7090
J. Phys. Chem. B 2010, 114, 7090–7097
Contributions of Components in Guanidine Hydrochloride to Hemoglobin Unfolding Investigated by Protein Film Electrochemistry Zhibin Mai,† Xiaojuan Zhao,†,‡ Zong Dai,*,† and Xiaoyong Zou*,† School of Chemistry and Chemical Engineering, Sun Yat-Sen UniVersity, Guangzhou 510275, People’s Republic of China, and College of Light Industry and Food Science, Zhongkai UniVersity of Agriculture and Engineering, Guangzhou 510225, People’s Republic of China ReceiVed: February 4, 2010; ReVised Manuscript ReceiVed: April 12, 2010
The contribution of the chloride anion (Cl-) and guanidinium cation (Gdn+) to the denaturing efficiency of guanidine hydrochloride (GdnCl) upon the unfolding of hemoglobin (Hb) was investigated electrochemically. Hb was entrapped in a didodecyldimethylammonium bromide (DDAB)-film-modified glassy carbon electrode and unfolded by the components of GdnCl. The changes of the direct electrochemical behaviors of Hb, including peak current (Ip), formal potential (Eo′), and peak-to-peak separation (∆E), were utilized to characterize different unfolded states of Hb. UV-vis, circular dichroism, and fluorescence spectroscopy were also used to verify the structural information of Hb during the unfolding process. The results indicated that the denaturing efficiency of GdnCl was contributed to by Gdn+ and Cl- in synchronization, and the portions of such contributions were concentration-dependent. In addition, the presence of Gdn+, Cl-, or GdnCl can enhance the reversibility of the redox reaction of Hb to the same degree. The method provides not only an easy way to better understand the conformational changes of Hb but also a strategy to control its conformation. Introduction Investigations of the folding/unfolding of protein have drawn increasing interest in recent years because they reveal insight into the prediction of the structure of the protein from the polypeptide sequence, constituting the protein folding/unfolding pathway.1 Hemoglobin (Hb), which is the main protein in red blood cells, is well-known for its function of transporting oxygen from lungs or gills to peripheral tissues in the vascular system of animals.2 Abnormal conformation of Hb can lead to the perturbation of the vascular system. Therefore, the investigation of the structural transition of Hb is of great biological importance. It is known that under different denaturing conditions, Hb molecules can undergo different conformational transitions such as aggregation,3 disassembling of subunits,4 and exposure/burial of amino acid surface area.5,6 According to UV-vis and mass spectroscopy, the hydrogen cation (H+) has been confirmed to be able to alter the pro-oxidative activity of Hb to lipid oxidation and dissociate the subunits of Hb.4,7-11 Allosteric effectors, such as the chloride anion12 and adenosine-5′-triphosphate (ATP),13 can influence the equilibrium between R (oxystrucutre) and T (deoxystructure) states of Hb and thereby the oxygen-binding ability. The subunit interactions and structural alterations of the heme cavity in the unfolding process of Hb were probed by fluorescence.12,14,15 Temperature and ligand binding can also cause the structural transition of Hb. Nuclear magnetic resonance (NMR) in combination with hydrogen-deuterium (H-D) exchange of peptide NH atoms or circular dichroism spectroscopy (CD) were used to monitor such unfolding events.16-18 Polar molecules, such as urea, can unfold Hb by changing the strength of hydrophobic interactions within Hb.19,20 * To whom correspondence should be addressed. E-mail: ceszxy@ mail.sysu.edu.cn (X. Y. Zou);
[email protected]. (Z. Dai). Tel: +86-020-84114919. Fax: +86-020-84112901. † Sun Yat-Sen University. ‡ Zhongkai University of Agriculture and Engineering.
Guanidine hydrochloride (GdnCl), which is also a commonly used denaturant, is found to have higher denaturing ability than many denaturants.21,22 For instance, with respect to urea and the chloride anion (Cl-), GdnCl was reported to split the chains of the tetrameric Hb molecule in a more effective way.22,23 The origination of the high denaturing effect of GdnCl was investigated by using equimolar units of GdnCl, Cl-, and the guanidinium cation (Gdn+) to unfold model peptide helices.21 The results showed that the denaturing efficiency of Gdn+ was identical to that of Cl-.21 However, the investigations on the contribution of Gdn+ and Cl- to the denaturing efficiency of GdnCl only focused on small proteins or peptides. Bovine hemoglobin (Hb), which is a representative metalloprotein with a multisubunit, comprises two pairs of R- and β-subunits, each of which contains a heme group.2 The conformational transitions and the loss of tertiary or secondary structures of Hb can occur within every subunit during the inducement of GdnCl, which makes the unfolding of Hb more complicated. Although sedimentation velocity and gel electrophoresis have been performed to investigate the unfolding of Hb by GdnCl, the effects of unique denaturing components of GdnCl (including Gdn+ and Cl-) on the unfolding of Hb and their contributions to the denaturing efficiency of GdnCl are still ambiguous.22,23 Electrochemistry plays an important role in investigating the structural transformation of Hb by monitoring the redox signals of heme irons.13,24,25 Compared with spectral techniques,4,10-12,14-18 electrochemistry has unique abilities in achieving the kinetic and thermodynamic information of the redox process by simple protocols26 and probing the structural alterations around heme irons of Hb molecules. It has been confirmed that the changes of electrochemical parameters can provide structural information on the unfolded Hb.25 The change of peak current (Ip) after denaturation is closely related to the exposure/burial of heme irons or the dissembling/assembling of subunits within Hb molecules. The change of peak-to-peak separation (∆E) reflects
10.1021/jp101082d 2010 American Chemical Society Published on Web 05/05/2010
Guanidine Hydrochloride and Hemoglobin Unfolding the promotion/suppression of the electron transfer of Hb, which indicates the variations of the distance between the heme irons and the electrode surface. The previously reported electrochemical methods for investigating the folding/unfolding of Hb are commonly performed in Hb solutions based on the redox reaction of heme irons at bare or modified electrodes.24,25 However, the high concentration of denaturants may increase the viscosity of the protein solution and restrict the diffusion of protein molecules to the electrode surface. Meanwhile, when eliminating the traces of oxygen from protein solution by nitrogen, the foaming phenomenon may also cause a perturbation to the detection and a waste of the protein sample. Protein film electrochemistry, which investigates the protein molecules on the electrode surface, is an effective strategy to obtain the electrochemical signal of protein.27 Didodecyldimethylammonium bromide (DDAB) film, which has been used to form phospholipid vesicles with Hb to mimic the artificial red blood cell,28 was documented to promote electron transfer from Hb to the electrode surface.29-32 Since Hb can diffuse rapidly through the DDAB layer, a relatively fast electrontransfer rate can be achieved, and the adsorption of macromolecular adsorbents can be inhibited at the same time.33-35 Moreover, because the detection of the redox signal of Hb entrapped in the DDAB film is carried out in the buffer solution without proteins, traces of oxygen can be effectively eliminated without foam. In this work, the unfolding of Hb induced by Gdn+, Cl-, and GdnCl was investigated electrochemically based on the redox reaction of heme irons within Hb, which was encapsulated in a DDAB-film-modified glassy carbon electrode (GCE/DDABHb). The penetration of the denaturants into the DDAB film led to the structural alterations of entrapped Hb. The corresponding electrochemical parameters during this process were used to indicate the contributions of Gdn+ and Cl- to the denaturing efficiency of GdnCl on Hb. The proposed method not only specified the unfolding events of Hb induced by GdnCl but also provided a strategy to modulate the structural transition of Hb in a bionic condition. Experimental Section Reagents. Bovine hemoglobin (Hb) and GdnCl were purchased from Sinopharm Chemical Reagent Co., Ltd., and used as received. DDAB was obtained from Alfa Aesar. Guanidine phosphate (GdnPA) was purchased from Aladdin Reagent Company. All other chemicals were of analytical grade. The buffer solution used in this work was 0.1 mol L-1 phosphate buffer solution (PBS, pH 7.0), which was prepared from the stock solutions of 0.1 mol L-1 NaH2PO4 and Na2HPO4. The denaturing solutions of GdnCl, Cl-, and Gdn+ were prepared by dissolving a desirable amount of denaturants in 0.1 mol L-1 PBS (pH 7.0). All solutions were prepared with doubly distilled water. Preparation of a DDAB-Hb-Film-Modified Glassy Carbon Electrode (GCE/DDAB-Hb). The glassy carbon electrode (GCE, 3 mm in diameter) was polished to a mirror finish mechanically with 0.3 and 0.05 µm alumina powders. The wellpolished GCE was cleaned in absolute ethanol and doubly distilled water by sonication for 1 min, respectively. A 10 mmol L-1 DDAB suspension was prepared in doubly distilled water and sonicated for 30 min to produce a homogeneous vesicle dispersion. The DDAB suspension was then mixed with a 5 mg mL-1 Hb solution (in a 0.1 mol L-1 pH 7.0 PBS) for 5 min at a ratio of 1:1/V:V. After that, 5 µL of the mixture was spread on a well-polished GCE and dried at 4 °C for 1.5 h.
J. Phys. Chem. B, Vol. 114, No. 20, 2010 7091
Figure 1. Typical cyclic voltammograms of GCE/DDAB and GCE/ DDAB-Hb in the absence (a and b) and presence (c and d) of 3 mol L-1 GdnCl. The scan rate was 0.5 V s-1.
Apparatus. All electrochemical experiments were carried out with a CHI760c electrochemical workstation (Shanghai, China) equipped with a conventional three-electrode cell. A GCE/ DDAB-Hb was used as a working electrode. A platinum wire and a Ag/AgCl electrode (3 mol L-1 KCl) were used as the counter and reference electrodes, respectively. The denaturing solutions of GdnCl, Cl-, and Gdn+ were prepared by dissolving a desirable amount of denaturants in 0.1 mol L-1 PBS (pH 7.0). The cyclic voltammograms (CVs) of unfolded Hb were obtained in the denaturing solutions where GCE/DDAB-Hb was incubated for 45 min. The scan rate of the cyclic voltammetry was 0.5 V s-1 to avoid the perturbation of autoxidation of Hb-Fe(II) by residual oxygen.36 Prior to all electrochemical experiments, buffer solutions/denaturants were purged with high-purified nitrogen for 30 min, and a nitrogen atmosphere was then maintained above the solution during all electrochemical measurements. The UV-vis spectra of Hb in So¨ret band were recorded with a Shimadzu UV-3150 spectrophotometer in the range of 300-500 nm at a medium scan rate. The intrinsic fluorescence of tryptophan (Trp) of Hb in the range of 300-400 nm was measured with a Shimadzu RF-5301PC spectrofluorimeter at the excitation wavelength of 280 nm. In order to obtain better spectra, a series of Hb stock solutions with the concentration of 2500 mg L-1 was prepared by dissolving Hb in different denaturing solutions of 0-7 mol L-1 GdnCl, 3 mol L-1 Gdn+, and 3 mol L-1 Cl-, separately. These Hb stock solutions were left for 45 min and then diluted immediately into 50 and 200 mg L-1 for UV-vis and fluorescence measurements, respectively. All spectral measurements were carried out at room temperature. Results and Discussion Unfolding of Hb by GdnCl. The penetration of GdnCl to the DDAB-Hb film can lead to the structural alterations of Hb and thus its electrochemical properties. In order to verify the ability of the electrochemical method in investigating the Hb unfolding, the unfolding of Hb induced by GdnCl was investigated by electrochemistry, UV-vis spectrometry, and fluorescence. As shown in Figure 1, when DDAB-modified GCEs (GCE/ DDAB) were incubated in pH 7.0 PBS in the absence or presence of 3 mol L-1 GdnCl for 45 min, no redox peaks were found (Figure 1, curves a and c). However, after Hb was entrapped in the DDAB film (GCE/DDAB-Hb) and incubated in pH 7.0 PBS, a pair of quasi-reversible redox peaks with a
7092
J. Phys. Chem. B, Vol. 114, No. 20, 2010
Mai et al.
Figure 2. (A) CVs of GCE/DDAB-Hb after incubation in PBS containing different concentrations of GdnCl. (B) UV-vis spectra and (C) intrinsic tryptophan fluorescence spectra of Hb in the presence of different concentrations of GdnCl. The concentrations of GdnCl from (a) to (h) in (A), (B), and (C) were 0, 1, 2, 3, 4, 5, 6, and 7 mol L-1, respectively. The concentrations of Hb in (B) and (C) were 50 and 200 mg L-1, respectively. (D) Unfolding curves of Hb assessed by cathodic currents of GCE/DDAB-Hb (b), intrinsic tryptophan fluorescence intensity (9), and So¨ret band absorption of UV-vis spectra (2) in the presence of different concentrations of GdnCl.
Eo′ of -165 mV and ∆E of 87 mV were obtained (Figure 1, curve b). In agreement with the previous reports on the direct electron transfer of Hb in the DDAB film, the observed peaks here were attributed to the redox reaction of heme irons, and the entrapped Hb molecules can retain their native conformations in DDAB films.29-32,37 The native Hb molecule has an approximately spherical shape with four subunits within which the heme group is buried in a hydrophobic pocket with a fifth coordinative bond to the residue of the polypeptide (His-F8);38,39 the large steric hindrance between the heme irons of native Hb and the surface of the electrode resulted in quasi-reversible electrochemical behaviors of Hb. When GCE/DDAB-Hb was incubated in GdnCl, a significant enhancement of Ip along with a negative shift of Eo′ and a decrease of ∆E with respect to those of native Hb (GCE/DDABHb in pH 7.0 PBS) was observed (Figure 1, curve d, and Figure 2A). According to previous reports, GdnCl induced the unfolding of Hb by increasing the water solubility of its hydrophobic chains.7-9,40,41 Therefore, the exposure of the deep buried heme iron during the denaturing process was expected to increase the electroactive amount of Hb and facilitate the direct electron transfer between Hb and the electrode. The conformational changes of Hb induced by GdnCl were also investigated by UV-vis and fluorescence. UV-vis absorption in the So¨ret band is a sensitive conformational probe for monitoring the interaction between the heme irons and the globins.42,43 As shown in Figure 2B, a tight So¨ret band was observed at around 405 nm in 50 mg L-1 native Hb (Figure 2B, curve a), which was assigned to the heme monomer coordinated to His-F8 of native Hb.25,44 However, with increasing concentration of GdnCl from 3 to 7 mol L-1, the spectra showed a broad So¨ret band at 390 nm and a shoulder peak at 370 nm, which was ascribed to the dissociation of heme groups
from the hydrophobic pockets in the high concentrations of GdnCl (Figure 2B, curves c-h).25 The fluorescence emission spectrum of Trp can sensitively probe the changes of the tertiary structure of the protein.45-48 In native Hb, the fluorescence of RTrp14, βTrp15, and βTrp37 was quenched to the nonfluorescent hemes by Fo¨rster resonance energy transfer (Figure 2C, curve a).49-51 However, the unfolding of Hb induced by GdnCl caused the increase of the distance between the heme and Trp; thereby, the energy-transfer efficiency was reduced, and the Trp emission intensity at 340 nm was enhanced consequently (Figure 2C, curves b-h).52 According to eq 1, the unfolding percentage was used to estimate the degree of Hb unfolding induced by GdnCl
Unfolding/% )
f - fN × 100% fU - fN
(1)
where f represents the spectral/electrochemical values in a particular unfolding condition and fN and fU are the optical/ electrochemical values in the native and unfolded states.7,10,53 As manifested in Figure 2D, the unfolding percentage of native Hb was 0%, while that of fully unfolded Hb was 100%. The degree of Hb unfolding achieved from electrochemistry was well-correlated with that established from UV-vis and fluorescence spectra. These results revealed that the changes of the direct electrochemical response of Hb exhibited the ability to monitor the conformational transitions of Hb in a DDAB film. It should be noted that when investigating the unfolding of Hb from the electrochemical technique, the amount of Hb in the DDAB film must be constant to ensure the dependence of current response on the conformational change of Hb. Therefore, the possible leakage of Hb from GCE/DDAB-Hb during the
Guanidine Hydrochloride and Hemoglobin Unfolding
J. Phys. Chem. B, Vol. 114, No. 20, 2010 7093
Figure 3. (A) CVs of GCE/DDAB-Hb at different pHs. (B) Intrinsic tryptophan fluorescence spectra of Hb at different pHs. The concentration of Hb was 200 mg L-1. (C) UV-vis characterization in the So¨ret band of Hb at different pHs. The concentration of Hb was 50 mg L-1. The lines represent (solid line) pH 7.0, (dashed line) pH 5.0, and (dotted line) pH 4.6 PBS.
incubation processes was assessed by UV-vis. GCE/DDABHb was incubated in certain volumes of denaturing solution for 45 min, and the denaturing solution was measured by UV-vis at 405 nm. For reference, 50 mg L-1 of Hb dissolved in the denaturing solution was measured in the same manner to estimate the concentrations of leaked Hb. The percentages of concentrations of leaked Hb to the concentrations of surfaceentrapped Hb (L%) were roughly estimated from eq 2
L% )
AL × 50 × 100% Aref × 2500
(2)
where AL and Aref represent the absorptions of leaked Hb and the referential Hb solutions, respectively. The terms of 50 and 2500 are the concentrations of the referential Hb solution and surface-entrapped Hb. As shown in Table S1 (Supporting Information), the maximal L% was less than 0.07%, indicating that during the incubation process, the leakage of Hb from GCE/ DDAB-Hb into the bulk solution can be neglected. Consequently, the changes of the electrochemical responses of GCE/ DDAB-Hb induced by different denaturants were ascribed to the structural alterations of the Hb which was entrapped in the DDAB film. Effects of the Apparent pH of GdnCl on the Conformation of Hb. In strong acidic conditions, the dissociation of subunits within Hb molecules is promoted.54 Because of the hydrolysis of GdnCl, the effects of the resulting pH on the conformation of Hb should be considered. The apparent pH values of 3.0 and 6.0 mol L-1 GdnCl were 5.0 and 4.6, respectively. Therefore, two PBS with pH values of 5.0 and 4.6 were prepared to investigate the effect of H+ on the conformational transition of Hb. The pHs of PBS were regulated by H3PO4, via which the influences of other anions can be precluded. Meanwhile, it has been confirmed that the presence of Na+ caused relatively little effect on the conformational transition of Hb.55 Consequently, the changes of the electrochemical properties of Hb were solely caused by H+. In the acidic conditions, the Ip of Hb decreased slightly, concomitant with the positive shift of the Eo′ when compared with those in pH 7.0 PBS (Figure 3A). The electrochemical results suggested that the redox reaction of Hb was protoninvolved, and the Eo′ of entrapped Hb was insensitive to the changes of pH 4.6 and 5.0.30 However, the entry of water molecules into the heme cavity increased the barrier for the electron transfer by ∼50%, thereby causing the decrease of Ip.56,57 Meanwhile, in such pHs, the intrinsic fluorescence of Trp within Hb showed no obvious changes with respect to that of
native Hb (Figure 3B), and the UV-vis spectra of the So¨ret band slightly decreased (Figure 3C). These results indicated that the heme-imidazole bond in Hb did not break.58 From the spectral and electrochemical results, it was implied that H+ was not the main contributor to the unfolding signal of Hb induced by GdnCl. Since the structure around the heme irons was not altered significantly, the slight decrease of Ip and positive shift of Eo′ in these pHs were due to the electrochemical properties of the heme irons but not the unfolding of Hb. In order to avoid the influences of the pHs, hereafter, the pHs of denaturants (including GdnCl, Cl-, and Gdn+) were controlled at 7.0. Unfolding of Hb by the Components of GdnCl. As known, different denaturants can unfold proteins through different ways. However, unlike those common single-component denaturants, such as acid, alkali, and urea, GdnCl contains two effective components of Gdn+ and Cl-, both of which can induce the conformational transition of Hb. Therefore, the comparisons of the unfolding efficiencies of Cl-, Gdn+, and GdnCl to Hb can be helpful to understand the contributions of the components of GdnCl to Hb unfolding. As a complement to the electrochemical results, UV-vis and fluorescence were also imported to obtain the structural information of Hb. The electrochemical results of unfolded Hb by GdnCl reached a plateau at the concentration of 3 mol L-1 (Figure 2D). Aiming to obtain structural information of Hb in such unfolded states, Hb incubated with 3 mol L-1 denaturants was used in the spectral studies. Unfolding of Hb by Cl-. As a part of GdnCl, Cl- has been well-documented as an allosteric effector in modulating the quaternary structure of Hb.59-63 Meanwhile, Cl- is one of the chaotropic salts that can be used as a protein-dissociating agent for proteins which self-interact via the formation of intermolecular hydrogen bonds.64 Aiming to evaluate the contribution of Cl- to the unfolding efficiency of GdnCl, a series of PBS solutions containing 1, 2, 3, and 4 mol L-1 NaCl was prepared. GCE/DDAB/Hb was incubated into these NaCl-contained PBS solutions for 45 min, and the corresponding electrochemical behaviors were recorded. UV-vis and fluorescence were also used to obtain the structural information of Hb in the presence of 3 mol L-1 NaCl. The concentrations of Hb for UV-vis and fluorescence measurements were 50 and 200 mg L-1, respectively. As shown in Figure 4A, with increasing proportions of Clin PBS, the GCE/DDAB-Hb showed an obvious enhancement of Ip, which was in agreement with the earlier reports.24,65 However, the intrinsic Trp fluorescence (Figure 4B) showed an apparent increase of intensity and a blue shift of λmax, indicating an alteration of the microenvironments around Trp residues of Hb. The results may be from the aggregation of Hb
7094
J. Phys. Chem. B, Vol. 114, No. 20, 2010
Mai et al.
Figure 4. (A) CVs of GCE/DDAB-Hb after incubated in PBS solutions containing (a) 0, (b) 1, (c) 2, (d) 3, and (e) 4 mol L-1 NaCl. (B) Intrinsic fluorescence of Trp within Hb in the absence (solid line) and presence (dash line) of 3 mol L-1 Cl-; the concentration of Hb was 200 mg L-1. (C) So¨ret band absorption of Hb in the absence (solid line) and presence (dashed line) of 3 mol L-1 Cl-; the concentration of Hb was 50 mg L-1.
Figure 5. (A) CVs of GCE/DDAB-Hb in different concentrations of Gdn+; from (a) to (e), the concentrations of Gdn+ were 0, 1, 2, 3, and 4 mol L-1, respectively. (B) Intrinsic fluorescence of Trp and So¨ret band absorption of Hb (C) in the absence (solid line) and presence of 3 mol L-1 Gdn+ (dashed line). The concentrations of Hb for UV-vis and fluorescence measurements were 50 and 200 mg L-1, respectively.
when it was dissolved in 3 mol L-1 Cl-. The UV-vis absorption in the So¨ret band (Figure 4C) of Hb in 3 mol L-1 Cl- showed no obvious alterations when compared with those of Hb in pH 7.0 PBS, which indicated that Cl- did not cause observable secondary and tertiary structure changes around the heme pockets.24 As reported previously, the dissociating action of Clwas accounted by for its preferential binding to the dimer species,64 and during this process, no major shifts in protein conformation occurred because Cl- need not completely dissociate proteins from polymer to monomer. Therefore, the increase of Ip in the presence of Cl- was related to the formation of dimers of Hb. According to the Faradaic equation, Ip was proportional to the electroactive amount of dissociated/unfolded Hb. In this work, the normalized peak current (Inor) was used to evaluate the dissociating ability of Cl- to Hb. Inor is defined in eq 3
Inor ) Iunfolded /Inative
(3)
where Iunfolded is Ip under certain unfolding conditions and Inative is the Ip of Hb in its native state. When the amount of Cl- was 4 mol L-1 (more NaCl cannot be thoroughly dissolved in pH 7.0 PBS), the Inor was 1.5. The experimental results suggested that half of amount of Hb was split into dimers by Cl-. However, it should be noted that if all of the Hb was dissociated into dimers, the ideal value of Inor would have been 2. The bias from the ideal value of Inor in this work can be ascribed to the presence of DDAB, which may partially prevent the binding between Cl- and Hb. In the presence of Cl-, better reversibility and a negative shift of Eo′ of the redox process of Hb were obtained (Figure 4A). The reversibility and Eo′ of the redox reaction of Hb were related
to the propensity of the processes of donating or accepting electrons.13,66 All of them will be sensitively affected by the alterations of the globin surrounding the active heme in Hb. Cl- is able to form salt bridges within the central cavity in Hb and lead to alterations of the quaternary conformation.13 These conformational changes will alter the electron affinity at the active site heme by shortening the distance between the iron and porphyrin nitrogens and the proximal histidine residues. Therefore, the redox reaction of Hb was facilitated.24 Unfolding of Hb by Gdn+. Because the pKa of GdnCl is 13.61,67 guanidine is fully protonated at the pH of 7.0. Since Gdn+ can also influence the stability of proteins and enzymes,1 a series of PBSs (pH 7.0) containing 1, 2, 3, and 4 mol L-1 GdnPA was prepared. GCE/DDAB/Hb was incubated into these GdnPA-contained PBSs for 45 min, respectively, and the corresponding electrochemical behaviors were recorded. Spectral characterizations, including UV-vis absorption and fluorescence, were also introduced to obtain the structural information of Hb in the presence of 3 mol L-1 GdnPA. The concentrations of Hb for UV-vis and fluorescence measurement were 50 and 200 mg L-1, respectively. As manifested in Figure 5A, Gdn+ can promote the electron transfer of entrapped Hb with an increase of Ip and nearly no shift of the Eo′ of Hb (Figure 5A). The fluorescence of Trp induced by Gdn+ showed a maximal emission at 337.8 nm (Figure 5B, dash line), indicating the exposure of the Trp which was deeply buried in the hydrophobic core of Hb. UV-vis of Hb in 3 mol L-1 Gdn+ showed a broad So¨ret band at 390 nm and a shoulder peak at 370 nm, which was comparable to those of GdnCl and ascribed to the dissociation of heme groups from the hydrophobic pockets (Figure 5C, dashed line).25 The presence of Gdn+ can destroy the protein’s quaternary, tertiary, and secondary structures by
Guanidine Hydrochloride and Hemoglobin Unfolding
J. Phys. Chem. B, Vol. 114, No. 20, 2010 7095
Figure 6. (A) The dependences of (A) Inor, (B) ∆E, and (C) Eo′ of Hb on [Denaturants] (A and B) and log[Denaturants] (C), where the symbols represented (9) GdnCl, (2) Cl-, and (b) Gdn+; the dashed line in (A) is the simulated curve of the total contribution of Gdn+ and Cl-.
competing hydrogen bonds in the proteins and enhancing the water solubility of hydrophobic side chains.40,41 During this process, the detachment of heme irons from their native sites in Hb may decrease the electron-transfer resistance and thereby enhance the direct electron transfer of entrapped Hb, causing the increase of Ip and the electron-transfer rate of Hb. Separation of Denaturant and Salt Effects in GdnCl. Figure 6 illustrates the overall changes of the Ip, ∆E, and Eo′ of GCE/DDAB-Hb after being induced by different components of GdnCl. From the denaturing curves, the contributions of Cland Gdn+ to the denaturing effect of GdnCl were obtained. As shown in Figure 6A, both Cl- and Gdn+ caused the unfolding of Hb along with an increase of Inor. For Gdn+, Inor slightly increased by 1.25 at 1 mol L-1 Gdn+ and then leveled off. In the case of Cl-, Inor increased linearly with the concentrations of Cl-, and the rate of increment was relatively faster than that of Gdn+ when the concentrations of the denaturants were larger than 2 mol L-1. As for GdnCl, the increase of Inor was obviously larger than that in the same concentration of Gdn+ and Cl-. A plateau of Inor was found when GdnCl was 3 mol L-1. Assuming that the contributions of Gdn+ and Cl- to the GdnCl were equivalent and concentration-independent, the simulated unfolding curve induced by the contribution of Gdn+ and Cl- is shown in Figure 6A (dashed line). Obviously, the values of Inor induced by GdnCl in the real situation were much larger than the simulated ones, which reflected that the unfolding process of GdnCl involved the synchronizing contribution of Gdn+ and Cl-, and the portions of the contributions of Gdn+ and Cl- to the denaturing efficiency of GdnCl were concentration-dependent. Gdn+ decreased the hydrophobic chains of Hb, which led to the decrease of the tertiary and secondary structures of Hb and partially released heme irons. Meanwhile, Cl- split the tetrameric structure of Hb into dimers that increased the electroactive amount of Hb without alterations to the tertiary and secondary structures. The decrease of the quaternary and tertiary structures of Hb may accelerate the Cl- binding to Hb. Also, the split of the subunits of Hb may be helpful for the release of heme from the hydrophobic pocket. Therefore, Cl- and Gdn+ acted in synchronization during the unfolding of Hb by GdnCl and significantly increased the Ip of Hb. The ∆E of Hb was associated with the concentration of denaturants as well. As illustrated in Figure 6B, ∆E was decreased and leveled off when Gdn+ or GdnCl was 1 mol L-1. However, in the case of Cl-, the change of ∆E was slower, and a plateau was shown when Cl- was 2 mol L-1. The minimal ∆E values of Hb induced by Gdn+, GdnCl, and Cl- were all around 31 mV. The results indicated that all three denaturants can finally enhance the electron-transfer rate of entrapped Hb
to the same degree. However, Gdn+ and GdnCl likely improved the electron transfer more effectively. As discussed above, the unfolding of Hb by Gdn+ and Cl- proceeded in different ways. Gdn+ destroyed the tertiary and secondary structures of the proteins, while Cl- split the tetrameric Hb into dimers without alterations to the tertiary and secondary structures. The unfolding of Hb by Gdn+ exposed the hydrophobic core of Hb, which made Hb more flexible and effective to interact with the DDAB film.7 However, for the case of Cl-, the split of the tetrameric structure of Hb into dimers may lower the compactness of Hb molecules and thus enhance the mobility of Hb in the DDAB film. Both of these reasons were expected to enhance the electron-transfer rate of entrapped Hb to the same degree. The dependences of the Eo′ of Hb on the concentrations of the denaturants are shown in Figure 6C. In comparison with the Eo′ of native Hb, no obvious changes of Eo′ were found when Hb was unfolded by Gdn+. However, unfolding Hb by Cl- caused a negative shift of Eo′. The amount of the shifted Eo′ was the same as that caused by the same concentration of GdnCl. The results suggested that the Cl- in GdnCl was the main contributor to alter the Eo′ of Hb. Because Cl- can bind onto Hb specifically,64 the presence of Cl- in the denaturing solution not only affected the electrochemical behaviors of Hb in the DDAB film by changing Hb’s conformation but also provided an additional Donnan potential on the DDAB surface.68,69 Ideally, the slope of an ion-selective electrode for a univalent ion is 59 mV/log[Cl-] at 25 °C. When taking the Eo′ of GCE/ DDAB-Hb versus log[Denaturants], the Eo′ decreased linearly against the log[Denaturants] (Figure 6C). The slopes of the plots were -39.9, -42.2, and -12 mV L mol-1 for GdnCl, Cl-, and Gdn+, respectively. The bias of the slopes from the ideal value of 59 mV L mol-1 in the presence of salt may influence the electrochemistry of Hb by altering the interfacial Donnan potential and changing the Hb’s conformation.68 Conclusions A specific investigation was presented on the denaturing event induced by GdnCl on the DDAB-Hb film. The denaturing efficiency of GdnCl was contributed to by Gdn+ and Cl- in synchronization. The portions of such contributions were concentration-dependent. Meanwhile, both Gdn+ and Cl- can improve the mobility of unfolded Hb from different interactions, and Gdn+, Cl- or GdnCl can each enhance the reversibility of the redox reaction of Hb to the same degree. Cl- was the main contributor to the negative shift of the Eo′ of Hb incubated in GdnCl. The proposed method provided not only a strategy for investigating the contributions of the denaturing components of GdnCl to the unfolding of Hb but also possibilities of controlling the conformation of the protein manually.
7096
J. Phys. Chem. B, Vol. 114, No. 20, 2010
Acknowledgment. We gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 20805059, 20975117), the Natural Science Foundation of Guangdong Province (No. 7003714), the Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars of State Education Ministry, and the Ph.D. Programs Foundation of Ministry of Education of China (No.20070558010). Supporting Information Available: The evaluation of the leakage percent (L%) of Hb from GCE/DDAB-Hb. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Staniforth, R. A.; Bigotti, M. G.; Cutruzzol, F.; Allocatelli, C. T.; Brunori, M. Unfolding of apomyoglobin from Aplysia limacina: the effect of salt and pH on the cooperativity of folding. J. Mol. Biol. 1998, 275, 133–148. (2) Scheller, F. W.; Bistolas, N.; Liu, S.; Janchen, M.; Katterle, M.; Wollenberger, U. Thirty years of haemoglobin electrochemistry. AdV. Colloid Interface Sci. 2005, 116, 111–120. (3) Briehl, R. W. The relation between the oxygen equilibrium and aggregation of subunits in lamprey hemoglobin. J. Biol. Chem. 1963, 238, 2361–2366. (4) Simmons, D. A.; Wilson, D. J.; Lajoie, G. A.; Doherty-Kirby, A.; Konermann, L. Subunit disassembly and unfolding kinetics of hemoglobin studied by time-resolved electrospray mass spectrometry. Biochemistry 2004, 43, 14792–14801. (5) Bucci, E.; Fronticelli, C.; Gryczynski, Z.; Razynska, A.; Collins, J. H. Effect of intramolecular cross-links on the enthalpy and quaternary structure of the intermediates of oxygenation of human hemoglobin. Biochemistry 1993, 32, 3519–3526. (6) Johnson, C. R.; Angeletti, M.; Pucciarelli, S.; Freire, E. Oxygen binding to fallow-deer (Dama dama) hemoglobin: stepwise enthalpies at pH 7.4. Biophys. Chem. 1996, 59, 107–117. (7) Kristinsson, H. G. Acid-induced unfolding of flounder hemoglobin: evidence for a molten globular state with enhanced pro-oxidative activity. J. Agric. Food Chem. 2002, 50, 7669–7676. (8) Kristinsson, H. G.; Hultin, H. O. The effect of acid and alkali unfolding and subsequent refolding on the pro-oxidative activity of trout hemoglobin. J. Agric. Food Chem. 2004, 52, 5482–5490. (9) Kristinsson, H. G.; Hultin, H. O. Changes in trout hemoglobin conformations and solubility after exposure to acid and alkali pH. J. Agric. Food Chem. 2004, 52, 3633–3643. (10) Boys, B. L.; Konermann, L. Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system. J. Am. Soc. Mass Spectrom. 2007, 18, 8–16. (11) Boys, B. L.; Kuprowski, M. C.; Konermann, L. Symmetric behavior of hemoglobin R- and β-subunits during acid-induced denaturation observed by electrospray mass spectrometry. Biochemistry 2007, 46, 10675–10684. (12) Schay, G.; Smeller, L.; Tsuneshige, A.; Yonetani, T.; Fidy, J. Allosteric effectors influence the tetramer stability of both R- and T-states of hemoglobin A. J. Biol. Chem. 2006, 281, 25972–25983. (13) Peng, W.; Liu, X.; Zhang, W.; Li, G. An electrochemical investigation of effect of ATP on hemoglobin. Biophys. Chem. 2003, 106, 267–273. (14) Gottfried, D. S.; Juszczak, L. J.; Fataliev, N. A.; Acharya, A. S.; Hirsch, R. E.; Friedman, J. M. Probing the hemoglobin central cavity by direct quantification of effector binding using fluorescence lifetime methods. J. Biol. Chem. 1997, 272, 1571–1578. (15) Pin, S.; Royer, C. A.; Gratton, E.; Alpert, B.; Weber, G. Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques. Biochemistry 1990, 29, 9194–9202. (16) Abaturov, L.; Nosova, N.; Shlyapnikov, S.; Faizullin, D. Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: I. Effects of pH, temperature, and ligand binding. Mol. Biol. 2006, 40, 284–297. (17) Sugita, Y.; Nagai, M.; Yoneyama, Y. Circular dichroism of hemoglobin in relation to the structure surrounding the heme. J. Biol. Chem. 1971, 246, 383–388. (18) Artmann, G. M.; Burns, L.; Canaves, J. M.; Temiz-Artmann, A.; Schmid-Scho¨nbein, G. W.; Chien, S.; Maggakis-Kelemen, C. Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature. Eur. Biophys. J. 2004, 33, 490–496. (19) O’Brien, E. P.; Dima, R. I.; Brooks, B.; Thirumalai, D. Interactions between hydrophobic and ionic solutes in aqueous guanidinium chloride and urea solutions: lessons for protein denaturation mechanism. J. Am. Chem. Soc. 2007, 129, 7346–7353.
Mai et al. (20) Cacace, M. G.; Landau, E. M.; Ramsden, J. J. The Hofmeister series: salt and solvent effects on interfacial phenomena. Q. ReV. Biophys. 1997, 30, 241–277. (21) Scholtz, J. M. Guanidine hydrochloride unfolding of peptide helices: separation of denaturant and salt effects. Biochemistry 1996, 35, 7292– 7297. (22) Kawahara, K.; Kirshner, A. G.; Tanford, C. Dissociation of human CO-hemoglobin by urea, guanidine hydrochloride, and other reagents. Biochemistry 1965, 4, 1203–1213. (23) Bhattacharya, J.; GhoshMoulick, R.; Choudhuri, U.; Chakrabarty, P.; Bhattacharya, P. K.; Lahiri, P.; Chakraborti, B.; Dasgupta, A. K. Unfolding of hemoglobin variantssinsights from urea gradient gel electrophoresis photon correlation spectroscopy and zeta potential measurements. Anal. Chim. Acta 2004, 522, 207–214. (24) Sun, Y.; Liu, X.; Fan, C.; Zhang, W.; Li, G. Electrochemical investigation of the chloride effect on hemoglobin. Bioelectrochemistry 2004, 64, 23–27. (25) Li, X.; Zheng, W.; Zhang, L.; Yu, P.; Lin, Y.; Su, L.; Mao, L. Effective electrochemical method for investigation of hemoglobin unfolding based on the redox property of heme groups at glassy carbon electrodes. Anal. Chem. 2009, 81, 8557–8563. (26) Guo, L.; Qu, N. Chemical-induced unfolding of cofactor-free protein monitored by electrochemistry. Anal. Chem. 2006, 78, 6275–6278. (27) Armstrong, F. A.; Heering, H. A.; Hirst, J. Reaction of complex metalloproteins studied by protein-film voltammetry. Chem. Soc. ReV. 1997, 26, 169–179. (28) Takeoka, S. Developmental trend of artificial blood (artificial red blood cells). Jpn. Med. Assoc. J. 2005, 48, 135–139. (29) Ivanova, E. V.; Magner, E. Direct electron transfer of haemoglobin and myoglobin in methanol and ethanol at didodecyldimethylammonium bromide modified pyrolytic graphite electrodes. Electrochem. Commun. 2005, 7, 323–327. (30) Lu, Z.; Huang, Q.; Rusling, J. F. Films of hemoglobin and didodecyldimethylammonium bromide with enhanced electron transfer rates. J. Electroanal. Chem. 1997, 423, 59–66. (31) Mimica, D.; Zagal, J. H.; Bedioui, F. Electroreduction of nitrite by hemin, myoglobin and hemoglobin in surfactant films. J. Electroanal. Chem. 2001, 497, 106–113. (32) Boussaad, S.; Tao, N. J. Electron transfer and adsorption of myoglobin on self-assembled surfactant films: an electrochemical tappingmode AFM study. J. Am. Chem. Soc. 1999, 121, 4510–4515. (33) deGroot, M. T.; Merkx, M.; Koper, M. T. M. Heme release in myoglobin-DDAB films and its role in electrochemical NO reduction. J. Am. Chem. Soc. 2005, 127, 16224–16232. (34) Nassar, A. E. F.; Willis, W. S.; Rusling, J. F. Electron transfer from electrodes to myoglobin: facilitated in surfactant films and blocked by adsorbed biomacromolecules. Anal. Chem. 1995, 67, 2386–2392. (35) Rusling, J. F.; Nassar, A. E. F. Enhanced electron transfer for myoglobin in surfactant films on electrodes. J. Am. Chem. Soc. 1993, 115, 11891–11897. (36) Lei, C.; Wollenberger, U.; Bistolas, N.; Guiseppi-Elie, A.; Scheller, F. Electron transfer of hemoglobin at electrodes modified with colloidal clay nanoparticles. Anal. Bioanal. Chem. 2002, 372, 235–239. (37) Guto, P. M.; Rusling, J. F. Myoglobin retains iron heme and nearnative conformation in DDAB films prepared from pH 5 to 7 dispersions. Electrochem. Commun. 2006, 8, 455–459. (38) Rogers, P. H.; Arnone, A.; Mueser, T. C. Interface sliding as illustrated by the multiple quaternary structures of liganded hemoglobin. Biochemistry 2000, 39, 15353–15364. (39) Simmons, D. A.; Wilson, D. J.; Lajoie, G. A.; Doherty-Kirby, A.; Konermann, L. Subunit disassembly and unfolding kinetics of hemoglobin studied by time-resolved electrospray mass spectrometry. Biochemistry 2004, 43, 14792–14801. (40) Hargrove, M. S.; Krzywda, S.; Wilkinson, A. J.; Dou, Y.; IkedaSaito, M.; Olson, J. S. Stability of myoglobin: a model for the folding of heme proteins. Biochemistry 1994, 33, 11767–11775. (41) Gupta, R.; Yadav, S.; Ahmad, F. Protein stability: urea-induced versus guanidine-induced unfolding of metmyoglobin. Biochemistry 1996, 35, 11925–11930. (42) Shen, L.; Hermans, J., Jr. Kinetics of conformation change of spermwhale myoglobin. I. Folding and unfolding of metmyoglobin following pH jump. Biochemistry 1972, 11, 1836–1841. (43) Wang, S.; Chen, T.; Zhang, Z.; Shen, X.; Lu, Z.; Pang, D.; Wong, K. Direct electrochemistry and electrocatalysis of heme proteins entrapped in agarose hydrogel films in room-temperature ionic liquids. Langmuir 2005, 21, 9260–9266. (44) Hargrove, M. S.; Olson, J. S. The stability of holomyoglobin is determined by heme affinity. Biochemistry 1996, 35, 11310–11318. (45) Liu, C.; Bo, A.; Cheng, G.; Lin, X.; Dong, S. Characterization of the structural and functional changes of hemoglobin in dimethyl sulfoxide by spectroscopic techniques. Biochim. Biophys. Acta 1998, 1385, 53–60. (46) Eftink, M. R. The use of fluorescence methods to monitor unfolding transitions in proteins. Biophys. J. 1994, 66, 482–501.
Guanidine Hydrochloride and Hemoglobin Unfolding (47) Su kowska, A.; Roˇwnicka, J.; Bojko, B.; Po ycka, J.; Zubik-Skupien´, I.; Su kowski, W. Effect of guanidine hydrochloride on bovine serum albumin complex with antithyroid drugs: fluorescence study. J. Mol. Struct. 2004, 704, 291–295. (48) Nichols, N. M.; Matthews, K. S. P53 unfolding detected by CD but not by tryptophan fluorescence. Biochem. Biophys. Res. Commun. 2001, 288, 111–115. (49) Gryczynski, Z.; Lubkowski, J.; Bucci, E. Intrinsic fluorescence of hemoglobins and myoglobins. Methods Enzymol. 1997, 278, 538–569. (50) Hirsch, R. E. Front-face fluorescence spectroscopy of hemoglobins. Methods Enzymol. 1994, 232, 231–246. (51) Chiu, F.; Vasudevan, G.; Morris, A.; McDonald, M. J. Fluorescence studies of hyuman semi-β-hemoglobin assembly. Biochem. Biophys. Res. Commun. 1998, 242, 365–368. (52) Crespin, M. O.; Boys, B. L.; Konermann, L. The reconstitution of unfolded myoglobin with hemin dicyanide is not accelerated by fly-casting. FEBS Lett. 2005, 579, 271–274. (53) Haezebrouck, P.; Joniau, M.; Van Dael, H.; Hooke, S. D.; Woodruff, N. D.; Dobson, C. M. An Equilibrium Partially Folded State of Human Lysozyme at Low pH. J. Mol. Biol. 1995, 246, 382–387. (54) Hasserodt, U.; Vinograd, J. Dissociation of human carbonmonoxyhemoglobin at high pH. Proc. Natl. Acad. Sci. U.S.A. 1959, 45, 12–15. (55) Herskovits, T. T.; Cavanagh, S. M.; San George, R. C. Lightscattering investigations of the subunit dissociation of human hemoglobin A. Effects of various neutral salts. Biochemistry 1977, 16, 5795–5801. (56) Marcus, R. A.; Sutin, N. Electron transfers in chemistry and biology. Biochim. Biophys. Acta 1985, 811, 265–322. (57) Fedurco, M.; Augustynski, J.; Indiani, C.; Smulevich, G.; Antalik, M.; Bano, M.; Sedlak, E.; Glascock, M. C.; Dawson, J. H. Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions. J. Am. Chem. Soc. 2005, 127, 7638–7646. (58) Allis, J. W.; Steinhardt, J. Acid denaturation of carbonylhemoglobin. Protein unfolding without heme detachment. Biochemistry 1970, 9, 2286– 2293. (59) De Rosa, M. C.; Castagnola, M.; Bertonati, C.; Galtieri, A.; Giardina, B. From the Arctic to fetal life: physiological importance and
J. Phys. Chem. B, Vol. 114, No. 20, 2010 7097 structural basis of an ‘additional’ chloride-binding site in haemoglobin. Biochem. J. 2004, 380, 889–896. (60) Marta, M.; Patamia, M.; Colella, A.; Sacchi, S.; Pomponi, M.; Kovacs, K. M.; Lydersen, C.; Giardina, B. Anionic binding site and 2,3DPG effect in bovine hemoglobin. Biochemistry 1998, 37, 14024–14029. (61) Beretta, S.; Chirico, G.; Arosio, D.; Baldini, G. Role of ionic strength on hemoglobin interparticle interactions and subunit dissociation from light scattering. Macromolecules 1997, 30, 7849–7855. (62) Taboy, C. H.; Faulkner, K. M.; Kraiter, D.; Bonaventura, C.; Crumbliss, A. L. Concentration-dependent effects of anions on the anaerobic oxidation of hemoglobin and myoglobin. J. Biol. Chem. 2000, 275, 39048– 39054. (63) Bonaventura, C.; Tesh, S.; Faulkner, K. M.; Kraiter, D.; Crumbliss, A. L. Conformational fluctuations in deoxy hemoglobin revealed as a major contributor to anionic modulation of function through studies of the oxygenation and oxidation of hemoglobins A0 and deer lodge β2(NA2) HisfArg. Biochemistry 1998, 37, 496–506. (64) Sawyer, W. H.; Puckridge, J. The dissociation of proteins by chaotropic salts. J. Biol. Chem. 1973, 248, 8429–8433. (65) Chen, Y.; Jin, B.; Guo, L.; Yang, X.; Chen, W.; Gu, G.; Zheng, L.; Xia, X. Hemoglobin on phosphonic acid terminated self-assembled monolayers at a gold electrode: immobilization, direct electrochemistry, and electrocatalysis. Chem.sEur. J. 2008, 14, 10727–10734. (66) Perutz, M. F.; Wilkinson, A. J.; Paoli, M.; Dodson, G. G. The stereochemical mechanism of the cooperative effects in hemoglobin revisited. Annu. ReV. Biophys. Biomol. Struct. 1998, 27, 1–34. (67) Dawson, R. M. C.; et al. Data for Biochemical Research, 3rd ed.; Oxford University Press: New York, 1986; pp 322-323. (68) Nassar, A. E. F.; Rusling, J. F.; Kumosinski, T. F. Salt and pH effects on electrochemistry of myoglobin in thick films of a bilayer-forming surfactant. Biophys. Chem. 1997, 67, 107–116. (69) Naegeli, R.; Redepenning, J.; Anson, F. C. Influence of supporting electrolyte concentration and composition on formal potentials and entropies of redox couples incorporated in Nafion coatings on electrodes. J. Phys. Chem. 1986, 90, 6227–6232.
JP101082D
