Effective Electrochemical Method for Investigation of Hemoglobin

Sep 16, 2009 - Effective Electrochemical Method for Investigation of Hemoglobin Unfolding Based on the Redox Property of Heme Groups at Glassy Carbon ...
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Anal. Chem. 2009, 81, 8557–8563

Effective Electrochemical Method for Investigation of Hemoglobin Unfolding Based on the Redox Property of Heme Groups at Glassy Carbon Electrodes Xianchan Li, Wei Zheng,† Limin Zhang, Ping Yu, Yuqing Lin, Lei Su, and Lanqun Mao* Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences (CAS), Beijing 100190, China This study demonstrates a facile and effective electrochemical method for investigation of hemoglobin (Hb) unfolding based on the electrochemical redox property of heme groups in Hb at bare glassy carbon (GC) electrodes. In the native state, the heme groups are deeply buried in the hydrophobic pockets of Hb with a five-coordinate highspin complex and thus show a poor electrochemical property at bare GC electrodes. Upon the unfolding of Hb induced by the denaturant of guanidine hydrochloride (GdnHCl), the fifth coordinative bond between the heme groups and the residue of the polypeptides (His-F8) is broken, and as a result, the heme groups initially buried deeply in the hydrophobic pockets dissociate from the polypeptide chains and are reduced electrochemically at GC electrodes, which can be used to probe the unfolding of Hb. The results on the GdnHCl-induced Hb unfolding obtained with the electrochemical method described here well coincide with those studied with other methods, such as UV-vis spectroscopy, fluorescence, and circular dichroism. The application of the as-established electrochemical method is illustrated to study the kinetics of GdnHClinduced Hb unfolding, the GdnHCl-induced unfolding of another kind of hemoprotein, catalase, and the pHinduced Hb unfolding/refolding. Increasing interest has been drawn in the investigations on the thermodynamics and kinetics of protein folding/unfolding because of their great importance in manifestation of biological functions from genetic information.1 The misfolded proteins accumulated in inclusion bodies could result in the development of many disorders, such as Alzheimer’s disease, Parkinson’s disease, systemic amyloidoses, and many other abnormalities.2 To this end, a large number of methods have so far been developed to investigate the protein unfolding, such as UV-vis * Corresponding author. Fax: +86-10-62559373. E-mail: [email protected]. † Current address: Center for Biomedical Materials and Engineering, Harbin Engineering University, Harbin 150001, China. (1) (a) Kim, P. S.; Baldwin, R. L. Annu. Rev. Biochem. 1990, 59, 631. (b) Radford, S. E.; Dobson, C. M. Cell 1999, 97, 291. (c) Dobson, C. M. Nature 2003, 426, 884. (2) Fink, A. L. Folding Des. 1998, 3, R9. 10.1021/ac9015215 CCC: $40.75  2009 American Chemical Society Published on Web 09/16/2009

spectroscopy, fluorescence, circular dichroism, mass spectrometry, and infrared/Raman spectroscopy.3 Due to their ease in operation, low-cost instrumentation, and high sensitivity, recent decades have witnessed wide applications of electrochemical methods not only in the fundamental studies but also in the practical applications.4 Although substantial progress has so far been achieved with the electrochemical methods, particularly through interfering with materials sciences and life sciences as well as energy conversion and storage technologies,4 studies with such methods have been mainly limited for small molecules and remain pretty difficult for biomacromolecules, such as proteins.4a,b,e On the other hand, while great efforts have been made to facilitate the direct electron transfer of redox-active proteins at an electrode and, to this end, recent years have witnessed a rapid progress in the protein electrochemistry and thereby the development of so-called thirdgeneration electrochemical biosensors,5 it remains a challenge to investigate the protein unfolding with the protein electron transfer properties, in spite of the fact that most kinds of proteins are chemically redox active and thereby electrochemically active from the thermodynamic point of view. (3) (a) Hargrove, M. S.; Krzywda, S.; Wilkinson, A. J.; Dou, Y.; Ikeda-Saito, M.; Olson, J. S. Biochemistry 1994, 33, 11767. (b) Dong, S.; Zhao, Z.; Ma, H. J. Proteome Res. 2006, 5, 26. (c) Wilson, A. J.; Groves, K.; Jain, R. K.; Park, H. S.; Hamilton, A. D. J. Am. Chem. Soc. 2003, 125, 4420. (d) Konishi, Y.; Feng, R. Biochemistry 1994, 33, 9706. (e) Holzbaur, I. E.; English, A. M.; Ismail, A. A. Biochemistry 1996, 35, 5488. (f) Chah, S.; Kumar, C. V.; Hammond, M. R.; Zare, R. N. Anal. Chem. 2004, 76, 2112. (g) Ragona, L.; Fogolari, F.; Romagnoli, S.; Zetta, L.; Maubois, J. L.; Molinari, H. J. Mol. Biol. 1999, 293, 953. (h) Gupta, R.; Yadav, S.; Ahmad, F. Biochemistry 1996, 35, 11925. (4) For example, see: (a) Willner, I. Science 2002, 298, 2407. (b) Mano, N.; Yoo, J. E.; Tarver, J.; Loo, Y.; Heller, A. J. Am. Chem. Soc. 2007, 129, 7006. (c) Watanabe, T.; Ivandini, T. A.; Makide, Y.; Fujishima, A.; Einaga, Y. Anal. Chem. 2006, 78, 7857. (d) Li, X.; Zhou, H.; Yu, P.; Su, L.; Ohsaka, T.; Mao, L. Electrochem. Commun. 2008, 10, 851. (e) Lu, Y.; Li, X.; Zhang, L.; Su, L.; Mao, L. Anal. Chem. 2008, 80, 1883. (f) Xiao, H.; Liu, L.; Meng, F.; Huang, J.; Li, G. Anal. Chem. 2008, 80, 5272. (g) Lin, Y.; Zhu, N.; Yu, P.; Su, L.; Mao, L. Anal. Chem. 2009, 81, 2067. (h) Kato, D.; Sekioka, N.; Ueda, A.; Kurita, R.; Hirono, S.; Suzuki, K.; Niwa, O. J. Am. Chem. Soc. 2008, 130, 3716. (i) Kurita, R.; Yokota, Y.; Ueda, A.; Niwa, O. Anal. Chem. 2007, 79, 9572. (5) (a) Jo ¨nsson, G.; Gorton, L. Electroanalysis 1989, 1, 465. (b) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G. J. Am. Chem. Soc. 1996, 118, 10321. (c) Patolsky, F.; Weizmann, Y.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 2113. (d) Yan, Y.; Zheng, W.; Su, L.; Mao, L. Adv. Mater. 2006, 18, 2639. (e) Zhou, H.; Gan, X.; Wang, J.; Zhu, X.; Li, G. Anal. Chem. 2005, 77, 6102.

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Scheme 1. Heme Active Site Structures in Hb from Bovine Blood (A), Cat. from Bovine Liver (B), and Cyt c from Horse Heart (C)a

a Pink dotted lines indicate the coordinative bonds between the iron atom and porphyrin or neighboring amino acid residues. Red circles in (C) indicate the thioether bonds between heme and amino acid residues in cyt c. Atoms: carbon (gray), oxygen (red), nitrogen (blue), sulfur (yellow), iron (wine).

Hemoproteins are one class of proteins including hemoglobin (Hb), peroxidases, cytochrome c (cyt c), catalase (cat.), and so forth. This kind of protein has long been used as the paradigm for understanding the structure-function relationships of proteins.6 Moreover, the unfolding of this kind of protein is closely associated with physiological abnormalities. For example, under very acidic conditions, the cooperative oxygen binding property of Hb is decreased and the pro-oxidative activity is dramatically increased mainly due to the significant conformational changes in its structure and heme crevice.7 While hemoproteins generally contain one or more heme groups in the hydrophobic pockets, their structures are different in terms of the linkage between the heme groups and the polypeptides. For instance, the heme groups in some kinds of hemoproteins, such as Hb and cat., bind with the polypeptides through a coordinative bond between the heme iron and neighboring amino acid residues,8 which could be readily destroyed under the denaturing conditions, resulting in the dissociation of these groups from the polypeptides (Scheme 1A,B).3a,7a,9 Differently, the heme group in cyt c is covalently linked to the polypeptide chain via thioether linkages to Cys-14 and Cys-17, leading to a strong coordinative bond between the heme iron and His-18 even under denaturing conditions (Scheme 1C).10 While some excellent electrochemical methods have been previously used to investigate the unfolding of horse heart cyt c with the facilitated electrochemistry of this kind of protein on (6) (a) Badghisi, H.; Liebler, D. C. Chem. Res. Toxicol. 2002, 15, 799. (b) Choi, J.; Terazima, M. J. Phys. Chem. B 2002, 106, 6587. (c) Sagle, L. B.; Zimmermann, J.; Dawson, P. E.; Romesberg, F. E. J. Am. Chem. Soc. 2004, 126, 3384. (d) Robinson, V. L.; Smith, B. B.; Arnone, A. Biochemistry 2003, 42, 10113. (7) (a) Kristinsson, H. G. J. Agric. Food Chem. 2002, 50, 7669. (b) Kristinsson, H. G.; Hultin, H. O. J. Agric. Food Chem. 2004, 52, 3633. (c) Kristinsson, H. G.; Hultin, H. O. J. Agric. Food Chem. 2004, 52, 5482. (8) (a) Mueser, T. C.; Rogers, P. H.; Arnone, A. Biochemistry 2000, 39, 15353. (b) Reid, T. J., III; Murthy, M. R. N.; Sicignano, A.; Tanaka, N.; Musick, W. D. L.; Rossmann, M. G. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4767. (c) Tosha, T.; Uchida, T.; Brash, A. R.; Kitagawa, T. J. Biol. Chem. 2006, 281, 12610. (9) (a) Hargrove, M. S.; Olson, J. S. Biochemistry 1996, 35, 11310. (b) Simmons, D. A.; Wilson, D. J.; Lajoie, G. A.; Doherty-Kirby, A.; Konermann, L. Biochemistry 2004, 43, 14792. (10) (a) Sivakolundu, S. G.; Mabrouk, P. A. J. Biol. Inorg. Chem. 2003, 8, 527. (b) Pineda, T.; Sevilla, J. M.; Roma´n, A. J.; Bla´zquez, M. Biochim. Biophys. Acta 1997, 1343, 227. (c) Fisher, W. R.; Taniuchi, H.; Anfinsen, C. B. J. Biol. Chem. 1973, 248, 3188. (d) Hamada, D.; Hoshino, M.; Kataoka, M.; Fink, A. L.; Goto, Y. Biochemistry 1993, 32, 10351.

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surface-functionalized electrodes,10b,11 and bovine serum albumin with the oxidation of amino acid residues through the use of electrocatalysts12a or special electrode materials,12b the structural differences between Hb and those kinds of proteins unfortunately make it difficult to apply the as-established electrochemical methods to investigate the unfolding of Hb. As a consequence, studies on Hb unfolding with an electrochemical method still remain a challenge. This study demonstrates a facile and effective electrochemical approach to probing Hb unfolding based on the electrochemical redox property of heme groups in Hb at conventional electrodes. Hb is known to possess a heterotetrameric structure composed of four subunits, each of which has a heme group deeply buried in the hydrophobic pocket with a five-coordinate high-spin complex.8a In its native state, Hb shows a poor electrochemical property at the conventional glassy carbon electrode. The unfolding of Hb induced by the denaturant of guanidine hydrochloride (GdnHCl) could result in the break of the fifth coordinative bond between the heme groups and the residue of the polypeptides (i.e., His-F8), leading to the dissociation of the heme groups from the polypeptide chains and thus resulting in a good electrochemical response at the electrodes that could be used for investigation on the unfolding of such kind of protein. Compared with the existing methods, such as UV-vis spectroscopy, fluorescence, and circular dichroism used for the study of Hb unfolding, the electrochemical method demonstrated here may be advantageous in terms of its ease in operation and cost-effective feature. This study essentially offers a facile and effective electrochemical route to the unfolding of the hemoproteins with five-coordinate highspin heme groups. EXPERIMENTAL SECTION Chemicals. Hb (MW 64 500) from bovine blood and cat. (MW 250 000) from bovine liver were purchased from Sigma and used without further purification. GdnHCl was purchased from Prome(11) (a) Bixler, J.; Bakker, G.; McLendon, G. J. Am. Chem. Soc. 1992, 114, 6938. (b) Barker, P. D.; Mauk, A. G. J. Am. Chem. Soc. 1992, 114, 3619. (c) Ferri, T.; Poscia, A.; Ascoli, F.; Santucci, R. Biochim. Biophys. Acta 1996, 1298, 102. (d) Zhu, Y.; Dong, S. Bioelectrochem. Bioenerg. 1996, 41, 107. (e) Fedurco, M.; Augustynski, J.; Indiani, C.; Smulevich, G.; Antalı´k, M.; Ba´no´, M.; Sedla´k, E.; Glascock, M. C.; Dawson, J. H. J. Am. Chem. Soc. 2005, 127, 7638. (12) (a) Guo, L. H.; Qu, N. Anal. Chem. 2006, 78, 6275. (b) Chiku, M.; Nakamura, J.; Fujishima, A.; Einaga, Y. Anal. Chem. 2008, 80, 5783.

ga Corp. Free hemin was purchased from ABCR GmbH & Co. KG, Germany. The aqueous solutions of hemin in 0.10 M phosphate buffer (pH 7.0) were prepared with the coexistence of 5.0 M GdnHCl. Other chemicals were of at least analytical grade and used as received. Aqueous solutions were prepared with doubly distilled water. Unfolding of Hb and Cat. Aqueous solutions of GdnHCl with different concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 M were prepared in 0.10 M phosphate buffer (pH 7.0), and the pH values of the GdnHCl solutions were adjusted to 7.0 with KOH solution. To induce the unfolding of Hb and cat. by GdnHCl, 100 µL of the stock solutions of Hb and cat. was added to the GdnHCl solutions and the resulting solutions were allowed to stand by for 1 h at 4 °C prior to the measurements. The concentrations of Hb used for electrochemical, UV-vis absorbance, fluorescence, and circular dichroism (CD) measurements were 25, 2.0, 2.0, and 0.63 µM, respectively. Acid- or alkaline-induced unfolding/refolding of Hb was studied in 0.10 M citric acid buffer (pH 3.0) and 0.10 M phosphate buffer (pH 11.0), respectively. Apparatus and Measurements. Cyclic voltammetry (CV) was performed with a computer-controlled electrochemical analyzer (CHI 660B, Chenhua, Shanghai, China) in a two-compartment and three-electrode cell. Glassy carbon (GC) electrodes (3 mm diameter, BAS) were used as working electrodes, and a platinum spiral wire was used as the counter electrode. All potentials were biased versus a Ag/AgCl electrode (saturated with KCl). Prior to use, GC electrodes were first polished with emery paper and then with aqueous slurries of fine alumina power (0.3 and 0.05 µm). The electrodes were finally rinsed with acetone and doubly distilled water under an ultrasonic bath, each for 5 min. For the electrochemical experiments conducted under anaerobic conditions, the solutions were bubbled with pure N2 gas for more than 30 min, and N2 gas was kept flowing over the solution during the electrochemical measurements. To avoid the adsorption of Hb or heme groups onto the electrode surface, freshly prepared electrodes were used in each electrochemical measurement and the electrochemical measurements were carried out immediately after the electrodes were immersed into the solutions. UV-vis spectra were recorded on a UV-1601PC spectrometer (Shimadzu, Japan). Fluorescence measurements were conducted on an F-2500 fluorescence spectrometer with xenon lamps (Hitachi Ltd., Japan). CD spectra were recorded on a J-810 CD spectrometer with xenon lamps (Jasco Corp. Japan). Unless stated otherwise, all measurements were performed at ambient temperature. RESULTS AND DISCUSSION GdnHCl-Induced Unfolding of Hb. Figure 1 depicts typical cyclic voltammograms of 25 µM Hb in 0.10 M phosphate buffer (pH 7.0) in the absence or presence of 5.0 M GdnHCl as the denaturant. In the absence of GdnHCl, no redox peak was recorded for Hb (black curve). In the presence of 5.0 M GdnHCl in the Hb solution, a pair of well-defined redox waves was clearly recorded at a formal potential (E°′) of -0.35 V (blue curve), calculated by averaging the cathodic and anodic peak potentials. The peak-to-peak separation was 55 mV (at 500 mV s-1), and the ratio of cathodic-to-anodic peak current was near unity. Since the GdnHCl denaturant is electroinactive (red curve), the observed redox wave was ascribed to the reversible redox

Figure 1. Typical cyclic voltammograms obtained at a GC electrode in N2-saturated 0.10 M phosphate buffer (pH 7.0) containing 25 µM Hb (black curve), 5.0 M GdnHCl (red curve), or 25 µM Hb + 5.0 M GdnHCl (blue curve). The blue curve was recorded after the mixture was prepared for 1 h. The pH values of the solutions were adjusted to 7.0 after addition of GdnHCl. The scan rate was 0.50 V s-1.

process of heme groups in Hb at GC electrodes. According to the previous reports,8a,9b Hb in its native state is approximately spherical in shape with dimensions of 65 × 55 × 50 Å and has a heterotetrameric structure composed of four subunits that are referred to as R1 and R2 subunits with eight helices and β1 and β2 subunits with seven helices. In each subunit, the heme group is buried in the hydrophobic pocket with a fifth coordinative bond with the residue of the polypeptide (His-F8), as shown in Scheme 1A. In addition to the covalent bond between iron and His-F8, there are van der Waals bindings between atoms on the porphyrin ring and about 60 atoms of the polypeptide chain in each subunit. The long distance between the heme groups and electrode and the large steric hindrance of native Hb essentially result in the poor electrochemical response of Hb at the bare GC electrodes (Figure 1, black curve). As addressed previously,3a,h,7 typical denaturants, such as GdnHCl, urea, acid, alkali, and salts, for most kinds of proteins could denaturalize the proteins probably through destroying their quaternary, tertiary, and secondary structures by competing hydrogen bonds in the proteins and by enhancing the water solubility of hydrophobic side chains. Similarly, the presence of GdnHCl denaturant may also destroy the structure and enhance the water solubility of the hydrophobic side chains of Hb, both resulting in the dissociation of the heme groups from the hydrophobic pockets and thus constituting one of the main consequences for the well-defined redox wave at the GC electrode, as depicted in Figure 1 (blue curve). To ensure the ascription of the observed redox wave obtained for the GdnHCl-denatured Hb at GC electrodes to the heme groups dissociated from the hydrophobic pockets in Hb, we have conducted control experiments with free hemin since this compound is structurally identical with the heme groups in Hb. As shown in Figure 2, one pair of well-defined, reversible redox waves was recorded at GC electrodes with an E°′ of -0.36 V for free hemin with a peak-to-peak separation of 58 mV (blue curve), characteristic of the reversible electrode process of the free hemin. This CV response for 100 µM free hemin was actually similar to that for 25 µM Hb denatured by 5.0 M GdnHCl, implying the GdnHCl-denatured unfolding of Hb may lead to the dissociation of the heme groups from the hydrophobic pockets in Hb. Besides, the GdnHCl-induced unfolding of Hb was studied with UV-vis Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

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Figure 2. Cyclic voltammograms obtained at a GC electrode in 0.10 M N2-saturated phosphate buffer (pH 7.0) containing 25 µM Hb + 5.0 M GdnHCl (black curve) or 100 µM free hemin + 5.0 M GdnHCl (blue curve). Cyclic voltammograms were recorded after the mixtures were prepared for 1 h. The pH values of the GdnHCl solutions were adjusted to 7.0. The scan rate was 0.50 V s-1.

Figure 3. UV-vis spectra of 0.10 M phosphate buffer containing 2.0 µM Hb only (black curve), 2.0 µM Hb + 5.0 M GdnHCl (blue curve), and 8.0 µM free hemin + 5.0 M GdnHCl (red curve). The green curve represents the UV-vis spectrum of 5.0 M GdnHCl in 0.10 M phosphate buffer (pH 7.0) containing no Hb. The pH values of the GdnHCl solutions were adjusted to 7.0.

spectroscopy, as displayed in Figure 3. Without the presence of GdnHCl, a tight Soret band at 406 nm was obtained (black curve), which was assigned to the heme monomer coordinated to His-F8 in the native state of Hb.9a With the presence of 5.0 M GdnHCl in Hb solution, the spectrum of Hb exhibits a broad Soret band at about 390 nm and a shoulder at around 365 nm (blue curve), which was almost identical with that of free hemin (red curve). The similarities in the cyclic voltammograms (Figure 2) and the UV-vis spectra (Figure 3) for GdnHCl-denatured Hb and free hemin strongly suggest that the redox wave recorded for the GdnHCl-denatured Hb (Figure 1, blue curve) could be ascribed to the redox process of heme groups dissociated from the hydrophobic pockets in Hb. As another support for this ascription, the mixture of 25 µM Hb and 5.0 M GdnHCl denaturant was filtered with a filter with a 3000 MW cutoff and CV measurements were conducted with GC electrodes in the ultrafiltrate (data not shown). The appearance of the well-defined redox wave at an E°′ of -0.35 V in the ultrafiltrate indicates the coordinative bond between the heme groups and His-F8 was broken and thus the heme groups dissociated from the hydrophobic pockets of Hb. 8560

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On the other hand, a close inspection of the cyclic voltammograms obtained for 100 µM free hemin and for 25 µM GdnHCldenatured Hb (Figure 2) reveals that the redox peak currents were almost the same, presumably demonstrating that all heme groups in Hb were dissociated from the hydrophobic pockets under the present denaturing conditions. The fact that the GdnHCl-induced denaturation of Hb essentially occurs with the dissociation of heme groups from the hydrophobic pockets of Hb and the good electrochemical reactivity of the dissociated heme groups at bare electrodes substantially form a straightforward basis for the electrochemical approach to Hb unfolding, as described below. Figure 4A displays cyclic voltammograms in 0.10 M phosphate buffer (pH 7.0) containing Hb with the presence of GdnHCl with different concentrations. The peak current of the redox wave clearly increases with increasing GdnHCl concentration and becomes almost stable when the GdnHCl concentration is higher than 4.0 M. This demonstrates that the concentration of heme groups dissociated from Hb increases with increasing GdnHCl concentration, and all of the heme groups in 25 µM Hb were dissociated when the GdnHCl concentration was higher than 4.0 M. It may be noted that the increased viscosity of the buffer caused by the addition of high concentrations of GdnHCl denaturant did not greatly affect the electrochemical response of the heme groups, which was reasoned from the almost unchanged reversibility of the redox wave of the heme groups with increasing concentration of GdnHCl denaturant in the buffer, as displayed in Figure 4A. These studies essentially demonstrate that the electrochemical method described here based on the redox process of the heme groups dissociated from the hydrophobic pockets in Hb could be effectively used to study the Hb unfolding induced by GdnHCl denaturant. To further validate the electrochemical approach to Hb unfolding, the GdnHCl-induced Hb unfolding was synchronously studied with the methods previously employed for Hb unfolding including UV-vis spectroscopy, fluorescence, and CD. By taking advantage of the heme absorption in the UV-vis spectra, as mentioned above, one may investigate the Hb unfolding with UV-vis spectroscopy. As shown in Figure 4B, the Soret absorption at 406 nm was largely decreased as the GdnHCl concentration was increased. As the GdnHCl concentration was higher than 3.0 M, the spectra with a broad Soret band near 390 nm and a shoulder near 365 nm became quite similar to those of free hemin and the absorption at 406 nm was almost unchanged as a function of the GdnHCl concentration.13 In addition, Hb contains a number of tryptophan residues that are deeply buried in the hydrophobic areas close to the heme groups. In the native state of Hb, the fluorescence of these residues is quenched by surrounding groups (i.e., heme groups), and as a consequence, the native Hb shows a weak fluorescence signal at around 335 nm. However, upon the unfolding induced by the GdnHCl denaturant, the heme groups dissociate from the polypeptide chain, and as a result, the initially buried tryptophan residues are exposed to the solvent. As a consequence, Hb shows a high fluorescence emission intensity and a slight bathochromic shift. The change in the fluorescence intensity essentially formed a basis for the Hb unfolding with a fluorescence method, as reported previously.7a,b As shown in (13) Lebrun, F.; Bazus, A.; Dhulster, P.; Guillochon, D. J. Agric. Food Chem. 1998, 46, 5017.

Figure 4. (A) Cyclic voltammograms obtained at a GC electrode in 0.10 M N2-saturated phosphate buffer (pH 7.0) containing 25 µM Hb in the absence and presence of GdnHCl with different concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 M (from inner to outer). The scan rate was 0.50 V s-1. (B) UV-vis spectra, (C) fluorescence spectra, and (D) CD spectra of the solutions of Hb and GdnHCl with concentrations of 0 M (black curves), 1.0 M (red curves), 2.0 M (green curves), 3.0 M (blue curves), 4.0 M (orange curves), and 5.0 M (pink curves) in 0.10 M phosphate buffer (pH 7.0). Dotted curves show the spectra of 3.0 M GdnHCl in 0.10 M phosphate buffer (pH 7.0). The concentrations of Hb were 25 µM (A), 2.0 µM (B, C), and 0.63 µM (D). For fluorescent measurements (C), the excitation wavelength was 280 nm, and the inset shows the excitation spectrum of Hb in 0.10 M phosphate buffer (pH 7.0). All signals were recorded after the mixing of GdnHCl into the phosphate buffer for 1 h. The pH values of the GdnHCl solutions were adjusted to 7.0.

Figure 4C, the fluorescence intensity recorded for GdnHCldenatured Hb increases with increasing GdnHCl concentration and becomes stable when the GdnHCl concentration is higher than 4.0 M. On the other hand, there are many helices in Hb molecules, and hence, Hb possesses a well-defined CD spectrum of helices. The GdnHCl-induced unfolding of Hb could lead to local perturbations in the helices including the proximal and distal sides of the heme pockets and thus to the loss of helical structure and proximal and distal histidine interactions, resulting in a decrease in the ellipticity.7a,b Such a property could be used for monitoring Hb unfolding with a CD spectrum. For instance, the ellipticity around 222 and 208 nm clearly decreased with increasing GdnHCl concentration, and both peaks almost disappear when the concentration of GdnHCl is higher than 3.0 M (Figure 4D). The results obtained with UV-vis spectroscopy, fluorescence, and CD were well consistent with those of previous reports9a and coincide with the electrochemical results shown in Figure 4A, demonstrating that the electrochemical method described here based on the redox property of heme groups could be effective for the use of Hb unfolding. Figure 5 summarizes the results obtained with electrochemistry, UV-vis spectroscopy, fluorescence, and CD for Hb unfolding as a function of the GdnHCl denaturant concentration. The unfolding percentage of Hb obtained with the four methods was calculated by using the value obtained in the absence of GdnHCl denaturant as 0% unfolding and the that obtained in the presence

Figure 5. Comparison of UV-vis absorbance (3), fluorescence (2), CD (0), and CV (9) changes resulting from the unfolding of Hb induced by different concentrations of GdnHCl. The plots were obtained from the reciprocals of absorbance at 406 nm for UV-vis spectra, the intensity at 335 nm for fluorescence, the reciprocals of ellipticity at 222 nm for CD, and the cathodic current at -0.38 V (vs Ag/AgCl) for CV, against the concentration of GdnHCl.

of GdnHCl denaturant (5.0 M for CV, fluorescence, and CD and 3.0 M for UV-vis) as 100% unfolding. The good consistency in the unfolding curves obtained with CV and UV-vis, fluorescence, and CD further substantially validates the electrochemical method based on the redox process of the heme groups at conventional Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

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Figure 7. Typical cyclic voltammograms obtained at a GC electrode in N2-saturated 0.10 M phosphate buffer (pH 7.0) containing 25 µM cat. (black curve), 3.0 M GdnHCl (red curve), or 25 µM cat. + 3.0 M GdnHCl (blue curve). The blue curve was recorded after cat. was added into the GdnHCl solution for 1 h. The pH values of the GdnHCl solutions were adjusted to 7.0. The scan rate was 0.50 V s-1.

Figure 6. (A) Cyclic voltammograms obtained at GC electrodes in 0.10 M N2-saturated phosphate buffer (pH 7.0) containing 25 µM Hb in the absence (dashed blue curve) and presence (black curves) of 2.0 M GdnHCl. The cyclic voltammograms were recorded after Hb was dissolved into GdnHCl solutions for different times of (from inner to outer) 5, 10, 20, 40, 90, and 180 min. The pH values of the GdnHCl solutions were adjusted to 7.0. The scan rate was 0.50 V s-1. (B) Current responses obtained at GC electrodes in 0.10 M N2-saturated phosphate buffer (pH 7.0) containing different concentrations of GdnHCl as a function of time duration for cyclic voltammogram recording after the addition of 25 µM Hb into the GdnHCl solutions. The concentrations of GdnHCl were 0 M (b), 1.0 M (9), and 2.0 M (2). Data were taken from the cathodic currents at -0.38 V in the cyclic voltammograms.

bare GC electrodes as a new tool for the study of the unfolding processes of Hb induced by GdnHCl. Application for Kinetic Study of GdnHCl-Induced Hb Unfolding. With the electrochemical method, the kinetics of Hb unfolding induced by GdnHCl was investigated, as typically shown in Figure 6A. The redox peak currents increase with increasing time duration after Hb was added into the GdnHCl solutions, suggesting the dissociation of the heme groups from the hydrophobic pockets of the native Hb during the GdnHCl-induced unfolding process of Hb. As displayed in Figure 6B, the kinetics for the unfolding process was essentially dependent on the concentration of the GdnHCl denaturant; a higher concentration of GdnHCl eventually results in a faster kinetic unfolding of Hb, which could be evident from the higher slope obtained with 2.0 M GdnHCl, as compared with that obtained at 1.0 M GdnHCl. Moreover, a close inspection of the plots reveals that the Hb unfolding within the initial 40 min was biphasic, one within 20 min and the other ranging from 20 to 40 min. These biphasic processes have different kinetics; the kinetics of the former was obviously faster than that of the latter. This demonstration presumably suggests that a biphasic process was possibly involved 8562

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in the GdnHCl-induced Hb unfolding with a fast phase for the heme group dissociation from β subunits and a slow one for the heme group dissociation from R subunits.14 Applications for GdnHCl-Induced Cat. Unfolding and pHInduced Hb Unfolding/Refolding. With the aim to extend the electrochemical method demonstrated here for the study of unfolding of other kinds of hemoproteins with five-coordinate highspin heme groups, we studied the GdnHCl-induced unfolding of cat., another kind of hemoprotein with a covalent bond between the heme groups and the polypeptide chain (Scheme 1B) similar to that in Hb with the electrochemical method employed for Hb unfolding. As depicted in Figure 7, similar to the case of Hb, no redox wave was recorded for cat. or GdnHCl at the bare GC electrodes under the present conditions. The addition of cat. into the GdnHCl solution leads to a pair of redox waves at an E°′ of -0.35 V, which was also ascribed to the redox processes of the heme groups (blue curve), implying that the electrochemical method based on the redox property of the heme groups dissociated from the hydrophobic pockets of cat. could also be used for the study of GdnHCl-induced cat. unfolding. Nevertheless, a comparison of the peak currents recorded for cat. and Hb reveals that the former currents were obviously smaller than the latter ones under the same unfolding conditions, which could be presumably understood in terms of the different structures of both proteins. Similar to Hb, bovine liver cat. also has four subunits, and each subunit has one heme-binding pocket.8c Nevertheless, cat. is dumbbell-shaped with a length of 90 Å and a waist diameter of 50 Å and is bigger than Hb in size.8b More importantly, the heme groups in cat. are deeply buried in the molecule, 20 Å below the molecular surface.8b In other words, the heme groups are more deeply buried in the hydrophobic pockets in the interior of cat., as compared with those in Hb (10 Å below the molecular surface8a). As a consequence, under the same unfolding conditions, fewer heme groups were dissociated from the cat. molecules to produce an electrochemical signal at the electrodes, as compared with those from Hb molecules. (14) Hargrove, M. S.; Singleton, E. W.; Quillin, M. L.; Ortiz, L. A.; Phillips, G. N.; Olson, J. S.; Mathews, A. J. J. Biol. Chem. 1994, 269, 4207.

unfolding/refolding process is reversible, consistent with those reported previously.15 Compared with those recorded for GdnHCl-denatured Hb (Figure 4 A), the currents for aciddenatured Hb were much smaller, suggesting that fewer heme groups were dissociated from the polypeptides in the aciddenatured Hb. Similarly, the changes in the cyclic voltammograms of Hb at alkaline pH were quite close to those at acidic pH (Figure 8 B), with the exception of the potential of the heme groups due to the pH dependence of the redox process of these groups.16 The results demonstrated here are well consistent with those obtained with other methods reported previously,9,15 suggesting that the electrochemical method could be potentially employed for the investigations on the Hb unfolding/refolding induced by pH.

Figure 8. (A) Typical cyclic voltammograms obtained at a GC electrode in N2-saturated 0.10 M citric acid buffer containing 25 µM Hb when the buffer pH was adjusted to pH 7.0 (black curve), pH 3.0 adjusted from pH 7.0 (blue curve), and pH 7.0 adjusted back from pH 3.0 (red curve). (B) Typical cyclic voltammograms obtained at a GC electrode in N2-saturated 0.10 M phosphate buffer containing 25 M Hb after the buffer pH was adjusted to pH 7.0 (black curve), pH 11.0 adjusted from pH 7.0 (blue curve), and pH 7.0 adjusted back from pH 11.0 with H3PO4 solution (red curve). The blue and red curves were recorded after the buffer pH was adjusted to the desired values for 1 h. The scan rate was 0.50 V s-1.

In addition, the applicability of the electrochemical method demonstrated here was explored for Hb unfolding/refolding induced by pH, as typically shown in Figure 8. At pH 7.0, no redox wave was recorded at GC electrodes for Hb (black curve). However, after the solution pH was adjusted to 3.0 with citric acid solution, a pair of redox waves was recorded at a formal potential of -0.18 V (blue curve) with a peak-to-peak separation of 90 mV (at 500 mV s-1). This redox wave was ascribed to the redox process of heme groups in the acid solution. After the solution pH was adjusted back to 7.0 with NaOH solution, the redox peak of the heme groups disappeared (red curve). This phenomenon reveals that the heme groups also dissociate from polypeptides during the acid-induced unfolding process, and could be re-embedded into the polypeptide chains after the pH value was adjusted back to be neutral. In other words, this

CONCLUSIONS By utilizing the fact that the Hb unfolding induced by common denaturants generally occurs with the dissociation of heme groups from the hydrophobic pockets of Hb and the good electrochemical reactivity of the dissociated heme groups at bare glassy carbon electrodes, we have demonstrated here a facile and effective electrochemical method for investigation of Hb unfolding induced by typical denaturants, such as GdnHCl. The good consistency of the results obtained with the electrochemical method demonstrated here with those of other methods currently employed for the study of protein unfolding substantially validates our electrochemical method based on the redox process of the heme groups as a new tool for the study of Hb unfolding. Compared with other electrochemical methods previously employed for the study of protein unfolding, the method demonstrated here could be advantageous in terms of, for example, its simple uses of bare electrodes to monitor the signal during Hb unfolding. This study essentially paves a facile but effective approach to investigation of protein unfolding. ACKNOWLEDGMENT The present research was financially supported by the NSF of China (Grant Nos. 20625515, 90813032, 20935005, and 20721140650), National Basic Research Program of China (Grant Nos. 2010CB933502 and 2007CB935603), Chinese Academy of Sciences, and Institute of Chemistry.

Received for review July 9, 2009. Accepted August 29, 2009. AC9015215 (15) (a) Steinhardt, J.; Zaiser, E. M. J. Am. Chem. Soc. 1953, 75, 1599. (b) Steinhardt, J.; Zaiser, E. M.; Beychok, S. J. Am. Chem. Soc. 1958, 80, 4634. (c) Shen, L. L.; Hermans, J. Biochemistry 1972, 11, 1836. (d) Shen, L. L.; Hermans, J. Biochemistry 1972, 11, 1842. (16) (a) Sun, Y.; Wang, S. Bioelectrochemistry 2007, 71, 172. (b) Liu, J.; Qiu, J.; Sun, K.; Chen, J.; Miao, Y. Helv. Chim. Acta 2009, 92, 462.

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