak, so a reduced decreased background signal is expected. The bispecific aptamer showed binding abilities to Mb and inactivated SARS-CoV-2.Using these detection components, we successfully detected inactivated SARS-CoV-2 by only mixing of the components, and the linear detection range met the clinically required level. Owing to the sequential enzymatic reactions only on the viral surface, the rapid detection system without any washing procedures was established. By exchanging a set of antibody and aptamer, it was applied to inactivated influenza A virus detection.Finally, hemin and hemin-binding aptamer were used as the acceptor and applied it to the inactivated SARS-CoV-2 visualization. As a result, not only the viruses in a 96-well plate but also on a nitrocellulose membrane was visualized by luminol-derived chemiluminescence through a conventional smartphone camera. This was a first report of the on-site virus visualization. In addition, the use of sequential enzymatic reaction provides the signal amplification when applied to electrochemical detection3), leading to more rapid and convenient detection using established portable devices. This system can be a platform for control of infection in public space in the future. References 1) D. Miura et al., Talanta, 234, 122638 (2021).2) K. Tsukakoshi et al., Nucleic Acid Res., 49, 6069-6081 (2021).3) B. Jeong et al., Anal. Chem., 85, 1784-1791 (2013). Figure 1

Decreased peroxidase activity of glycated hemoglobin inversely correlated with levels of glycated hemoglobin in type 2 diabetes patients

Background and objectives: Diabetes mellitus is a metabolic disorder with diverse etiological factors. Imbalances between oxidative stress and antioxidant defenses play critical roles in the pathogenesis and progression of diabetes. peroxidase, an antioxidant enzyme similar to superoxide dismutase and catalase, is involved in the etiology and progression of diabetes. This study aimed to elucidate the relationship between peroxidase activity of hemoglobin and glycated hemoglobin with fasting blood glucose levels in patients with type-2 diabetes mellitus. Methods: Sixty-four patients with type-2 diabetes mellitus and an equal number of healthy controls were recruited for the study. Peroxidase activity of hemoglobin and glycated hemoglobin was assayed using L. Goth's method for serum catalase assay, with further modification and standardization. Results: Peroxidase activity of hemoglobin was significantly reduced (P<0.0001) in patients with type 2 diabetes compared to healthy controls, as was peroxidase activity of glycated hemoglobin (P< 0.001). In both patients and controls, peroxidase activity of glycated hemoglobin was significantly higher than normal hemoglobin. In patients with type 2 diabetes, glycated hemoglobin showed a significant negative correlation (r=- 0.308, P= 0.013) with its peroxidase activity, indicating increased activity is associated with poorer glycemic control. Furthermore, peroxidase activiy of normal hemoglobin positively correlated with peroxidase activity of glycated hemoglobin (mainly HbA1c )(r= 0.305, P=0.014). Conclsions: Peroxidase activity of glycated hemoglobin increases with glycated hemoglobin levels, indicating deteriorating glycemic control in patients with type 2 diabetes.

Peroxidase activity of erythrocytes hemoglobin under action of low-frequency vibration

Background. Hemoglobin is a hemoprotein which in the presence of oxidative equivalents, such as H2O2, can act as peroxidase with a very high oxidative potential. Hemoglobin oxidation is accompanied by generation of highly oxidized forms of iron and globin radicals that have high oxidative activity and are toxic to cells. In addition, peroxidase activity may indicate structural changes that occur in the hemoglobin molecule as a result of chemical modification. Materials and Methods. Erythrocyte suspension was subjected to vibration for 3 h within the frequency range from 8 to 32 Hz with amplitudes of 0.5 ± 0.04 and 0.9 ± 0.08 mm. At certain intervals, hemoglobin peroxidase activity was determined together with the content of its ligand forms in the hemolysates of cells. Additionally, experiments were performed to investigate the mechanism and calculate the kinetic parameters of peroxidase reaction. Results and Discussion. Experimental data on low-frequency vibrations effect on erythrocyte hemoglobin peroxidase activity were analyzed. The kinetics of the oxidation reaction of p-phenylenediamine by hemoglobin in erythrocytes was studied. It was found that peroxidase oxidation has a ping-pong mechanism. The kinetic parameters of the peroxidase reaction involving hemoglobin were determined. The change of kinetic parameters after two-hour exposure to the incubation medium and low-frequency vibration was studied. A possible mechanism of action of hemoglobin in oxidation reactions involving H2O2 was proposed. Conclusion. Any effect that initiates the formation of methemoglobin leads to an increase in the peroxidase activity of hemoglobin due to the involvement of the latter in the pseudoperoxidase cycle and the formation of toxic reactive globin radicals. The high content of oxyhemoglobin in the cell, observed under vibrations within the frequency range of 16–32 Hz with an amplitude of 0.9 ± 0.08 mm, can prevent its oxidation and involvement in the pseudoperoxidase cycle.

Peroxidase activity of hemoglobin and heme destruction in the presence of hydrogen peroxide and CT-DNA

The aim of this study was to investigate the peroxidase activity of Hb with different concentrations of hydrogen peroxide and compare it with hypochlorous acid effect on Hb. Hypochlorous acid at higher concentrations decomposed Hb and heme, releasing fee iron ion from the metal center. High concentrations of hydrogen peroxide switched the peroxidase activity of Hb towards the partial Hb and heme destruction. Heme alone was degraded showing that the Hb conformation and protein environment protects Hb from the distraction in the presence of highly increased hydrogen peroxide concentration that occurs as a result of oxidative stress. In the presence of CT-DNA acted inhibition of the peroxidase activity of Hb was observed signaling inhibited hydrogen peroxide consumption.

Peptide Inhibitor of Complement C1 Inhibits the Peroxidase Activity of Hemoglobin and Myoglobin.

Hemoglobin is the natural carrier of oxygen in red blood cells (RBCs). While intracellular hemoglobin provides life-sustaining oxygen transport, extracellular free hemoglobin displays toxicity due to inherent peroxidase activity generating reactive oxygen species that subsequently react with the hemoglobin molecule to produce toxic heme degradation products resulting in free radicals, oxidative stress damage, and lipid peroxidation. We have recently demonstrated that Peptide Inhibitor of Complement C1 (PIC1) inhibits peroxidase activity of the heme-based enzyme myeloperoxidase. To elucidate whether PIC1 could inhibit peroxidase activity of hemoglobin, we evaluated the consequence of PIC1 on RBC lysates, methemoglobin, and myoglobin using tetramethylbenzidine (TMB) as an oxidation target. PIC1 reversibly and dose-dependently prevented TMB oxidation to tetramethylbenzidine diimine by RBC lysates, methemoglobin, and myoglobin, having comparable activity to the inhibitor 4-aminobenzoic acid hydrazide. PIC1 inhibited TMB oxidation of RBC lysates similar to L-cysteine suggesting that the two cysteine residues contained in PIC1 may mediate peroxidase activity. PIC1 also inhibited heme destruction by NaOCl for RBC lysates, hemoglobin, and myoglobin as assayed by preservation of the Soret absorbance peak in the presence of NaOCl and reduction in free iron release. In conclusion, PIC1 inhibits peroxidase activity of hemoglobin and myoglobin likely via an antioxidant mechanism.

Molecular mechanisms of bio-catalysis of heme extraction from hemoglobin

Red blood cell hemolysis in sickle cell disease (SCD) releases free hemoglobin. Extracellular hemoglobin and its degradation products, free heme and iron, are highly toxic due to oxidative stress induction and decrease in nitric oxide availability. We propose an approach that helps to eliminate extracellular hemoglobin toxicity in SCD by employing a bacterial protein system that evolved to extract heme from extracellular hemoglobin. NEAr heme Transporter (NEAT) domains from iron-regulated surface determinant proteins from Staphylococcus aureus specifically bind free heme as well as facilitate its extraction from hemoglobin. We demonstrate that a purified NEAT domain fused with human haptoglobin β-chain is able to remove heme from hemoglobin and reduce heme content and peroxidase activity of hemoglobin. We further use molecular dynamics (MD) simulations to resolve molecular pathway of heme transfer from hemoglobin to NEAT, and to elucidate molecular mechanism of such heme transferring process. Our study is the first of its kind, in which simulations are employed to characterize the process of heme leaving hemoglobin and subsequent rebinding with a NEAT domain. Our MD results highlight important amino acid residues that facilitate heme transfer and will guide further studies for the selection of best NEAT candidate to attenuate free hemoglobin toxicity.

Enhancing effect of cimetidine on peroxidase activity of human erythrocyte hemoglobin.

Hemoglobin is released to the serum after erythrocyte lyses. Haptoglobin is responsible for carrying hemoglobin into the serum. In hemolytic disease, the amount of hemoglobin which is released to the serum is high; however, the amount of haptoglobin is not enough for binding all the released hemoglobins. Free hemoglobin has peroxidase activity (a pseudoenzyme) and has been indicated to be harmful for patients. This study is focused on the effect of cimetidine on peroxidase activity of hemoglobin. Erythrocytes were lysed to obtain hemoglobin. Peroxidase activity of hemoglobin was detected using o-dianisidine and H(2)O(2) as substrates. Our results showed that the drug operated as an activator for the pseudoenzyme. Cimetidine bound to the pseudoperoxidase in an un-competitive manner and decreased the Km. Half maximal effective concentration (EC(50)) of cimetidine was determined to be about 12.5 mM. Alkaline pH increased the rate of reaction. Arrhenius plot showed that the activation energies of reactions in the absence and presence of drug were about 10.5 kJ/mol and 7.65 kJ/mol, respectively. The results demonstrated that cimetidine activates the peroxidase activity of free hemoglobin. Hence, it is suggested that the prescription of cimetidine for the patients with hemolyses diseases may enhance the harmful effects of free hemoglobin in these patients.

Redox reactivity in propolis: direct detection of free radicals in basic medium and interaction with hemoglobin

Propolis is one of many natural products with known antioxidant properties. The present work aims to investigate the intimate molecular-level mechanisms of this antioxidant reactivity. Electron paramagnetic resonance (EPR)-detectable free radical signals are described here for the first time in propolis extracts. The shape of these signals and the conditions in which they were obtained, point to polyphenolic flavonoids as the sites of the radicals. An inverse correlation between antioxidant capacity and free radical signal intensity is shown. The free radical reactivity of propolis is also illustrated by the effect it exerts on the biologically-relevant peroxide reactivity of hemoglobin. A new test of antioxidant ability in natural extracts such as propolis is proposed, based on modulation of the ascorbate peroxidase activity of hemoglobin (HAPX). Results of this test correlate well with those obtained via traditional methods such as those based on DPPH (2,2-diphenyl-1-picrylhydrazyl), or on ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid).

Determination of serum haptoglobin reference value in clinically healthy Iranian fat-tailed sheep.

Summary Blood samples were collected from the jugular vein of 101 clinically healthy Iranian fat-tailed sheep according to their age ( 5 years) and sex. Serum haptoglobin (Hp) was measured based upon the preservation of the peroxidase activity of haemoglobin, which is directly proportional to the amount of Hp. The reference value for serum Hp of apparently healthy Iranian fat-tailed sheep was 0.05-0.18 g/l. Age and sex had no significant effect on the serum concentration of Hp in examined animals.

Peroxidase activity of hemoglobin towards ascorbate and urate: A synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC)

Acellular hemoglobins developed as oxygen bridging agents with volume expanding properties (“blood substitutes”) are prone to autoxidation and oxidant-mediated structural changes in circulation. In the presence of hydrogen peroxide and either ascorbate or urate we show that ferric hemoglobin functions as a true enzymatic peroxidase. The activity saturates with both substrates and is linearly dependent on protein concentration. The activity is enhanced at low pH with a p K a of 4.7, consistent with protonation of the ferryl species (Fe(IV)−OH) as the active intermediate. To test whether these redox reactions define its behaviour in vivo we exchanged transfused guinea pigs with 50% polymerized bovine Hb (PolyHbBv) and monitored plasma levels of endogenous ascorbate and urate. Immediately after transfusion, met PolyHbBv levels increased up to 30% of total Hb and remained at this level during the first 24 h post transfusion. Plasma ascorbate decreased by 50% whereas urate levels remained unchanged after transfusion. A simple kinetic model, assuming that ascorbate was a more active ferric heme reductase and peroxidase substrate than urate, was consistent with the in vivo data. The present finding confirms the primary and secondary roles of ascorbate and urate respectively in maintaining the oxidative stability of infused Hb.

Reaction between Gas-phase Hydrogen Atom and Chemisorbed Bromine Atoms on a Silicon(001)-(2X1) Surface

Electron transfer of a redox protein at a bare gold electrode is too slow to observe the redox peaks. A novel Nafion-riboflavin functional membrane was constructed during this study and electron transfer of cytochrome c, superoxide dismutase, and hemoglobin were carried out on the functional membrane-modified gold electrode with good stability and repeatability. The immobilized protein-modified electrodes showed quasireversible electrochemical redox behaviors with formal potentials of 0.150, 0.175, and 0.202 V versus Ag/AgCl for the cytochrome c, superoxide dismutase and hemoglobin, respectively. Whole experiment was carried out in the 50 mM MOPS buffer solution with pH 6.0 at 25 oC. For the immobilized protein, the cathodic transfer coefficients were 0.67, 0.68 and 0.67 and electron transfer-rate constants were evaluated to be 2.25, 2.23 and 2.5 s?1, respectively. Hydrogen peroxide concentration was measured by the peroxidase activity of hemoglobin and our experiment revealed that the enzyme was fully functional while immobilized on the Nafion-riboflavin membrane.

Electrochemical Behavior of Redox Proteins Immobilized on Nafion-Riboflavin Modified Gold Electrode

Electron transfer of a redox protein at a bare gold electrode is too slow to observe the redox peaks. A novel Nafion-riboflavin functional membrane was constructed during this study and electron transfer of cytochrome c, superoxide dismutase, and hemoglobin were carried out on the functional membrane-modified gold electrode with good stability and repeatability. The immobilized protein-modified electrodes showed quasi-reversible electrochemical redox behaviors with formal potentials of 0.150,0.175, and 0.202 V versus Ag/AgCl for the cytochrome c, superoxide dismutase and hemoglobin, respectively. Whole experiment was carried out in the 50 mM MOPS buffer solution with pH 6.0 at 25 °C. For the immobilized protein, the cathodic transfer coefficients were 0.67, 0.68 and 0.67 and electron transfer-rate constants were evaluated to be 2.25, 2.23 and 2.5 s -1 , respectively. Hydrogen peroxide concentration was measured by the peroxidase activity of hemoglobin and our experiment revealed that the enzyme was fully functional while immobilized on the Nafion-riboflavin membrane.

A Hydrogen Peroxide Biosensor Based on Peroxidase Activity of Hemoglobin in Polymeric Film

A Hydrogen peroxide (H2O2) biosensor, based on hemoglobin (Hb) and ortho-phenylenediamine (o-PD) gold electrode, was fabricated. Hb was immobilized onto the electrode surface by electrochemical polymerize method with o-PD. The designed biosensor showed a well defined redox peak which was attributed to the direct electrochemical response of Hb. The immobilized Hb exhibited an excellent electrocatalytical response to the reduction of hydrogen peroxide, enabling the sensitivity determination of H2O2. Factors and performances such as pH, potential, influencing the designed biosensor, were studied carefully. The amperometric detection of H2O2 was carried out at -300 mV in phosphate buffer solution (PBS) (0.1 M) with pH 6.0. This biosensor showed a fast amperometric response (less then 5 s) to H2O2. The levels of the (Relative standard deviation) RSDs (< 3.5%) for the entire analyses reflected a highly reproducible sensor performance. Using the optimized conditions, the detection limit of the biosensor was 1 x 10(-7) M and linear range was from 5 x 10(-6) to 1.25 x 10(-4) M. In addition, this sensor showed long-term stability and good sensitivity.

Evaluation of serum haptoglobin in clinically healthy Iranian camels (Camelus dromedarius)

Blood samples were collected from the jugular vein of 70 clinically healthy dromedary camels according to their age (1–2, 2–3, 3–4, 4–5 and >5 years) and sex. Serum Hp was measured according to preservation of the peroxidase activity of haemoglobin, which is directly proportional to the amount of haptoglobin. Age and sex had no significant effect on the serum concentration of Hp in clinically healthy Iranian dromedary camels.

Review article: population screening for colorectal cancer

Colorectal cancer is a common cancer and common cause of death. The mortality rate from colorectal cancer can be reduced by identification and removal of cancer precursors, adenomas, or by detection of cancer at an earlier stage. Pilot screening programmes have demonstrated decreased colorectal cancer mortality; as a result many countries are developing colorectal cancer screening programmes. The most common modalities being evaluated are faecal occult blood testing, flexible sigmoidoscopy and colonoscopy. Implementation of screening tests has been hampered by cost, invasiveness, availability of resources and patient acceptance. New technologies such at computed tomographic colonography and stool screening for molecular markers of neoplasia are in development as potential minimally invasive tools. This review considers who should be screened, which test to use and how often to screen.

Effect of nano cadmium sulfide on the electron transfer reactivity and peroxidase activity of hemoglobin

Hemoglobin (Hb) is immobilized with cadmium sulfide (CdS) nanoparticles (NPs) on pyrolytic graphite (PG) electrode to characterize the electrochemical reactivity and peroxidase activity of the protein. The result demonstrates that fine redox waves of Hb can be achieved after this protein is entrapped in CdS NPs. Meanwhile, the protein can exhibit nice catalytic activity towards hydrogen peroxide (H 2O 2). Linear relationship between the reductive peak current and the H 2O 2 concentration has been obtained from 5.0 × 10 − 6 to 4.0 × 10 − 4 mol/L, on the basis of which a new kind of H 2O 2 biosensor might be developed in the future.

Development of a time-resolved fluorometry based immunoassay for the determination of canine haptoglobin in various body fluids

A time-resolved immunofluorometric assay (TR-IFMA) was developed for the determination of haptoglobin (Hp) in canine serum. Haptoglobin was purified from canine acute phase serum by ammonium sulphate precipitation followed by gel filtration. This isolated dog Hp was used as the standard to calibrate the assay. Intra- and inter-assay coefficients of variation of the assay were, respectively, 5.7% and 16.6% at 0.51 mg/mL, 2.4% and 10.6% at 2.1 mg/mL and 10.5% and 11.9% at 32.5 mg/mL. The dilution of serum samples with high Hp concentrations resulted in linear regression equations with R2 of 0.99 and 0.97. A high correlation was found in serum Hp measurements by TR-IFMA and a commercial assay based on peroxidase activity of haemoglobin bound to haptoglobin (R2 = 0.96). The limit of detection for the TR-IFMA method was 0.002 microg/mL. The addition of fresh haemolysate to serum samples did not affect the haptoglobin concentration (P = 0.694). Statistical differences (P < 0.003) were found between healthy dogs and dogs with different pathological processes. In whole blood, Hp concentrations were much lower than in serum but closely related (R2 = 0.84) whereas saliva Hp concentrations were poorly related with serum concentrations (R2 = 0.53). However, the concentration of Hp in saliva was significantly (P < 0.039) higher in dogs with pathological processes compared to healthy dogs. The assay sensitivity was adequate to also be applied to whole blood and saliva specimens.

A hydrogen peroxide sensor based on the peroxidase activity of hemoglobin immobilized on gold nanoparticles-modified ITO electrode

A novel ITO electrode surface modified with spherical and rod-shaped gold nanoparticles was prepared by a surfactant-assisted seeding growth approach, which provided a biocompatible matrix for the immobilization of hemoglobin (Hb). By electrochemical impedance measurements, gold nanoparticles modification and Hb immobilization on the electrode surfaces were characterized using [Fe(CN) 6] 3−/[Fe(CN) 6] 4− redox probe. Owing to the promoted electron transfer of Hb by gold nanoparticles, the Hb immobilized gold nanoparticles-modified ITO (Hb/Au/ITO) electrode exhibited an effective catalytic response to the reduction of H 2O 2 with good reproducibility and stability. The linear relationship existed between the catalytic current and the H 2O 2 concentration in the range of 1 × 10 −5 to 7 × 10 −3 M. The detection limit (S/N = 3) was 4.5 × 10 −6 M.

Enhanced peroxidase activity of hemoglobin in a DNA membrane and its application to an unmediated hydrogen peroxide biosensor.

Hemoglobin can exhibit not only a direct electron transfer reacting after being entrapped in a DNA membrane, but also a greatly enhanced peroxidase activity toward the reduction of hydrogen peroxide. Based on the direct electrochemical property and nice enzymatic activity of the protein in a DNA membrane, a reagentless hydrogen peroxide biosensor was prepared. The peak current related to hydrogen peroxide was linearly proportional to its concentration in the range of 1.9 x 10(-6)-6.8 x 10(-4) mol L(-1). The detection limit was 1 x 10(-6) mol/L.