Electrochemical investigation of immobilized hemoglobin: Redox chemistry and enzymatic catalysis

J. Biochem. Biophys. Methods 68 (2006) 87 – 99 www.elsevier.com/locate/jbbm

Electrochemical investigation of immobilized hemoglobin: Redox chemistry and enzymatic catalysis Hui-Hong Liu a,b , Guo-Lin Zou a,⁎ a

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China b Department of Chemistry and Biology, Xiangfan University, Xiangfan, 441053, China Received 13 December 2005; received in revised form 26 March 2006; accepted 9 April 2006

Abstract Hb entrapped in the Konjak glucomannan (KGM) film could transfer electrons directly to an edge-plane pyrolytic graphite (EPG) electrode, corresponding to the redox couple of FeIII/FeII. The redox properties of Hb, such as formal potential, electron transfer rate constant, the stability of the redox state of protein and redox Bohr effect, were characterized by cyclic voltammetry and square wave voltammetry. The stable HbKGM/EPG gave analytically useful electrochemical catalytic responses to oxygen, hydrogen peroxide and nitrite. © 2006 Elsevier B.V. All rights reserved. Keywords: Hemoglobin; Konjak glucomannan; Redox chemistry; Enzymatic catalysis

1. Introduction Hemoglobin (Hb) has been widely studied because of its physiological importance. It was the first protein to have its three-dimensional structure determined in the late 1950s. Since then hemoglobin has served as a model for understanding the relationship between a protein's structure and function [1]. Hb does not function in biological electron transfer chains, but redox reactions of hemoglobin have gained importance because of the general interest of the role of oxidative stress in diseases and the possible role of red blood cells in oxidative stress [2,3]. Although no catalytic reactions are normally associated with hemoglobin, modest peroxidase and cytochrome P450 activity capable of oxidizing a variety of biological substrates has been identified [4]. In addition to O2, ⁎ Corresponding author. Tel.: +86 7103903236; fax: +86 7103591876. E-mail addresses: [email protected] (H.-H. Liu), [email protected] (G.-L. Zou). 0165-022X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2006.04.001

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the ferrous heme iron in Hb can bind other diatomic molecules, such as carbon monoxide and nitric oxide, so Hb plays the role of a nitric oxide scavenger in blood [5]. All these processes are related to the redox reaction of Hb. From these points of view, electrochemical studies of Hb might provide new insight into its physiological functions and be helpful in fabricating biosensors. Cyclic voltammetry is widely used to characterize the redox properties of proteins or enzymes. This technique can provide information about the kinetics of the electron transfer reactions, any coupled chemical reactions and in addition thermodynamic data (i.e., redox potentials) [6,7]. However, Hb displays poor redox kinetics for the following reasons: (1) heme is buried deep inside the globin, resulting in a long electron transfer distance between the redox active site and electrode surface, (2) these proteins require high activation energy for conformational changes associated with the redox transition [8], (3) the redox process instigated by dissociation and association of the bound H2O molecule in the reduced and oxidased form [9], (4) macromolecular impurities or denatured proteins adsorbed on electrodes create a passive layer that blocks the electron transfer between heme and electrodes [10], and (5) proteins locate at electrode surface resulting in unfavorable orientations for electron transfer [11]. Due to the development of modified electrodes, the rate of direct electron transfer between Hb and electrodes is enhanced. Various types of films have been constructed, such as self-assemble monolayer [8,12], surfactant films [10], nano-materials [13–15], and polysaccharide hydrogel [16,17]. Konjak glucomannan (KGM) is a high molecular weight polysaccharide classified as a glucomannan. The molecular structure is comprised of mannose and glucose chains in a molar ratio of 1.6 to 1 respectively with beta 1–4 linkages. The average molecular weight is typically 1 million Da, which accounts for konjac's high pseudoplastic viscosity. The physical properties and the gelation behavior of KGM have been thoroughly explored [18,19]. In this paper, we show that Hb can be immobilized stably on the surface of an edge-plane pyrolytic graphite electrode using KGM hydrogel. To our knowledge, this is the first time KGM hydrogel as an immobilized material has been used for studying direct electrochemistry of Hb; particularly concerning the redox chemistry and enzymatic properties of Hb. 2. Materials and methods 2.1. Materials Bovine hemoglobin (Fluka) was used as received and was dissolved in 10 mM phosphate buffer solution (pH 7.0). The concentration of Hb 1.34 × 10− 4 M was determined by UV–Vis spectroscopy using ε406 = 41,000 M− 1 cm− 1 [1]. Konjak glucomannan (KGM) was extracted from the tuber of Amorphallus revieri supplied by Zhuxi Konjac Institute, Hubei. Its hydrogel was first prepared by dissolving 0.4 g of KGM in 100 mL of boiling water, and then allowed to cool at room temperature. N,N-dimethylformamide (DMF) was added to the KGM hydrogel before mixing with the Hb solutions. The ratio of DMF to KGM (v/v) is 1:4 in all experiments. A stock solution of H2O2 was diluted from 30% prior to use. Other chemicals were of at least analytical reagent grade. The concentrations of H2O2 and NO2− were determined by titration with a potassium permanganate standard solution. Water used was doubly distilled and sterilized.

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2.2. Preparation of modified electrode The area of the EPG was determined by cyclic voltammetry using the Randles-Sevcik equation for a reversible redox couple [Fe(CN)6]3−/[Fe(CN)6]2−, giving a value of 0.28 ± 0.06 cm2. The EPG was first polished with 0.05 μm alumina polishing suspension (Buehler) and then cleaned ultrasonically in acetone and then water, respectively, for 3 min. 10 μL of protein–KGM solution was pipetted onto the EPG surface and air dried for about 8 h. The modified electrode is denoted as Hb-KGM/EPG in the text. 2.3. Measurements and apparatus Cyclic voltammetry (CV) was carried out with a computer-controlled electrochemical workstation (CHI 660A, USA) in a standard two-compartment glass cell with a conventional three-electrode configuration, which consisted of a saturated calomel electrode (SCE) as the reference, a piece of platinum gauze as the counter and an edge-plane pyrolytic graphite as working electrode (EPG, Le Carbone-Lorraine, France). Buffers were purged with purified nitrogen for at least 20 min prior to use and all experiments were done under a nitrogen atmosphere. All potentials given below were relative to SCE. Reflection-absorption infrared (RAIR) spectra of the hemoglobin and KGM-hemoglobin films on the EPG surface were obtained using a Magna-IR 500 Spectrometer (Nicolet Instrument Corporation, USA). Films were prepared by transferring KGM-hemoglobin solution or a solution of protein alone onto the EPG electrode surface. A spectrum of the bare EPG surface was subtracted as background. UV–visible absorption (UV–Vis) spectra were recorded on a Tu-1901 UV–Vis spectrophotometer (Purkinje General Instrument Co. LTD. Beijing, China). The KGM-hemoglobin films for UV–Vis were prepared by dropping a 1:1 mixture of protein–KGM gel solution. A film only containing hemoglobin on a glass slide was used as a control. The reference was an uncoated glass slide. 3. Results 3.1. UV–Vis spectroscopy The position of the Soret absorption band of heme iron provides structure information about the heme pocket. Fig. 1 shows the UV–Vis spectra of both Hb films and Hb in KGM films on glass slides immersed in solution (PBS pH 7.0). The Soret bands of 410 nm for Hb film shifted to 413 nm when Hb was entrapped in KGM hydrogel. A shift of 3 nm in the Soret absorption band of Hb suggests that the environment of heme pocket may be altered slightly in KGM hydrogel films. 3.2. RAIR spectroscopy RAIR is a surface infrared technique ideally suited for exploring the secondary structure of protein on a solid substrate surface. The shapes of amide I (1700–1600 cm− 1) and amide II (1600–1500 cm− 1) infrared absorption bands of proteins provide detailed information on the secondary structure of the polypeptide chain. The amide I and II bands in the RAIR spectra of Hb in KGM film (Fig. 2) had shapes similar to that of the Hb film alone but the bands had small blue

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KGM-Hb

0.20 0.18

A

0.16 0.14 0.12 0.10

Hb

0.08 0.06 300

350

400

450

500

550

λ / nM Fig. 1. UV–Vis spectra of Hb and KGM-Hb films on glass slides.

shift (1656 to 1648 cm− 1 and 1549 to 1548 cm− 1), which indicated that Hb entrapped in KGM film keeps its original conformation. 3.3. Redox chemistry of immobilized Hb Neither bare EPG nor KGM-modified EPG showed any electrochemical response in either buffer solution or protein solution. However, when Hb-KGM/EPG was immersed into the 0.1 M PBS (pH 7.0), a pair of well-defined and reversible redox peaks was observed (Fig. 3). The cathodic peak potential Epc = − 0.361 V and the anodic peak potential Epa = − 0.295 V at a scan rate of 0.5 V s− 1, in 0.1 M PBS (pH 7.0). The formal potential E°′ (− 0.328 V versus SCE) was evaluated by taking the mean value of the cathodic and anodic peak potentials, e.g. E°′ = (Epc +

Fig. 2. RAIR spectra of Hb and KGM-Hb films on the EPG electrode surfaces.

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-10

b -5

i / μA

a 0

5

10 -0.8

-0.6

-0.4

-0.2

0.0

0.2

E / V vs SCE Fig. 3. Cyclic voltammograms of KGM/EPG (a), Hb-KGM/EPG (b) in 0.1 M pH 7.0 PBS, scan rate 0.5 V s− 1.

Epa) / 2. These results indicate that the reversible redox peaks result from the Fe(III)/Fe(II) couple of Hb [10] and the electron transfer rates between Hb and the electrode surface are facilitated by KGM hydrogel. The surface coverage (Γ) of 4.26 × 10− 11 mol cm− 2 was estimated from integration of the reduction peak in the CVs according to Γ = Q/nFA, where Q is the charge involved in the reaction, n is the number of electron transferred, F is the Faraday's constant, and A is the electrode area. Average formal potentials (E°′ = −0.336 V), apparent coverage (Γ = 5.12 × 10− 11 mol cm− 2), electron transfer coefficient (α = 0.51), and apparent heterogeneous electron transfer rate constants (ks = 42 ± 6) were also estimated by nonlinear regression analysis of square wave voltammetry (SWV) forward and reverse curves [20,21]. A linear dependence of anodic and catholic peak currents versus the scan rate ranging from 0.1 V s− 1 to 5 V s− 1 was observed, which is different from that for heme proteins in surfactant films [22,23]. The charges (Q) involved in oxidative and reductive processes remained nearly constant with an increasing scan rate. These are characteristic of thin-layer electrochemical behavior of immobilized heme proteins, rather than freely-diffusing proteins. These indicate that Hb is confined by the KGM hydrogel. The potential separation between anodic and cathodic peaks (ΔEp = Epa − Epc) is 66 mV at a scan rate of 0.5 V s− 1 that is larger than the theoretical value of 0 mV for a surface process, which is probably attributed to the long distance from the heme of the protein to the electrode surface due to the deep-buried electroactive centers. 3.4. Redox Bohr effect Nearly reversible voltammograms with stable and well-defined redox peaks were obtained in the pH range of 3.0–10.0 (Fig. 4A). The pH increase led to a negative shift in E°′ (Fig. 4B). The shift in E°′ depended on pH, suggesting that the redox reaction was accompanied by the transfer of a proton. The linear regression equation of E (V) = − 0.021 − 0.044pH was obtained, with a correlation coefficient of 0.991 (n = 8). The absolute value of the slope (44 mV/pH) is smaller than the theoretically expected value, 59 mV/pH for a one electron and one proton reaction [24]. The reason might be the influence of the protonation states of transligands to the heme iron and

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pH=9.0 7.0 5.0 3.0

A

i / μA

-5

0

5

10 -0.8

-0.6

-0.4

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0.0

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E / V vs SCE -0.10

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E / V vs SCE

-0.20 -0.25 -0.30 -0.35 -0.40 -0.45 2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0 11.0

pH Fig. 4. (A) Cyclic voltammograms of Hb-KGM/EPG in solution with different pH. Scan rate 0.5 V s− 1. (B) Plot of E°′ versus pH.

amino acids around the heme, or the protonation of the water molecule coordinated to the central iron [25]. Additionally, when the pH values were adjusted to below 4.0, the cathodic current increased remarkably compared to that obtained at the pH values above 4.0. The change was reversible. A well-defined CV of Hb-KGM/EPGs was obtained in pH 7.0 PBS. When the modified electrode was transferred from a pH 7.0 to a pH 4.0 solution, an asymmetric redox CV was observed. Subsequently, the same CV as obtained at pH 7.0 was again achieved when the same modified electrodes were transferred back to the pH 7.0 buffer. This is likely due to a reversible pH-induced conformational change at low pH. 3.5. Reduction of oxygen Hb is heme-based oxygen binding and delivery protein found in a wide variety of organisms, including most vertebrates and a number of invertebrate species. Although it is not an enzyme, hemoglobin provides oxygen transport from lungs to muscles via the principle of positive

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cooperative. Hemoglobin binds oxygen with positive cooperative in much the same way that the allosteric enzyme responses with positive cooperative to the binding of its substrate. Electrochemical catalytic reduction of oxygen by the Hb-KGM/EPG was explored by CV (Fig. 5). In a deoxygenated PBS (pH 7.0), KGM/EPG presents no electrochemical signal (Fig. 3a) and Hb-KGM/EPG presents a pair of redox peaks (Fig. 3b) within the surveyed potential range of 0.2 to − 0.8 V. But in PBS saturated oxygen with air, a reduction peak at − 0.706 V was observed using KGM/EPG. For Hb-KGM/EPG, a significant increase in reduction peak at − 0.284 V, accompanied by the decrease of the oxidation peak of HbFeIII/FeII redox couple, was observed. The positive shift of potential about 0.522 V is the typical characteristics of electrocatalysis. The increase of the reduction peak current and the shift of peak potential with an increase in the amount of oxygen in solution indicated that Hb entrapped in KGM film had reacted with oxygen. Oxygen can bind reversibly to heme to form HbFeII-O2, which can then undergo electrochemical reduction at the potential of HbFeIII reduction, producing HbFeII again [26]. The potential of the reduction peak is more positive about 100 mV than the HbFeIII/FeII redox couple. This could be explained if the reduction peak was associated with the reduction of HbFeIIO2 rather than HbFeIII. The reproducible reduction peak current could be obtained when the processes of deoxygenating and oxygenating the solution were carried out in turn. In other words, the reduction activity of Hb to oxygen did not decrease in the processes. Although the mechanism of catalytic reduction of oxygen by heme enzymes is not yet very clear, it may differ from that of reduction of H2O2 in which HbFeII is oxidated to HbFeIVfO by H2O2, resulting in the inactivation of Hb. 3.6. Reduction of hydrogen peroxide Hemoglobin reacts with hydrogen peroxide to catalyze the removal of hydrogen peroxide, without being consumed in the process (a property that qualifies Hb as a pseudoperoxidase enzyme), and to generate ferrylhemoglobins (FeIVfO) as a transient intermediate. Ferrylhemoglobin is a strong oxidizing agent that is believed to mediate the peroxidation of lipids, proteins, carbohydrates, and nucleic acids. -5

b

a

0

I / μA

5 10 15 20

c

d

25 -0.8

-0.6

-0.4

-0.2

0.0

0.2

E / V vs SCE Fig. 5. Cyclic voltammograms of KGM/EPG without oxygen (a) and saturated with air (c), Hb-KGM/EPG without oxygen (b) and saturated with air (d) in 0.1 M pH 7.0 PBS, scan rate 0.5 V s− 1.

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Electrochemical catalytic reductions of hydrogen peroxide using Hb-KGM/EPG were studied by voltammetry. For an Hb-KGM/EPG in a pH 7.0 PBS, a reduction peak at − 0.30 V was observed after addition of H2O2, and at the same time the HbFeII oxidation peak decreased (Fig. 6). However, direct reduction of H2O2 could not be obtained at the bare EPG or EPG coated with KGM hydrogel in the potential range scanned, indicating that the reduction of H2O2 was catalyzed by Hb entrapped in KGM film. The reduction peak current increased with an increasing concentration of H2O2. The calibration curve gradually tended to a plateau and then dropped down when adding H2O2, implying a progressive enzyme inactivation in the presence of higher concentration of H2O2. The extent of inactivation of Hb was dependent on the incubation time of the electrodes and the hydrogen peroxide concentration. The results are similar to the catalytic behaviors of horseradish peroxidase [27], because Hb has similar structure of peroxidase and intrinsic catalytic activities to peroxide compounds. The reduction peak currents were linearly proportional to H2O2 in the concentration of 4.89–66.00 μM. A linear regression equation of -4

A -2

I / μA

0

2

a 4

b c

6 -0.8

-0.6

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B

4.2

I / μA

4.0 3.8 3.6 3.4 3.2 3.0 2.8 0

10

20

30

40

50

60

70

C / μM Fig. 6. (A) Cyclic voltammograms of Hb-KGM/EPG in 0.1 M pH 7.0 PBS containing (a) 0, (b) 27.6 μM and (c) 58.6 μM H2O2, scan rate 0.2 V s− 1. (B) Plot of catalytic peak current versus the concentration of H2O2.

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Ip (μA) = 2.90 + 0.02C (H2O2, μM) was obtained, with a correlation coefficient of 0.997 (n = 9). Hb-KGM/EPG displayed good reproducibility with a relative standard deviation of 3.3% for eight independent determinations in 26.8 μM H2O2 solution. Moreover the current depended linearly on the scan rate in the range of 0.1 V s− 1 to 0.8 V s− 1, suggesting a reaction controlled by Hb confined in KGM hydrogel but not a H2O2 diffusion-controlled process. The result is similar to that reported elsewhere [28]. 3.7. Reduction of nitric oxide NO is found in vivo and regulates various physiological functions including blood pressure, platelet aggregation, and neurotransmission. NO binding to heme enzymes such as myoglobin

-4

A

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I / μA

0 2 4

a b c

6 8 10 12 -1.0

-0.8

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3.6

3.4

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3.0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

CNO2- / μM Fig. 7. (A) Cyclic voltammograms of Hb-KGM/EPG in 0.1 M pH 5.0 PBS containing (a) 0, (b) 0.48 and (c) 0.92 mM NO−2 , scan rate 0.2 V s− 1. (B) Plot of catalytic peak current versus the concentration of NO−2 .

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and hemoglobin has been linked to a number of important physiological processes. NO is one of the products of the disproportionation reaction of NO2− when it is present in the acidic solution [29]. Cyclic voltammograms of an Hb-KGM/EPG in pH 5.0 PBS shows a pair of reverse redox peaks located at approximately −0.220 V. When NO2− was added into the pH 5.0 PBS, scanning from +0.2 V to − 1.2 V, an irreversible cathodic peak at − 0.70 V was observed (Fig. 7A). The peak corresponds to the reduction of the nitric oxide adduct of Hb [30]. The peak was not observed when the solution pH was up to 6.0, indicating that NO cannot be released in the solution due to the lack of a proton. The peak reduction current was linearly proportional to NO2− within a range of concentration of 0.12–1.13 mM (Fig. 7B). A linear regression equation of Ip (μA) = 3.04 + 0.76C (NO2− , mM) was obtained, with a correlation coefficient of 0.989 (n = 6). 4. Discussion 4.1. Characterization of Hb-KGM film The visible absorption spectra show a similar shape but with small shift of Soret band for Hb and Hb-KGM films on glass slides (Fig. 1). The amide I and II bands in the RAIR spectrum of Hb-KGM also have a similar shape but with small band shift to that of the film alone (Fig. 2) which presents evidence of small perturbation of the second structure of Hb entrapped in KGM films. These results suggest that Hb in the KGM hydrogel is not grossly denatured, but that the environment in which Hb is embedded creates some changes. 4.2. Redox reaction of Hb in KGM hydrogel The electron transfer rate was greatly facilitated between GC electrode and Hb in KGM hydrogel, which suggested that KGM played a crucial role in the electrochemical process for Hb. The interaction between Hb and KGM can partially explain why there is favorable reorientation of Hb molecules in KGM hydrogel under the electrochemical driving force. The surface coverage (5.12 × 10− 11 mol cm− 2) of Hb entrapped on electrode surfaces is just about 10% or less of the total amount of protein deposited on the electrode surface. However, the values are as large as 39 times the theoretical monolayer coverage (counting in one hemecontaining chain) of 1.3 × 10− 12 mol cm− 2 for Hb, which were estimated taking into account the crystallographic dimensions of 6.4 × 5.5 × 5.0 nm3 (in 4 heme-containing chains) [31] assuming one molecule with the long axis parallel to the electrode surface. The results indicate that the electrochemical responses can be attributed to multilayers of proteins. The formal potential E°′ (− 0.328 V versus SCE) obtained using Hb-KGM/EPG shift negatively compared to the value obtained in solution (− 0.104 V versus SCE) [32], suggesting the presence of interactions between Hb and the immobilized material KGM. Comparing the results of this work with those reported elsewhere [33], it is obvious that the average formal potentials (E°′) and apparent heterogeneous electron transfer rate constants (ks) depend on the materials used to immobilize the proteins, suggesting that the electron transfer between proteins and the electrodes is affected by the local environment of the protein. As is well known, the electron transfer rate can be facilitated with favorable orientation of proteins, which could be adjusted by electrostatic force when the electrode surface is modified with films possessing positive or negative charge, due to the asymmetric distribution of charge of globin [34]. In addition, an attractive interaction between proteins and surfactant via aggression of long chain of

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surfactant into protein molecules and the hydrophobic force can also accelerate electron transfer rate according to Boussaad and Tao [35]. Furthermore, it has been demonstrated that small changes in the accessibility of the heme group to the protein exterior by exposing proteins to solvent dielectric can modulate the heme reduction potential dramatically [36]. All the interactions can decrease the reorganization energy of proteins, facilitating the electron transfer processes. Decreasing the solution pH will increase the formal potentials of many heme proteins that undergo proton-coupled electron transfer [37], which has been successfully reproduced in iron (III) protoporphyrin IX protein maquettes designed de novo [38]. This redox Bohr effect, a shift in the formal potential as a function of pH, results from the change in the protonation of a water molecule at the sixth coordination position in the heme iron and also protonation of the protolytic groups around the heme with changing pH [39]. Processes involving coupled electron and proton transfer are often tuned by the electrostatic interaction between the metal center and the charged sites in the vicinity of the heme group. A mount of DMF added into the KGM hydrogel can accelerate the gelation of KGM hydrogel and enhance the rate of electron transfer between Hb and electrode. Electrochemical responses of Hb-KGM/EPGs, prepared by the mixture of proteins and KGM hydrogel without DMF, were faint and unstable with an asymmetric cyclic voltammogram. Changing the ratio of DMF to KGM (v/v), from 1:9 to 1:3, causes the redox peaks to become more symmetric with the ratio of the redox peak currents Ipc/Ipa approaching 1. The reason is probably that trapping Hb in the hydrogel may have exposed the heme crevice to a more hydrophobic environment, which disrupts the distal-pocket H-bonding network, and thus lower the reorganization energy for electron transfer [40]. A similar function of DMF was found in the system of organohydrogel [41]. However, when the ratio was 1:2 or more, proteins would precipitate from the hydrogel solution. The ratio of DMF to KGM (v/v) of 1:4 was used in the experiments. The Hb-KGM/EPGs were relatively stable. During 1 day, 10 cycles were measured at an interval of 2 h. No significant decrease in electroactivity was found. Additionally, the electrochemical response of the proteins on the electrode surface decreased by less than 14% one month after the electrode was stored in 10 mM PBS (pH 7.0) at 4 °C. 5. Conclusions Hemoglobin form stable films with KGM hydrogel, which was used to immobilize and facilitate the electron transfer between Hb and the GC electrode. The formal potential dependence on pH indicated a reaction of electron transfer coupled with proton transfer. Visible absorption and reflectance absorption infrared spectra proved the heme environment of Hb entrapped in KGM hydrogel to be in its native status. The embedded Hb retained the electrocatalytic activities for oxygen, hydrogen peroxide and nitric oxide. 6. Simplified description of the method and its (future) applications Hb entrapped in the KGM hydrogel exhibits facile direct electrochemistry and demonstrates catalytic reactivity to oxygen, hydrogen peroxide and nitric oxide. This methodology described here would be applicable to determining redox parameters of proteins and preparing hydrogel based third generation of biosensors. Also an important implication of this work is that the electrochemical studies for other proteins in the native, hydrated form can be performed without requiring complex methodology.

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