Anal. Chem. 2001, 73, 2850-2854
Electron-Transfer Reactivity and Enzymatic Activity of Hemoglobin in a SP Sephadex Membrane Chunhai Fan,† Haiyan Wang,† Sai Sun,‡ Dexu Zhu,† Gerhard Wagner,*,§ and Genxi Li*,†
Department of Biochemistry and National Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P. R. China, Department of Computer Science, Nanjing University, Nanjing 210093, P. R. China, and Department of Biological Chemistry and Molecular Pharmacology, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115
Hemoglobin can exhibit a direct electron-transfer reaction after being entrapped in a SP Sephadex membrane. A pair of stable and well-defined redox waves are obtained at a hemoglobin-SP sephadex modified pyrolytic graphite electrode. The anodic and cathodic peak potentials are located at -0.244 and -0.336 V (vs SCE), respectively. On the other hand, the peroxidase activity of the protein in the membrane is also greatly enhanced. The apparent Michaelis-Menten constant is calculated to be 1.9 mM, which shows a large catalytic activity of hemoglobin in the SP Sephadex membrane toward hydrogen peroxide (H2O2). According to the direct electron-transfer property and enhanced peroxidase activity of Hb in the membrane, a Hb/SP Sephadex membrane-based H2O2 biosensor is prepared, with a linear range ∼5.0 × 10-6 to 1.6 × 10-4 mol/L. Hemoglobin (Hb) is probably the most studied protein in existence.1,2 Its function in the red blood cell is as an oxygen vehicle. Efforts have been taken to obtain its electrochemical response at solid electrode surfaces because, as is well-known, electrochemical methods are powerful tools for probing and evaluating the structure-function relationship of metalloproteins.3 Electrochemical studies of Hb might open new insights into its pysiological functions. However, unlike some other small heme proteins such as cytochromes,4-7 it is difficult for Hb to exhibit hetergeneous electron-transfer processes in most cases,8,9 which * Corresponding authors. Fax: +86-25-359-2510. E-mail:
[email protected]. Fax: 617-432-4383. E-mail:
[email protected]. † Department of Biochemistry and National Laboratory of Pharmaceutical Biotechnology, Nanjing University. ‡ Department of Computer Science, Nanjing University. § Harvard Medical School. (1) Stryer, L. Biochemistry, 3rd ed.; Freeman: New York, 1988. (2) Glanz, J. Science 1996, 271, 1670. (3) Armstrong, F. A.; Heering, H. A.; Hirst, J. Chem. Soc. Rev. 1997, 26, 169179. (4) Zhu, Y.; Li, J.; Dong, S. J. Chem. Soc., Chem. Commun. 1996, 51-52. (5) Pineda, T.; Sevilla, J. M.; Roman, A. J.; Blazquez, M. Biochim. Biophys. Acta 1997, 1343, 227-234. (6) Ferri, T.; Poscia, A.; Ascoli, F.; Santucci, R. Biochim. Biophys. Acta 1996, 1298, 102-108. (7) Rivera, M.; Seetharaman, R.; Girdhar, D.; Wirtz, M.; Zhang, X.; Wang, X.; White, S. Biochemistry 1998, 37, 1485-1494. (8) Ye, J.; Baldwin, R. P. Anal. Chem. 1988, 60, 2263-2268.
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means that the electron-transfer reactivity of Hb is very slow. It coincides with the fact that Hb is not designed as an electrontransfer protein, and its electroactive center is deeply buried in its electrochemically “insulated” peptide backbones.10 Many electron transfer proteins are strongly membraneassociated, and a kind of organized molecular architecture within the membrane environment is required for their efficient electron transfer.11-15 It thus implies that there exists some relationship between membranes and electron-transfer reactivity of proteins. SP Sephadex is usually employed to purify proteins in chromatography,16 providing a suitable environment for the entrapment of proteins. In this study, SP Sephadex is immobilized at a pyrolytic graphite (PG) electrode surface to mimic a membrane environment. As shown in our results, the SP Sephadex-entrapped Hb exhibits improved electron-transfer reactivity and, accordingly, gives nice electrochemical responses. Recent studies revealed that some proteins underwent certain functional conversions through noncovalent interactions with lipid membranes. For example, cytochrome c was observed to be functionally converted from an electron-transfer protein to a N-demethylase-like enzyme via the supramolecular formation with the artifical phosphate-lipid membrane.17,18 The studies on functional conversion of proteins not only reveal possible interaction mechanisms between proteins and membranes but also provide an alternative and convenient path to enzyme engineering, which is useful in many application areas, including developing new biosensoring materials. Because the entrapped Hb might also interact with the SP Sephadex membrane, the possible functional (9) Schlereth, D.; Mantele, W. Biochemistry 1992, 31, 7494-7502. (10) Stellwagen, E. Nature 1978, 275, 73-74. (11) Hamachi, I.; Fujita, A.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 90969102. (12) Bianco, P.; Haladjian, J. Electrochim. Acta 1997, 42, 587-594. (13) Salamon, Z.; Hazzard, J. T.; Tollin, G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6420-6423. (14) Lu, Z.; Huang, Q.; Rusling, J. F. J. Electroanal. Chem. 1997, 423, 59-66. (15) Fan, C.; Zhuang, Y.; Li, G.; Zhu, J.; Zhu, D. Electroanalysis 2000, 12, 11561158. (16) Wu, G.; Pan, H.; Wu, Y. Handbook of Biochemical and Molecular Biological Experiments; Scientific Press: Chinese, 1999. (17) Hamachi, I.; Fujita, A.; Kunitake, T. J. Am. Chem. Soc. 1994, 116, 88118812. (18) Fujita, A.; Senzu, H.; Kunitake, T.; Hamachi, I. Chem. Lett. 1994, 12191222. 10.1021/ac001397s CCC: $20.00
© 2001 American Chemical Society Published on Web 05/02/2001
conversion of the entrapped Hb is examined. It is observed that Hb showed enhanced peroxidase activity through its interaction with the membrane. EXPERIMENTAL SECTION Bovine Hb was purchased from Serva (Ottweiler, Germany) and used as received. Stock solutions were kept at a temperature of 4 °C. SP Sephadex c-50 was from Pharmacia (Peapack, NJ). All other chemical reagents were of analytical grade. Water was purified using a Milli-Q purification system (Barnstead; Bedford, MA) to a specific resistance >16 M cm-1 and used to prepare all solutions. Electrochemical experiments were performed using a PARC 263A potentiostat/galvanostat (EG&G; Princeton, NJ), using a three-electrode configuration. A multiblock heater (LAB-LINE Instruments Inc.; Hong Kong) was employed to maintain the temperature at 20 ( 0.5 °C. A saturated calomel electrode (SCE) and a platinum electrode served as reference and counter electrodes, respectively. Potentials are reported with respect to SCE unless specially stated. Digital simulation of voltammograms was carried out using a nonlinear regression analysis computer program, which was developed using MATLAB 5.3 (MatWorks, Natick, MA). The substrate electrode was a pyrolytic graphite (PG) disk electrode (A ) 6.38 mm2). The Hb-SP Sephadex- or SP Sephadexalone- or Hb-alone-modified PG electrode was prepared as follows. The substrate electrode was first polished using rough and fine sandpapers. Then it was polished to a mirror smoothness using an alumina (particle size ∼0.05 µm)/water slurry on silk. Finally, the electrode was thoroughly washed with water and then was treated in an ultrasonic bath for about 5 min. SP Sephadex was dissolved in N,N′-dimethylformamide. The SP Sephadex solution was then mixed with a Hb aqueous solution to a final solution containing 1 mg/mL SP Sephadex and 0.1 mM Hb. That solution (10 µL) was spread evenly onto the surface of the PG disk electrode using a microsyringe. Alternatively, only SP Sephadex or only Hb was cast onto the PG electrode surface. The membrane at the PG electrode surface was dried overnight at room temperature, and the electrode was thoroughly rinsed with water. The modified electrode was stored at 4 °C when not in use. The test solution was a HAc-NaAc (acetate) buffer solution, pH 5.5. It was first bubbled thoroughly with high purity nitrogen. Then a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout the experiment. Scanning electron micrographs (SEMs) were recorded by an X-650 scanning electron microanalyzer (Hitachi; Tokyo, Japan). A piece of PG block coated with either Hb-SP Sephadex membrane or SP Sephadex membrane alone was fixed onto the SEM mounting stage with two-side adhesive. UV-vis absorbance spectroscopy was performed using a UV2201 spectrophotometer (Shimadzu; Kyoto Japan). Fourier transformed infrared (FT-IR) spectra were obtained by using a 170SX FT-IR spectrometer (Nicolet; Madison, WI) at a 2 cm-1 resolution. Hb solution, or SP Sephadex solution, or Hb-SP Sephadex mixture was deposited onto a Teflon chip, respectively. After the membrane on the chip was dried in air, the membrane was stripped off and tabletted with KBr powder for measurement. To eliminate the interference of SP Sephadex, the spectrum of Hb
Figure 1. Top SEM views of the surface of the PG electrode immobilized with (a) SP Sephadex (b) Hb-SP Sephadex. ×150× magnification.
obtained from Hb-SP Sephadex membrane was subtracted from the spectrum of SP Sephadex. RESULTS AND DISCUSSION Figure 1a,b is the top SEM views of the surface of the PG electrode immobilized using SP Sephadex and Hb-SP Sephadex, respectively. It is observed that the top view of the SP Sephadexalone-modified PG electrode displays a lot of smooth, globular particles scattering at the flat surface of the PG electrode (Figure 1a). On the other hand, at the Hb-SP Sephadex-modified PG Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
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electrode surface, these particles are surrounded by some amorphous materials (Figure 1b). These observations suggest that Hb has been incorporated into the SP Sephadex membrane. Hb is not denatured after it is incorporated into the SP Sephadex membrane, as suggested by UV-vis spectra. The Soret band of Hb is located at 406 nm, which is sensitive to variations of the microenviroments around the heme site. Previous studies showed that the band will diminish if the protein is fully denatured.19,20 Our experimental results reveal that the Soret band for entrapped Hb is located at 410 nm, shifting only a few nanometers toward the red (see Supporting Information). This result suggests that there exists slight structural variations in the vicinity of the heme site, and no significant denaturation occurs. FT-IR spectroscopy is sensitive to the secondary structure of the protein. Our FT-IR studies show that Hb is not denatured after the protein is incorporated into the SP Sephadex membrane. As is well-known, the shapes of the amide I and amide II infrared absorbance bands of Hb provide detailed information on the secondary structure of the polypeptide chain.21,22 The amide I band (1700-1600 cm-1) is caused by CdO-stretching vibrations of peptide linkages in the protein’s backbone. The amide II band (1620-1500 cm-1) results from a combination of N-H bending and C-N stretching. In our experimental results, the amide I and II bands of Hb in the SP Sephadex membrane are located at 1653.2 cm-1 and 1539.7 cm-1, respectively, which are nearly the same as those obtained for the protein itself (1657.0 cm-1 and 1534.3 cm-1) (see Supporting Information). Because previous studies showed that amide I and II would significantly diminish if Hb was denatured,20,23 similarities of the characteristics in the FT-IR spectra suggest that Hb retains the essential features of its native structure in the SP Sephadex membrane. SP Sephadex might provide a desirable membrane environment for Hb to undergo facile electron-transfer reactions. The electrochemical reaction of entrapped Hb was examined with cyclic voltammetry. Panels a and b of Figure 2 are the cyclic voltammograms (CVs) of a 0.1 M HAc-NaAc buffer solution, pH 5.5, obtained at the PG electrode coated with Hb or Hb-SP Sephadex, respectively. They show that a pair of stable and welldefined redox waves can be obtained at the Hb-SP Sephadexcoated PG electrode (Figure 2b). The anodic and cathodic peak potentials are located at -0.244 and -0.336 V (vs SCE), respectively. In contrast, no corresponding waves are observable if the protein is not incorporated in the SP Sephadex membrane (Figure 2a). The possibility that SP Sephadex contributes to the observed redox waves in the CV should be excluded. Figure 2c is the CV curve obtained at a PG electrode that was coated with SP Sephadex alone, where no redox wave exists. Therefore, SP Sephadex itself is not electroactive in the potential range of interest. From the above discussion, it is obvious that direct
electron transfer of Hb is achieved through entrapment in the SP Sephadex membrane. Both the anodic and cathodic peaks currents (I) of Hb obtained at the Hb-SP Sephadex-membrane-modified PG electrode are proportional to the scan rate (ν), ranging from 0.05 to 2 V s-1 (linear regression equations y ) 0.0717-0.00612x, r ) 0.999; y ) 0.0386 + 0.00614x, r ) 0.999). The slopes obtained by linear regression of both log Ipa and log Ipc vs log ν are 1.09 for Ipa and 1.05 for Ipc, respectively. It is characteristic of thin-layer electrochemical behavior,24 that is, nearly all electroactive met-Hb in the membrane is converted to ferrous Hb on the forward CV scan and vice verse. The apparent standard potential (E°′), estimated from its midpoint potential (E1/2), is -0.290 V (vs SCE), which is close to the value obtained by Rusling et al.14,25 Experimental results also reveal that the Hb-SP Sephadexmodified PG electrode is very stable. No significant decrease of the peak currents is seen after at least one week’s storage at 4 °C. The electron-transfer reactivity of Hb at the SP Sephadex membrane was further studied by square wave voltammetry (SWV), because SWV is known to be powerful in characterizing the electrochemistry of interfacially confined redox moleules.26 The SWV curve of Hb in the SP Sephadex membrane is shown in Figure 3a. Obviously, it shows a better resolution and higher signal-to-noise ratio. The peak width at half-maximum ∼210 mV, higher than the theoretical values. Therefore, the redox reaction of Hb in the SP Sephadex membrane coincides with a kinetic dispersion phenomenon.27 Digital simulation of the SWV curve of Hb has been carried out to estimate the apparent heterogeneous electron-transfer rate constant (ks) of Hb in the membrane. A nonlinear regression analysis program is employed, which is based on a model developed by Osteryoung and Rusling et al. that combines the theory for SWV of a surface-confined species with a dispersion of apparent standard potential (E°′) values.28,29 (The equations and fitting parameters are provided in Supporting Information.) The
(19) George, P.; Hanania, G. Biochem. J. 1953, 55, 236-243. (20) Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 23862392. (21) Kauppinen, J. K.; Moffatt, D. J.; Mantsch, H. H.; Cameron, D. G. Appl. Spectrosc. 1981, 35, 271-276. (22) Rusling, J. F.; Kumosinski, T. F. Intell. Instrum. Comput. 1992, 10, 139145. (23) Song, Y. P.; Petty, M. C.; Yarwood, J.; Feast, W. J.; Tsibouklis, J.; Mukherjee, S. Langmuir 1992, 8, 257-261.
(24) Murray, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13. (25) Nassar, A.-E. F.; Rusling, J. F. J. Am. Chem. Soc. 1996, 118, 3043-3044. (26) Reeves, J. H.; Song, S.; Bowden, E. F. Anal. Chem. 1993, 65, 683-688. (27) Rowe, G. K.; Carter, M. T.; Richardson, J. N.; Murray, R. W. Langmuir 1995, 11, 1797-1806. (28) Nassar, A.-E. F.; Zhang, Z.; Hu, N.; Rusling, J. F.; Kumosinski, T. F. J. Phys. Chem. B 1997, 101, 2224-2231. (29) O’Dea, J. J.; Osteryoung, J. Anal. Chem. 1993, 65, 3090-3097.
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Figure 2. Cyclic voltammograms of a 0.1 M HAc-NaAc buffer solution, pH 5.5, obtained at a PG electrode coated with (a) Hb, (b) Hb-SP Sephadex, and (c) SP Sephadex. Scan rate, 50 mV/s.
Figure 3. (a)Square-wave voltammogram obtained at a Hb-SP Sephadex-modified PG electrode for a 0.1 M NaAc-HAc buffer solution, pH 5.5. Pulse height, 50 mV; frequency, 150 Hz; 2 mV step. (b) Simulated curve.
results are displayed in Figure 3. The simulated curve fit nicely with the experimental curve, indicating the effectiveness of this method. The apparent heterogeneous electron-transfer rate constant (ks) of Hb is thus calculated to be 102.12 s-1, which shows that the electron transfer of Hb in the membrane is fairly facile. It is well-known that Hb alone gives no direct electrochemical response, and even in the presence of electrochemical mediators, its ks is still several orders lower than that obtained here;30 therefore, it can be safely concluded that the electron-transfer reactivity of Hb is much improved through entrapment in the SP Sephadex membrane. The pH effect upon the electrochemical behavior of Hb in the SP Sephadex membrane has also been tested. Nearly reversible voltammograms are observed for all of the pH range tested (3.0∼9.0), with stable and well-defined peaks (see Supporting Information). Moreover, the pH-induced variations in the wave shape and potentials in voltammograms are reversible; that is, the same CV can be obtained if the Hb-SP Sephadex-modified electrode is transferred from a background solution with a different pH value to its original solution. Meanwhile, it is observed that an increase of pH in the background solution leads to a negative shift of E°′ of the protein. E°′ is found to be linearly proportional to pH value in the range of 3.0∼9.0, with a linear regression equation of y ) -66.3 - 46.5x, r ) 0.996. The above results show that Hb can exhibit electron-transfer reactivity in a SP Sephadex membrane. Further studies reveal that the enzymatic activity of Hb is also enhanced. It is known that Hb and myoglobin have some intrinsic peroxidase activities because of their close structural similarity to peroxidases.31 Efforts have been taken to improve their peroxidase activity through the method of mutagenesis.31,32 On the other hand, cytochrome c, also a heme protein, can undergo functional conversion from an electron-transfer protein to a N-demethylase-like enzyme through noncovalent interaction with a phosphate-lipid membrane. Because Hb has been incorporated within the SP Sephadex membrane and might interact with the (30) Song, S.; Dong, S. Bioelectrochem. Bioenerg. 1988, 19, 337-346. (31) Matsui, T.; Ozaki, S.; Liong, E.; Phillips, G. N., Jr; Watanabe, Y. J. Biol. Chem. 1999, 274, 2838-2844. (32) Alayash, A. I.; Ryan, B. A.; Eich, R. F.; Olson, J. S.; Cashon, R. E. J. Biol. Chem. 1999, 274, 2029-2037.
Figure 4. Cyclic voltammograms obtained at a Hb-SP Sephadexmodified PG electrode for a 0.1 M NaAc-HAc buffer solution, pH 5.5, (a) before and (b) after the addition of 0.15 mM H2O2 to the buffer solution. (c) Cyclic voltamogram obtained at a Hb-alone-modified PG electrode (free of SP Sehadex) for the H2O2 solution. Scan rate, 400 mV/s.
membrane, the possible functional conversion of Hb from an oxygen storage protein to peroxidase is examined in this work. Experimental results reveal that the peroxidase activity of Hb is greatly enhanced in the SP Sephadex membrane. Parts a and b of Figure 4, separately, are the CV curves obtained at the Hb-SP Sephadex-modified PG electrode for a 0.1 M NaAc-HAc buffer solution, pH 5.5, before and after the addition of 0.15 mM H2O2 in the buffer. It can be observed that the cathodic peak current apparently increases after the addition of H2O2, but the anodic peak disappears, which is characteristic of an electrochemically catalytic reaction.33 Noticably, no corresponding electrochemical signal is observable employing either a bare PG electrode or a SP Sephadex-alone-modified PG electrode (free of protein) in the same H2O2 solution; therefore, the catalytic process comes from the specific enzymatic catalytic reaction between Hb and H2O2, which indicates a large decrease in activation energy for the reduction of H2O2 in the presence of Hb. It is noticeable that SP Sephadex exerts an important effect on the catalytic ability of Hb. Figure 4c is the CV curve that was obtained from the same H2O2 solution employing a Hb-alone (free of SP Sephadex)-modified PG electrode. It obviously shows that the peak current obtained at the Hb-SP Sephadex-modified electrode is much higher than that obtained at the Hb-alonemodified electrode. This result clearly indicates that the peroxidase activity of Hb has been enhanced in the SP Sephadex membrane. A linear dependence between the catalytic peak current and the concentration of H2O2 is observed in the range of ∼5.0 × 10-6 to 1.6 × 10-4 mol/L H2O2 (Figure 5). The linear regression equation is y ) 3.48 + 0.18x, with a correlation coefficient of 0.999. Five independent determinations at a H2O2 concentration of 5.0 × 10-5 mol/L shows a relative standard deviation of 3.5%, which displays nice reproducibility of these measurements. The curve levels off at a concentration of 2.5 × 10-4 mol/L. It is consistent with an enzyme-like catalytic process of the substrate. The apparent Michaelis-Menten constant (KMapp), which gives an indication of the enzyme-substrate kinetics, can be calculated from the linear part of Figure 5, using the electrochemical version (33) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980.
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trapped Hb is greatly enhanced, which is comparable to or even higher than that of the native peroxidase, HRP.
Figure 5. Plot of the catalytic peak current versus the concentration of H2O2. Experimental conditions are the same as in Figure 4.
of the Lineweaver-Burk equation34.
1/Iss)1/Imax+KMapp/Imaxc
where Iss is the steady-state current after the addition of substrate (with the current derived from Hb subtracted), c is the bulk concentration of the substrate, and Imax is the maximum current measured under saturated substrate condition. The KMapp for the Hb-SP Sephadex-modified electrode is, thus, calculated to be 1.9 mM. As is well-known, the smaller KM shows the higher catalytic ability. The value of KM for Hb in this work is smaller than that obtained at a horseradish peroxidase (HRP)-based H2O2 sensor.34 Therefore, it clearly shows that the peroxidase activity of en(34) Li, J.; Tan, S. N.; Ge, H. Anal. Chim. Acta 1996, 335, 137-145. (35) Fan, C.; Chen, X.; Li, G.; Zhu, J.; Zhu, D.; Scheer, H. PCCP, Phys. Chem. Chem. Phys. 2000, 2, 4409-4413. (36) Armstrong, F. A.; Heering, H. A.; Hirst, J. Chem. Soc. Rev. 1997, 26, 169179. (37) Fan, C.; Li, G.; Zhu, J.; Zhu, D. Anal. Chim. Acta 2000, 423, 95-100.
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CONCLUSIONS The properties of Hb entrapped in a SP Sephadex membrane were characterized by both spectroscopic and electrochemical methods. It was observed that Hb could facilely exchange electrons with the electrode and at the same time, achieved a high peroxidase activity through the noncovalent interaction with SP Sephadex membrane. Biosensors are useful in both biochemical research and medical detection. It is one of the aims in the field of biosensors to widen the scope of biosensoring materials. Here, it is suggested that Hb acts as an ideal substitute for peroxidase in the field of biosensors because of its low cost and good enzymatic catalytic activity toward H2O2. This configuration for H2O2 determination also is advantageous in that it is based on the direct electron-transfer reaction of Hb, namely, it is a thirdgeneration biosensor. In addition, the protein-film-based technique employed in this and previous papers, because of its simple construction and good sensitivity, shows great promise in both protein characterization35,36 and biosensoring applications.37 ACKNOWLEDGMENT We greatly appreciate the support of the National Natural Science Foundation and the Science Foundation of Jiangsu Province, P. R. China, for this research. SUPPORTING INFORMATION AVAILABLE Three figures documenting additional experiments and equations for SWV simulation described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 29, 2000. Accepted March 6, 2001. AC001397S
