Direct Electron Transfer for Heme Proteins Assembled on

Direct Electron Transfer for Heme Proteins Assembled on Nanocrystalline TiO2FilmQingwen Li,Guoan Luo,*and Jun FengDepartment of Chemistry,Tsinghua University,Beijing100084,P.R.China;e-mail:galuo@Received:April4,2000Final version:June14,2000AbstractNanosized TiO2is now an attractive biocompatiable material widely used in toothpaste and cosmetics.Due to its unique physiochemical properties and its inclination to selectively combine with some groups of biomolecules,nanosized TiO2has been proposed as a promising interface for the immobilization of biomolecules.In this article,heme proteins involving cytochrome c,myoglobin and hemoglobin were attempted to be assembled onto a nanocrystalline TiO2®lm,their electrochemical behaviors on such interface were characterized with cyclic voltammetry.It was proved that TiO2®lm could not only offer a friendly platform to assemble protein molecules,but also enhance the electron transfer process between protein molecules and the electrode.The electrode kinetic parameters of each hemoprotein on such electrode as well as the effect of scan rate on the electrochemical behavior of each protein are discussed in details.Keywords:Nanocrystalline,TiO2®lm,Heme proteins,Cytochrome c,Myoglobin,Hemoglobin1.IntroductionHeme proteins are important in living cells which contain the porphyrin complex of iron(II)-heme or iron(III)-hemein as a prosthetic group.These proteins perform different physiologi-cal functions in the biological system[1,2].Cytochrome c (13000g mol71)contains a heme group and a polypeptide chain, its prosthetic group immersed in the protein molecule is combined with the protein through thioether bonds and through the coordination of the iron atom in the heme with the protein chain of amino acid radicals.A crevice-like fold formed by polypeptide chains is regarded as an active site for its redox reaction[3].Myoglobin is also a small single chain hemoprotein with molecule mass of16951g a mol,slightly larger than cyt c containing153amino acids[4].The globin folds into eight major a helical segments which serves to stabilize the conformation of iron heme through over sixty hydrophobic interactions and hydrogen bonds.As the heme group in myoglobin is much more buried with respect to the protein surface than in cyt c,the electron transfer process between protein and electrode is thus more inhibited.Hemoglobin(67000g mol71)is much larger than myoglobin and cyt c.It contains four polypeptide chains (two a and two b)and four heme prosthetic groups.In hemo-globin,the heme group is placed in a ligand pocket creating a hydrophobic environment.As its structure is now more clearly known than other kinds of globular proteins,it is often used as an instructive model for studying the function of many regulatory oligometric proteins.Electrochemical studies of these proteins have attracted considerable attention[5±10],since fundamental studies of the redox behaviors of these proteins may be of great help to further understand the relationship between their struc-tures and biological functions.Protein electrochemistry can be carried out in protein solution or with surface-con®ned techniques.Studies with protein solu-tion may often be confronted with such challenges as the dif®culty to achieve the direct electrochemistry of proteins on some bare electrodes as well as the poor reversibility of the direct heterogeneous electron transfer reactions between elec-trodes and biological molecules.The reasons that inhibit the direct electron transfer between electrodes and proteins may be mainly attributed to i)electroactive prosthetic groups deep within the protein structure,ii)adsorption denaturation of proteins onto electrodes and iii)unsymmetric distribution of surface charges on protein molecules and iv)low rate of mass transfer process of proteins.However,this inhibition can be overcome by modifying electrodes with some mediators such as FAD and methylene violet[11]etc.,or promoters including amino acids[12],carbohydrate molecule[13],inorganic compound[14],biological macromolecule[15],or polymer[16] etc.It has been generally considered that the introduction of mediators and promoters is likely to favor the orientation of protein molecules over the electrode surface or connect the electron communication between electrode and protein mole-cules by the presence of an``arm''.This arm has a structure like X.......Y,where X represents the functional group being easy to absorb onto an electrode surface,such as HS±,RS±,RSS±,4-pyridyl and R2N±etc.,Y can be some anions or alkali groups, such as±NH2,±COOH,±SO3H,±PO3H2and±OH etc.Since the X group is bonded with the electrode surface,the other group Y is able to combine with protein molecules through electrostatic action,ionic bond or hydrogen bond and stretch close to the active sites of proteins.Electrochemistry of protein solutions may also suffer other problems.The problems on one hand arise from the high cost and small amount of protein samples.On the other hand,as protein molecules have strong tendency to adsorb onto electrode surface,it is dif®cult to distinguish the electrochemical properties of native protein from the absorbed protein.Therefore,using surface-con®ned tech-niques,i.e.,immobilization of redox protein molecules onto electrode surfaces while retaining their biological and electro-chemical activities to the largest extent seems more competitive in this®eld.Additionally,it also serves to encourage the work in biosensor and bioenergy.Many efforts have been made to fabricate available protein electrodes,including to immobilize proteins on some metal oxide®lms such as In2O3[10],Al2O3 [17]etc.,in cryo-hydrogel[18],surfactant®lm[19],lipid®lm [20]recently,and investigated their electrochemical behaviors. While in this article,we chose nanocrystalline TiO2material as a solid matrix for protein immobilization.Nanosized TiO2 material is environmentally benign since it has been widely359Electroanalysis2001,13,No.5#WILEY-VCH Verlag GmbH,D-69469Weinheim,20011040-0397/01/0504±0359$17.50 .50a0used as additives in tooth-paste and cosmetics,it also has high protein loading due to the high speci®c surface area of nano-particles.It has been reported that Ti ion on the surface of TiO2 particles could favorably interact with some groups such as ±COOH,and less denaturation of absorbed proteins was found [21].As a result,cyt c,myoglobin and hemoglobin as hemo-protein samples were immobilized onto nanosized TiO2®lms modi®ed graphite electrodes,respectively,to fabricate protein electrodes.The promoted effect of nanosized TiO2and their electron transfer processes at such electrodes were investigated.2.Experimental2.1.Materials and ReagentsHorse heart cytochrome c,horse heart myoglobin and horse heart hemoglobin were purchased from Sigma.They were used directly without further puri®cation.Nanocrystalline TiO2powder was obtained from Shanghai Silicate Institute of China with particle size at about5nm.The graphite electrode material is a kind of pyrolytic graphite,obtained from Shanghai Graphite factory in China.Two kinds of buffer solutions with different ionic strength were prepared for measurements,one was phosphate buffer solution(PBS)of pH7.4with NaCl136.7mmol LÀ1,KCl 2.7mmol LÀ1,Na2HPO412H2O9.7mmol LÀ1and KH2PO4 1.5mmol LÀ1,the other was just50mmol LÀ1sodium dihydrogen phosphate aqueous solution.The pHs of the buffer solutions were adjusted by the addition of0.1M hydrochloric acid or0.1M sodium hydroxide solutions.All the reagents used were of analy-tical grade,and all of the solutions were prepared with doubly distilled water.2.2.Preparation of the TiO2Film Based ProteinElectrodeThe graphite electrodes were prepared by sealing a graphite rod into a Te¯on tube leaving the working area at about9mm2. The graphite electrode was polished with ultra®ne a-Al2O3 suspension solution,doubly distilled water and acetone succes-sively,and then dried under IR lamp for half an hour.In order to prepare protein electrodes,5mg of nanosized TiO2 powder was thoroughly dispersed in1mL absolute alcohol by an ultrasonic device®rst.An aliquot of this dispersed solution (10m L)was then spread over a rinsed graphite electrode surface, and then dried under IR lamp to evaporate the alcohol for 20min.The graphite electrode modi®ed with a layer of crys-talline TiO2®lm was thus achieved.TiO2®lm based protein electrode was prepared by depositing10m L protein solution (1mg a mL)onto the TiO2®lm electrode with a microsyringe, then kept in a refrigerator at about4 C overnight to evaporate the water.Prior to each measurement,the protein electrodes were rinsed with water thoroughly to remove unabsorbed protein molecules.2.3.ApparatusCyclic voltammetric experiments were carried out with a CHI660A electrochemical workstation(CH Instruments,USA). The three-electrode cell was composed of Ag a AgCl as refer-ence electrode,a Pt wire as counter electrode,and protein electrode as working electrode.Before each electrochemical measurement,the phosphate buffer in the cell was purged with highly puri®ed nitrogen gas for at least20min to remove dissolved oxygen and then kept under a nitrogen atmosphere. All experiments were carried out at room temperature (20Æ1 C).3.Results and Discussion3.1.V oltammetric Behavior of Cyt c Immobilized ontoNanocrystalline TiO2Film ElectrodeFigure1shows the cyclic voltammograms of the bare TiO2®lm electrode and the cyt c-TiO2®lm electrode in50mM phosphate buffer solution of pH6.0.A stable and well-de®ned redox curve of immobilized cyt c was obtained.However, nearly no response was observed when the bare graphite elec-trode was used to perform cyclic voltammetric experiments in a buffer solution containing5mg a mL cyt c,revealing that the electron transfer process between cyt c and the graphite elec-trode was enhanced by the presence of TiO2.Recent work has demonstrated that little denaturation was observed with cyt C immobilized onto TiO2thick®lm[21],so it suggested that the TiO2®lm would be useful to investigate the physical and chemical properties of protein molecules.Generally,since the isoelectric point of cyt c is about10and the isoelectric point of a metal oxide is often in the acid range,there probably is an electrostatic interaction between the protein molecules and metal oxide particles in the phosphate buffers.Other kinds of interaction may also exist,depending on the structural proper-ties of the metal oxides and proteins.It has been suggested that Ti ion can selectively chelate with some groups since TiO2 particles were found to absorb some kinds of dyes[22].As there are many kinds of functional groups in protein molecules, it may be the main reason why protein molecules have a strong tendency to assemble onto TiO2particles.When a TiO2 modi®ed electrode was immersed in a buffer containing cyto-chrome c,the protein molecules were spontaneously adsorbed onto the electrode surface.Different degrees of deformation of the protein molecules would consequently take place in the vicinity of the electrode.If their native conformations werenotFig.1.Cyclic voltammograms at0.1V a s in PBS buffers for a)bare TiO2®lm electrode;b)cyt c-TiO2®lm electrode.360Q.Li et al. Electroanalysis2001,13,No.5destroyed or their active sites were not inhibited,a well-de®ned redox curve could be observed.Indeed,no response or irre-versible redox response was often met at most of the unmodi-®ed metal electrodes [3,23],suggesting that the adsorption event deformed and inhibited the redox center of cyt c.,however,this situation may be improved with the TiO 2modi®ed graphite electrode.Figure 2shows the cyclic voltammetric curves of cyt c-TiO 2electrode at different scan rates.At low scan rate (`0.01V a s),the cathodic peak was relatively weaker and wider than the anodic peak.While at higher scan rate (b 0.05V a s),the cathodic peak became well-shaped and stronger.In addition,both the anodic peak potential (E pa )and cathodic peak potential (E pc )of cyt c were almost constant with different scan rates in the range of 0.05N 71V a s.Cathodic and anodic peak currents were found to be linearly proportional to scan rates from 0.02to 1V a s with correlation coef®cient at 0.999.The plot of logarithm of cathodic peak current vs.logarithm of scan rate also yielded a linear curve with correlation coef®cient R 1and slope at 0.92,indicating that the electrochemical behavior of the TiO 2®lm based cyt c electrode was in accord with the theory of thin layer voltammetry [24].The formal potential (E 0)of cyt c-TiO 2electrode was near 0.11V (0.32V vs.NHE)according to the general calculation method,i.e.,E 0 1a 2(E pc E pa ),a little larger than the value reported by Bowden [10].It may be attributed to the different absorbed state of protein molecules over the electrode surface.From Figure 3,it can be seen that the formal potential of cyt c hardly varied with the scan rate even at the scan rate up to 0.8V a s.The separation potential between the pair of redox peaks was about 0.085V ,suggesting such redox reaction was quarsireversible,as cyt c often works as a one-electron carrier.The electron transfer rate constant k et was estimated by the Laviron's method [25]for the quarsireversible case as 0.62s 71.3.2.V oltammetric Behavior of Myoglobin Immobilizedonto Nanocrystalline TiO 2Film Electrode Like cyt c,myoglobin also undergoes irreversible electron transfer reaction at bare metal electrodes [26].However a quasireversible electrochemical reaction of myoglobin can beachieved with puri®ed samples [27],by introduction of some promoters [28]such as methyl viologen,methylene blue,bril-liant cresyl blue and indium oxide etc.,or immobilizing it in cryo-hydrogel [18].Figure 4shows the CV curve of myoglobin immobilized onto TiO 2®lm electrode without further puri®ca-tion.A pair of well-de®ned redox peaks with the anodic and cathodic peak potentials more negative than those of cyt c obtained under the same experimental conditions can be seen.This result is in agreement with the fact that as the heme group is buried further in myoglobin,the reductive reaction from Fe(III)-heme to Fe(II)-heme is more dif®cult than that of cyt c.As the cathodic peak potential of myoglobin is located at À0.35V ,which is also the potential for the reduction of dissolved oxygen in the buffer solution,thorough removal of oxygen in the buffer solution is necessary to gain the native redox curve of myoglobin.It was also observed that both the anodic peak potential and cathodic peak potential hardly varied with the scan rate.The anodic peak was at ca.À0.35V ,while the cathodic peak was at about À0.23V ,so the peak separation was 0.12V and its formal potential was calculated as À0.27V ,close to the reported value [27].Although the redox centers of cyt c and myoglobin come from the heme group,their different positions in proteins may give rise to the different redox pared with the CV curves of cyt c,besides differences in their redox peak positions,there also existed a difference in the dependence of their redox behavior on the scan rate.In the case of cyt c,a higher scanrateFig.2.Effect of scan rate on CV curves (A)and anodic peak current(B).Fig.3.Effect of scan rate on the formal potential of cyt c assembled onto TiO 2electrodes.Heme Proteins on TiO 2Film 361Electroanalysis 2001,13,No.5seemed to be more favorable to its reversible behavior.While for myoglobin,symmetric CV curves with nearly equal reduction and oxidation peak heights can only be achieved at a scan rate lower than 0.15V a s.Higher scan rates caused the cathodic peak current to become larger and sharper than the anodic peak current.Figure 5shows the relationship between the scan rate and the cathodic peak current.It is obvious that there were two linear response regions,one in the range of the scan rate from 0.02a s to 0.15a s,the other from 0.15V a s to 0.5V a s,which is in agreement with the phenomenon described above.The biphasic behavior of myoglobin may be ascribed to the interference of oxygen.The plot of the logarithm of cathodic peak current vs.the logarithm of the scan rate gave a linear curve with corre-lation coef®cient R of 0.994and slope at 0.60,that deviated from the theoretical slope of thin layer voltammetry,suggesting that the redox reaction of Fe(III)-heme e ?Fe(II)-heme was quasireversible.According to the Laviron's method,the hetero-geneous electron transfer rate constant was approximately evaluated as 0.285s 71.3.3.V oltammetric Behavior of Hemoglobin Immobilizedonto Nanocrystalline TiO 2Film Electrode Since it can reversibly bind oxygen in aqueous solution,the important physiological function of hemoglobin is to transportoxygen.It has been demonstrated that although the bonding of oxygen does not alter the valence state of Fe ion in heme,it may bring about conformational changes in hemo-globin [2].In this regard,the dissolved oxygen in buffer would have a nonnegligible effect on the redox behaviors of hemoglobin.Figure 6shows the effect of dissolved oxygen in buffer solutions on the cyclic voltammograms of hemoglobin immobi-lized onto TiO 2®lm electrode.When the Hb-TiO 2electrode was cycled in the buffer containing dissolved oxygen,a strong reductive peak of hemoglobin appeared at À0.35V ,while there was no evidence of an oxidation peak.After the buffer was purged with highly pure nitrogen for 10min,the reductive peak at À0.35V decreased greatly,moreover,the corresponding oxidation peak emerged,indicating that the reductive peak corresponded to the reductive reaction of dissolved oxygen.If the buffer was continuously bubbled with nitrogen for a longer time a well-de®ned redox curve was observed with a reductive peak at À0.44V and an oxidation peak at 0.38V .This showed that removal of the dissolved oxygen involving binding oxygen helped the electron transfer process between electrode and hemoglobin which might be ascribed to its conformational change.As no reductive peak and oxidation peak appeared intheFig.4.Cyclic voltammograms of blank TiO 2®lm electrode (A)and myo-TiO 2electrode (B)at 0.1V as.362Q.Li et al.Electroanalysis 2001,13,No.5CV curves of hemoglobin solution even after bubbling with nitrogen for more than half an hour.It could be consequently concluded that the TiO2®lm as substrate for the immobilization of hemoglobin provided a friendly platform for the electro-chemistry of hemoglobin.Figure7shows the effect of scan rate upon the redox behavior of hemoglobin.It could also be observed that the redox peak potentials hardly changed with the scan rate.A pair of well-shaped redox peaks with nearly equal peak heights was observed at the scan rate higher than0.05V a s,but at lower sweep rates,the reductive peak appeared to be rather weak.As the cathodic peak current exhibited a linear relationship to the scan rate,implying that the behavior of such electrode agreed with the theory of the thin layer cell.The peak separation was within0.12V,and its heterogeneous electron transfer rate constant was evaluated as 0.137sÀ1.Based upon the formal potentials and electron transfer rate constants of three kinds of heme proteins,we could®nd that from cyt c to hemoglobin,with the increase of protein molecules,their formal potentials became more and more negative and their electron transfer rate constant became less and less.All this revealed is that their redox sites became more and more inhibited. However,their direct electron transfer processes could be obtained by assembling these proteins onto nanocrystalline TiO2®lms.4.ConclusionsThe surface-con®ned technique seems more convenient and effective to permit the absorbed state of biomolecules to be established,in¯uenced and measured simultaneously.Although some kinds of proteins including the proteins with disul®de-thiol groups,or prosthetic group may theoretically have electro-chemical activities,they rarely exhibit direct response owing to the deformation of proteins caused by strong adsorption on the electrode surface.As a result,development of a biocompatible or biomembrane-mimic electrode interface for protein electro-chemistry is still an active topic.In this work,a nanocrystalline TiO2®lm has been recommended to act as an effective alternative in this art.Immobilization of hemoproteins onto nanocrystalline TiO2®lm could combine several advantages of TiO2material involving:1)high biocompatiability of TiO2;2)speci®c binding with protein molecules;3)large speci®c surface area for high protein loading;4)promoting effect for electron transfer between electrode and protein.Although we are not clear about the interaction between the TiO2and protein molecules,the present study shows that the presence of TiO2®lm is favorable for the electron transfer between heme proteins and electrode.5.References[1]T.E.Creighton,Proteins±Structure and Molecule Properties,Freeman,New Y ork1984.[2]A.I.Lehninger,D.L.Nelson,M.M.Cox,Principles of Biochem-istry,2nd edn.,Worth,New Y ork1993.[3]D.D.Schlereth,W.Manrele,Biochem.1993,32,1118.[4]S.J.Dong,G.L.Che,Chemically Modi®ed Electrode,Science Pressof China1995.[5]S.Srinivasan,Y.A.Chizmadzhev,Comprehensive Treatise ofElectrochemistry,V ol.10,Plenum Press,New Y ork1985. 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Heme Proteins on TiO2Film363Electroanalysis2001,13,No.5。