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J. Phys. Chem. B 2010, 114, 7062–7070
Hemoglobin-Silver Interaction and Bioconjugate Formation: A Spectroscopic Study Mrityunjoy Mahato,† Prabir Pal,† Tapanendu Kamilya,†,‡ Ratan Sarkar,† Avinanda Chaudhuri,† and G. B. Talapatra†,* Department of Spectroscopy, Indian Association for the CultiVation of Science, JadaVpur, Kolkata-700 032, India, and Department of Physics, Narajole Raj College, Narajole, Paschim Medinipur-721 211, India ReceiVed: January 8, 2010; ReVised Manuscript ReceiVed: April 8, 2010
In this article, we report the results of the extent of interaction as well as the formation of a bioconjugate of human hemoglobin (Hb) with silver (Ag). The complexation process and conformational changes are characterized using different spectroscopic and microscopic techniques. The UV-vis study demonstrates the perturbation of the soret/heme band and generates conformational heterogeneity within the heme group in the presence of silver. A fluorescence study suggests that the Tryptophan (Trp) residues of Hb are in a more polar environment after conjugation. Initial fluorescence enhancement with addition of silver is due to metalenhanced fluorescence. Moreover, the fluorescence quenching after the formation of the Hb-Ag bioconjugate follows the modified Stern-Volmer (S-V) plot. The S-V plot along with the time-resolved fluorescence study indicates the presence of both static and dynamic types of quenching. In addition, the reduction potential values of the entities (Hb-heme, Ag+, and Trp) indicate the possible electron transfer. The secondary structure calculation from CD and FTIR spectra indicate R-helix to β-sheet conversion, and unfolding of Hb is also responsible for the bioconjugate formation. In addition, FE-SEM, phase contrast inverted microscopy (PCIM) images demonstrate the formation of the silver-protein bioconjugate. The overall data show that there is a change in the secondary as well as the tertiary structure of Hb after conjugation with silver. 1. Introduction Proteins are important biomolecules that play different roles in living beings. Their structure and function are strongly related. They possess selectivity and specificity, which arises from the confined nature of the microenvironment around their reaction center.1,2 The various specific and nonspecific interactions of ions with the residues of the proteins have important consequences to the biological function of the protein. In addition, protein folding is a hot topic in both biophysics and biochemistry that may be initiated due to the presence of ions in its environment.3,4 Moreover, proteins, especially blood and plasma proteins, are the major biomolecules in lives and the major target of many kinds of medicines,5 viruses,6 artificial drugs,7 metal ions,4,8 etc. Human adult hemoglobin (Hb) is a tetrameric blood protein, which contains four oxygen-binding hemes per monomer with uniquely bright red color.9 Silver has antiseptic and antimicrobial properties and is used in water recycling systems, in eye drops, and for many others medicinal uses.10,11 For the purpose of biomedical use of inorganic material (such as silver), the understanding of biocompatibility and toxicity is the key issue. Thus, the fundamental understanding of the conformational behavior of proteins in a protein-metallic conjugated system is of critical importance for the integration of biology with inorganic material. This can directly help in the advancement of bioinorganic conjugated systems, molecular diagnostics, therapeutics, molecular biology, material science, as well as bioengineering.12–18 Recently, a variety of methods have been developed to characterize the protein conformational changes.19–25 Among * To whom correspondence should be addressed. Tel.: +91-33-24734971. Fax: +91-33-24732805. E-mail:
[email protected]. † Indian Association for the Cultivation of Science. ‡ Narajole Raj College.
these, the most efficient spectroscopic methods include CD, FTIR, UV-vis absorption, as well as steady state and timeresolved emission spectroscopy.15,26 Since the changes in protein structure are crucial for their function, conformational change is the key thing to understand the structure-property relationship. In this study, we have tried to get a comprehensive understanding of the protein conformational behavior. The prime aim of this article is to study the interactions played in Hb with silver. In doing this, we have studied the formation of the Hb-Ag bioconjugate. The conformational change in Hb before and after conjugation is studied near the physiological pH (6.8) using a combination of spectroscopic techniques. 2. Experimental Section 2.1. Materials. Human hemoglobin, lyophilized and stored at 2-8 °C, was purchased from Sigma Chemical Co. and used as received. The silver nitrate, GR grade, was purchased from Merck. 2.2. Methods. 2.2 (A) Process of Substrate Cleaning. The glass and silicon substrate were cleaned and made hydrophilic by the process described elsewhere in our earlier literature.27 2.2 (B) Hemoglobin-SilWer Complex Formations. Stock solutions of Hb (1 mg/mL) and AgNO3 (10 mM) are prepared using deionized water having pH 6.8 and resistivity 18.2 MΩ cm (Milli-Q water, Millipore, USA) at room temperature (25 °C). Required amount of two solutions with/without further dilution are mixed together and kept aside for 30 min. Ag+ ions are formed and probably will attach to the electroactive sites of Hb, forming a protein-silver bioconjugate. In subsequent text, Ag+ concentration actually represents AgNO3 concentration. 2.2 (C) Spectroscopic Characterizations. The steady-state electronic UV-vis absorption and fluorescence emission spectra of pure Hb and Hb-Ag bioconjugate were recorded using a cuvette having a path length of 1 cm by means of an absorption
10.1021/jp100188s 2010 American Chemical Society Published on Web 04/30/2010
Hb-Ag Interaction and Bioconjugate Formation spectrophotometer (Shimadzu UV-vis 2401PC) and fluorescence spectrophotometer (Hitachi F-4500), respectively. The fluorescence lifetime measurements were done in a time correlation single photon counting (TCSPC) system. The samples were excited at 280 nm using a picoseconds diode laser (IBH Nanoled-07) in an IBH Fluorocube apparatus. The repetition rate is 1 MHz. The fluorescence decays were collected on a Hamamatsu MCP photomultiplier (C487802). The fluorescence decays were analyzed using IBH DAS6 software. The CD spectra of pure Hb and Hb-Ag bioconjugate were recorded at room temperature on a JASCO J-815 CD spectrometer (model No J-815-150 S) with 1 mm path length. In all the solutions, the concentration of Hb (CHb) was 0.1 mg/ mL. The concentrations of silver nitrate were (CAg) ) 0.1-10 mM. All spectra were collected in a triplicate from 190 to 250 nm to increase the signal-to-noise ratio of the CD spectra. The FTIR spectra of pure Hb and Hb-Ag bioconjugate on silicon wafers with CHb ) 0.1 mg/mL and CAg ) 10 mM were recorded by Magna-IR (model No 750 spectrometer, series II), Nicolet, USA. In all the cases, the data were averaged over 100 scans. The resolution of the instrument is 4 cm-1. 2.2 (D) Study of Morphology. The surface morphologies of all the drop-casted films on the ultrathin glass substrate of pure Hb and Hb-Ag conjugate were studied by a high-resolution field emission scanning electron microscope (FE-SEM, model No JEOL JSM-6700 F) and by a phase contrast inverted microscope (PCIM) (Motic model AE31 fitted with APEX MINI-LB2006C of Apex Instruments Co. India). The scale bar on PCIM images is drawn from image of the calibration slide at the same magnification. The concentrations of Hb and silver nitrate were 0.1 mg/mL and 10 mM, respectively. 2.2 (E) Secondary Structure Calculation of Hb Using CDPro and K2D2 Programs. The secondary structures of Hb such as R-helix, β-sheet, turn, unordered, etc. are calculated from the CD data using the SELCON3 (CD-pro) program. Also, the R-helix and β-sheet are calculated from CD data using the K2D2 program. For the SELCON3 program, we have chosen a reference set of 43 proteins since Hb is a soluble protein, and the CD data were in the range of 190-240 nm.28,29 3. Results and Discussion 3.1. UV-vis Absorption Spectroscopy. Figure 1 displays the recorded UV-vis absorption spectra of Hb and Hb-Ag conjugate with increasing concentration of CAg. The absorption spectra of pure Hb (Figure 1) show several electronic bands located at 279 (due to the phenyl group of Trp and tyrosine residues), 349 (ε band), 406 (heme or Soret band), 540, and 576 nm (oxy-band or Q-band as shown in inset A, Figure 1).30,31 The change in intensity of the Trp/tyrosine band, ε band, Soret band, as well as the Q-band with CAg implies that silver can access both the heme and the Trp residues. Boys et al.32 recently reported a similar type of observation. It is reported in the literature that the appearance of a strong Soret band (strongly allowed π-π* electronic transition) at 406 nm in UV-vis absorption spectra is due to the presence of Hb in its native form. This band originates from the heme group, embedded in a hydrophobic pocket formed by the protein’s backbone through appropriate folding.33,34 Study of the shape and position of the Soret absorbance band could provide important information about the possible unfolding and denaturation of protein during conjugation.30–35 Silver ion concentration dependent absorption spectra demonstrate a significant spectral change of the Soret band (inset B in Figure 1), in intensity, position, and shape. This indicates that the silver is
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Figure 1. UV-vis absorption spectra of (a) Hb-pure (CHb )0.1 mg/ mL) and (b-f) Hb-silver conjugate with CAg ) 0, 0.001, 0.01, 0.2, 0.5, and 1 mM, respectively. (g-h) Spectra of only silver nitrate with concentration of 1.0 and 0.1 mM. The inset A is a plot of absorption band (400-700 nm) of pure Hb with CHb ) 0.1 mg/mL (solid line) and 1.0 mg/mL (dashed line) and for CHb ) 1 mg/mL with CAg ) 40 mM silver (dotted line). The inset B is a plot of change in peak intensity (I/I0) and peak position (∆λmax) of the Soret band of Hb with various CAg.
directly involved in producing a disturbance of the structure and the exposure of the heme group to the aqueous medium.33 At low CAg (up to 0.2 mM) the Soret band intensity gets decreased and red-shifted. These spectral changes reflect a change in the chemical environment surrounding Hb that is not similar to its native state, suggesting that the heme group experiences a perturbation due to some kind of interaction of the heme group of the protein with the silver ions. In this regard, it is to be noted that according to A. Henglein the reduction potential of Ag+ is -1.8 V vs a natural hydrogen electrode (NHE), and this increases as the silver atom cluster increases up to 0.799 V for bulk silver.36 On the other hand, the reduction potential of Hb-heme is 0.106 V/NHE.37 Therefore, electron transfer may occur from Hb-heme to Ag+ to form Ag0. Thus, the oxidation of heme in the presence of silver perturbed its coordinate state, which is supposed to be responsible for the result. Any geometric distortion that changes a symmetry in the heme environment will cause a displacement of the nuclear coordinate, resulting in a shift in the Soret band.38,39 The out-of-plane distortions of the porphyrin are known to produce Soret red shifts,38,39 and changes are also expected from dielectric and electrostatic influences of the protein. However, with further increase of CAg, an increase in intensity with a shifting toward the UV region (blue-shifted) of the Soret band is noticed. The observation can be ascribed as the distortion of the iron atom in the heme-plane, which is caused along with the conjugation by disturbing the iron from heme-porphyrin moieties.40 Huang et al. showed the Soret band distortion as the denaturation in the basic environment.41 Zhou et al. showed that the {clay/Hb} film has a small blue shift and got broader and smaller at acidic environments. They explain it as the denaturation to some extent.42 With further increase of CAg (above 0.2 mM), the Soret band profile becomes broadened as well as shifts toward the UV region (blue-shifted). Regarding the broadening of the Soret band profile, Boffi et al. made an analysis.43 According to their analysis, the Soret band profile is described as the convolution of three terms
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A(ν) ) L(ν) X G(ν) X P(ν)
Mahato et al.
(1)
The first term L(ν) contains the usual Lorentzian line shape for an absorption band, accounting for the natural width of the electronic transition together with its coupling with high frequency modes. The coupling of the electronic transition with low frequency modes gives rise to a Gaussian line broadening, G(V). When the effects of coupling with both modes are taken into account, the spectral line shape will be the superposition of Voigtians (convolution of a Lorentzian with a Gaussian), which has explicit dependence on temperature. The last term, P(ν), represents the contributions to the spectral line width, originating solely from conformational heterogeneity of the heme group (i.e., on the coordinate of the iron atom in the heme-porphyrin system) due to the presence of conformational substates and different heme group environments. More precisely, according to Boffi et al., if the iron atom is located in the heme plane, the spectral transition at the Soret band maps Gaussian shape, and if it is out of the heme plane then the spectral transition maps the non-Gaussian Soret band and thus generates a conformational heterogeneity. The UV-vis data show the clear transition from near Gaussian to non-Gaussian shape of the Soret band, indicating the location of the iron atom moved from the in-plane toward the out-of-heme plane, and thus disturb the conformational homogeneity within the heme-porphyrin system. 3.2. Steady State and Time-Resolved Fluorescence Spectroscopy. Fluorescence spectroscopy is a useful tool to visualize information regarding the conformational changes of protein, degree of exposure of the fluorophore to the solvent, and the extent of its local mobility.19 We have studied the interaction of Hb with silver with varying CAg by examining the excitation (with λEM ) 340 nm) and emission (with λEX ) 280 nm) spectra of Trp as shown in Figure 2. The changes of excitation and emission intensity of Trp with CAg are represented in inset A of Figure 2. Generally, the fluorescence of protein arises from the three intrinsic fluorophores (Trp, tyrosine, and phenylalanine residues) present in the protein. The intrinsic fluorescence of many proteins is mainly contributed from Trp and tyrosine alone, due to the very low fluorescence quantum yield of phenylalanine. Moreover, the fluorescence of tyrosine is mostly quenched near an amino group, a carboxyl group, or a Trp.3 Hb has strong fluorescence, and there are three Trp units (R214Trp, β215Trp, β217Trp) in each R and β chain.44,45 When a fixed concentration of Hb (0.1 mg/mL) was titrated with different amounts of silver nitrate, a remarkable change in fluorescence intensity of Trp was observed (inset A of Figure 2). Furthermore, there is a slight red-shift of the maximum wavelength (λmax) of Trp fluorescence spectrum when the concentration of silver nitrate is increased, revealing the interaction with silver. The measurement of the emission maximum and the shift in its position due to changes of polarity around the fluorophore molecule are useful to understand the microenvironment of amino acid residues.46,47 Here the red-shifts suggest that the microenvironment of Trp residues has changed drastically, caused by solely the internal stark effect of Hb.48 The present data may be indicative of the feature that Trp is at or near the possible binding site. Also interestingly, a broad peak at ∼440-470 nm is observed along with the Trp emission upon excitation at 280 nm (as shown in inset B, Figure 2). This peak enhances in intensity along with the quenching of Trp at higher CAg. Recently, Weeks et al. reported a fluorescence band around this region (450-480 nm) in the case of heme containing human enzyme Cystathionin
Figure 2. Excitation (with λEM ) 340 nm) and emission (with λEX ) 280 nm) spectra of (a-i) pure Hb with CHb ) 0.1 mg/mL and Hb (CHb ) 0.1 mg/mL) with silver (CAg ) 0.01, 0.05, 0.1, 1, 4, 6, 10, and 25 mM), respectively. The inset A is a plot of change in Trp emission (b) and excitation (f) intensity (λ ∼ 340 nm) vs CAg. The inset B is a plot of emission spectra (λEX ) 280 nm) of Hb (CHb ) 1 mg/mL) (solid line) and for CHb ) 1 mg/mL with CAg ) 50 mM silver (dashed line).
beta Synthase (CBS).49 Although the origin of these emission peak/peaks is not clear to us, however, it may be due to the inner filter effect or reabsortion effect50 at 406 nm. Since the absorption band of heme is at 406 nm, it can absorb the emission energy and makes a valley around 406 nm. This effect is more prominent at higher concentration of Hb (as shown inset B, Figure 2) since the number of heme is more. For this reason, the tail part of the Trp emission is separated and seems to be another band around 440-470 nm. We have monitored the fluorescence of Trp with varying concentration of silver nitrate. At low concentration, an enhancement, and subsequently at higher concentration a decrement, of fluorescence intensity is observed. In this regard, the fluorescence intensity enhancement and saturation is found within CAg ) 1 mM and can be ascribed as the saturation of metal ion attachment that may shield the excited state from nonradiative decay processes.51 We have plotted the F0/F with CAg (as shown in Figure 3A) where F0 and F are the relative fluorescence intensity in the absence and in the presence of silver nitrate, respectively. The plot shows a nonlinear shape where F0/F decreases for CAg e 1 mM and increases for CAg > 1 mM. For most of the cases in the literature,52 linear quenching of fluorescence intensity in the presence of a quencher is found, even in the case of Hb in the presence of an antioxidant quencher.53 However, in the present case, the linear Stern-Volmer (S-V) plot is not expected as there are several differently lived tryptophans (discussed below) contributing to the total fluorescence. G. Patonay et al.54 used a modified form of the S-V equation to describe the fluorescence quenching and enhancement assuming the changes of the quantum yield (decrease in the case of quenching and increase in the case of enhancement) of the complex. Besides this, a few other nonlinear type S-V equations exist in the literature in the case of a biologically relevant metal complex55 and some antioxidants (vitamin E).56 However, in those cases, the nonlinearity is not much like ours, where a deep valley is present at CAg ) 1 mM, which is ascribed in the literature as the excited state reaching to the statistical limit.57,58 We failed to fit the F0/F versus CAg nonlinear data by various nonlinear S-V equations.52 In our case, the initial fluorescence
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Figure 4. Fluorescence decay curves of Hb in aqueous solution (CHb ) 0.1 mg/mL) associated with the lamp profile. Bottom: residual plot of fitting of eq 3 of Hb. The χ2 and DW parameter values are 1.02 and 1.87, respectively. The inset shows the plot of the reciprocal of the longest decay time (τ3) with CAg. Figure 3. Panel A represents the F0/F plot with the CAg. Panel B represents the modified Stern-Volmer plot (F0′/F) of the Hb complex in the presence of different CAg ′ .
intensity enhancement and slight red shifting within CAg ) 1 mM may be ascribed as metal enhanced fluorescence described by Lakowicz et al.,51 where the saturation of metal ion attachment occurs. Here, the metal ion increases the local incident field on the fluorophore. Moreover, the reduction potentials of Trp in the excited and ground states are 3.08 and -0.88 V with respect to NHE.59 In addition, the reduction potential of Ag+ is -1.8 V/NHE, and this increases as the silver atom cluster increases up to 0.799 V for bulk silver.36 Therefore, electron transfer may occur from ground and excited states of Trp to Ag+ to give atomic silver. Also, efficient electron transfer from excited Trp to Ag+ as well as to atomic silver or silver cluster may occur. Therefore, the electron transfer phenomenon that arises due to these reduction potentials may be responsible for such a spectral change in the emission spectra of the Trp ensemble. We have defined reduced ′ ) by subtracting with the value of concentration of silver (CAg CAg () 1 mM) where the fluorescence enhancement is at maximum. With addition of silver up to CAg ) 40 mM, the quenching of the excited state of the complex Hb molecule occurred. We have tried to fit the data (as shown in Figure 3B) with the modifed S-V equation in terms of a second-order polynomial of quencher concentration [Q] as represented in eq 2.60
F0′ /F ) (1 + KD[Q])(1 + KS[Q])
(2)
Here, F0′ is the reduced fluorescence intensity at CAg ′ ) 0, and F is the relative fluorescence intensity at CAg ′ > 0. KD and KS are the dynamic and static quenching constant. Figure 3B shows
a reasonably good fit with R2 value of 0.998. The values of KD and KS are 125 ( 2.1 and 20 ( 1.1 M-1, respectively, as obtained from the fitting results of the S-V plot. The observed nonlinear quenching (the upward curvature in Figure 3B) predicts that both the dynamic and static types of quenching are present in the same fluorophore.60 The first factor represents the dynamic type of quenching resulting from the encounter of the quencher with the excited state of the fluorophore, and the second factor is for static type of quenching resulting from the formation of a complex between the quencher and the fluorophore. Here we measure the Trp fluorescence decay average lifetime and other parameters of the Hb with λEX ) 280 nm for different samples. Figure 4 shows the representative Trp fluorescence decay of pure Hb with lamp profile. We have fitted the decay profiles using the following triple exponential function (eq 3) to fit the data.51 3
f(t) )
∑ Bi exp(-t/τi)
(3)
i)1
Bi′s and τi′s are the relative contributions and the lifetimes of the different components to the total decay. The residual plot as well as χ2 value indicate that these fluorescence decay profiles are triple-exponential in nature. The Trp fluorescence decay in Hb is reported to be triple-exponential.61 In addition, the relative contribution of each component to the total steady state fluorescence intensity can be defined as follows (eq 4)21,52 3
fi ) Biτi /
∑ Bjτj j)1
(4)
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TABLE 1: Fitting Parameters of Fluorescence Lifetime Measurements of Pure Hb (CHb ) 0.1 mg/mL) and Hb-Silver Conjugates at Different Silver Concentrations sample
CAg (mM)
B1
B2
B3
τ1 (ns)
τ2 (ns)
τ3 (ns)
f1
f2
f3
(ns)
pure Hb Hb-Ag Hb-Ag Hb-Ag
0.0 0.1 1.0 10.0
0.76 0.64 0.66 0.57
0.16 0.19 0.19 0.32
0.08 0.17 0.15 0.11
0.16 0.08 0.08 0.03
1.43 1.51 1.48 1.37
4.18 4.59 4.38 3.78
0.18 0.05 0.05 0.02
0.32 0.25 0.28 0.51
0.50 0.70 0.67 0.47
2.58 3.62 3.35 2.48
The values of f1, f2, and f3 are shown in Table 1. From these values, we can infer that there is a sufficient contribution of each component to the total fluorescence intensity. The average lifetime of fluorescence decay is calculated using the following formula (eq 5). All the values are reported in the Table 1. 3
〈τ〉 )
∑ i)1
3
Biτi2 /
∑ Biτi
(5)
i)1
The average lifetime of Hb is reported in the range 1.8-5.4 ns.62,63 We found the decay lifetime of Hb (CHb ) 0.1 mg/mL) in pure to be 2.58 ns which is well relevant with the previous literature. Here the lifetime data show that the average lifetime of Trp increases at low concentration of silver (0.1-1 mM) and decreases at high concentration of silver (10 mM). It is important to note that the observed triple exponential decay reflects the presence of several tryptophans in the protein, with different decay times, resulting from different environments. Thus, the decay times as such have no precise physical meaning and may be viewed as mere fitting parameters. However, the electron transfer between Trp and silver may accompany the lifetime change in the Trp fluorescence decay. The lifetime measurements along with the data shown in Figure 3B demonstrate the larger value of F0′/F than 〈τ0〉/〈τ〉 which again indicates static quenching at higher CAg.64 According to Noronha et al., the pre-exponential coefficient data in time-resolved fluorescence spectroscopic measurements can be applied to probe local conformational change in protein.65,66 The fluorescence quantum yield of the tryptophan ensemble increases when CAg is increased in the low CAg region (up to 1 mM). Table 1 shows that such increase is due to the decrease of the pre-exponential coefficient of the shortest decay time (B1), which in turn indicates that some heavily quenched Trp becomes less quenched at CAg ) 0.1 mM. This necessarily means either the suppression of a quenching site in the protein or the increase in the distance between Trp and the quencher site induced by the protein conformational change.65 Here also we have plotted the reciprocal of the longest decay time (τ3) with CAg, which can be assigned to be the unquenched Trp above CAg ) 0.1 mM (shown in inset of Figure 4). The slope of the linearly fitted plot gives the quenching rate constant value of 4.5 × 109 M-1 s-1.67 Here, for the Trp quenching to be close to diffusion control, the quencher should rapidly diffuse and interact with the Trp during the lifetime of the excited state. This can be easily recognized by the parallel drop of the fluorescence lifetime and the quantum yield of Trp with the quencher.68 Our fluorescence lifetime and the quantum yield data show the almost parallel drop indicating the Trp quenching to be diffusion limited. Another signature of this phenomenon is that the quenching rate constant should be comparable with the diffusion coefficient of the quencher within the protein. The obtained quenching rate constant is consistent with the quenching of Trp by other quenchers like oxygen and acrylamide.67
Figure 5. (A) FE-SEM images of drop-casted Hb-silver bioconjugate structures. Insets of the panel are in higher magnification. (B) Phase contrast inverted microscopic image in micrometer scale.
3.3. Study of Morphology. We have studied the Hb-silver conjugate by a high-resolution field emission scanning electron microscope (FE-SEM) and phase contrast inverted microscopy (shown in Figure 5). The sample of Hb (CHb ) 0.1 mg/mL) as conjugated with 10 mM silver is casted on a hydrophilic glass slide and allowed to dry in a desiccator. From FE-SEM images, it is seen to form globular as well as nodular structures with dimension of globular structures in a few micrometers (typically 5-10 µm). Thus, from microscopic data it can be stated that the Hb-silver can form bioconjugate structures, by the metal and protein surface. The bioconjugate structure is prominent from the FE-SEM and the PCIM images (shown in panel B, Figure 5). The nodular structures may originate due to the fusion of two or more globular structures at proximity. Therefore, from microscopic images coupled with UV-vis and fluorescence studies it can be stated that the easy access of silver into the cavities of the Hb is by the diffusion process, as occurs in the biomineralization processes.69 Here the formation of bioconjugate structure may also be due to some kind of self-assembly70 on the substrate as seen from FESEM and/or may be formed in solution also.71 3.4. CD Spectroscopy. CD spectroscopy was used to monitor the changes of conformation of the Hb-silver conjugate system in comparison to Hb pure (shown in Figure 6).15 The far-UV region (190-250 nm) of the CD spectrum in solution, corresponding to peptide bond absorption, was employed to study
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CAg (mM)
R
β
T
U
pure Hb Hb-Ag Hb-Ag Hb-Ag
0.0 0.1 1.0 10.0
80.14/84.70 72.79/68.40 75.86/65.99 70.36/68.41
1.75/0.57 4.39/1.84 1.12/2.63 1.14/1.84
4.84 10.34 10.55 11.37
13.27 12.47 12.48 16.91
a R, β, T, and U represent the R-helix, β-sheet, turn, unordered conformational elements in the secondary structure of protein, respectively.
Figure 6. Far UV-CD spectra of (a) Hb pure (CHb ) 0.1 mg/mL) and (b-d) Hb-silver conjugates with different silver concentrations. The inset represents the monomer structure of Hb collected from the Protein Data Bank.
the secondary structure of Hb. The CD spectra were done in solution and not in cast film as CD spectra of thin film are very weak and may not be quantitative regarding the determination of secondary structure of protein. It is known that when a polypeptide chain existed in the R-helical conformation, there are two negative minima at ∼209 and ∼222 nm, and a positive band at ∼190 nm is observed.70,72 The 209 nm band corresponds to π-π* transition of the R-helix, whereas the 222 nm band corresponds to π-π* transition for both the R-helix and random coil.73 On the other hand, for the existence of a β-sheet, a positive and a negative band appeared at 190 and 215 nm, respectively.70,72 The intensity of the three CD bands reflects the amount of helicity in the protein. Since the structure of Hb is predominately R-helical, we measured the CD spectra of different systems to estimate the extent of conformational change of the secondary structure.73 The intensity change of these absorption peaks clearly shows the change of secondary structure to some extent. The CD spectra indicate conformational changes up to CAg ) 0.1 mM with no further changes, which can be explained by the heme oxidation resulting from the electron transfer. The transition of the secondary structure can be evaluated by R-helix content from CD result.74 However, the CD method is not able to provide information on the three-dimensional structure of the protein.75 It is to be mentioned that the change in absorbance of the Soret band arises due to the interaction between the heme moiety and the well-defined tertiary structure. Hence, it has been used to monitor protein unfolding at the tertiary structure level.76 From our CD results it is seen that the peaks located at ∼193, ∼209, and ∼223 nm were decreased in the Hb-silver conjugate system indicating loss of R-helix and unfolding of Hb. However, there is no signature about the β-sheet as seen from the CD spectra. In this regard, we have calculated the secondary structural elements from CD data at pH ) 6.8 using SELCON3 as well as the K2D2 program, and they are represented in Table 2. It is seen that the amount of R-helix of pure Hb in both the programs is ∼80%, which is more or less close to the earlier study.29 Here the amount of R-helix decreases and β-sheet increases (though it is not in regular manner) along with the conjugation of silver. The change of R-helix and β-sheet as obtained from the SELCON3 and K2D2 programs is in the same trend at least qualitatively with the FTIR data (as discussed in
the next section). The secondary structures are also calculated using the deconvolution technique in FTIR spectroscopy. 3.5. FTIR Study. FTIR spectroscopy is one of the valuable tools to monitor the conformational changes at the secondary structure level in proteins that span different components such as R-helix, β-sheet, turns or coil, and intra- and intermolecular aggregates.74–82 The infrared spectra of protein is mainly comprised of amide-I (∼1600-1700 cm-1) and amide-II (∼1500-1600 cm-1). The amide-I band resulted from carbonyl stretching vibrations of the peptide backbone that depends on the strength of the hydrogen bond and the interactions between the amide units, and amide-II is due to the combination of N-H in-plane bending and C-N stretching vibrations of peptide groups.80 A critical and important step in the FTIR study of proteins is the assignment of the different components of secondary structure in the amide-I band. A rough assignment of the amide-I band as suggested in most protein studies is as follows: 1651-1658 cm-1 (R-helix); 1618-1642 cm-1 (βsheets); 1666-1688 cm-1 (turns); 1618-1623 cm-1 (intermolecular aggregates); and 1683-1689 cm-1 band (intramolecular aggregates), respectively.14,79–83 The deconvolution of the normalized amide-I band into components is useful to study the conformational status of the protein/enzyme.82 Generally, Lorentzian, Gaussian, and/or a mixture of these functions are used to describe the components.84 However, in our experiment, we have applied Gaussian function rather than Lorentzian and/or a mixture of these functions, and the reason behind it is described in our earlier literature.85 A multiple peak fitting technique with the Gaussian profile was employed to fit the normalized amide-I band which allows one to identify different components and in particular to determine the corresponding peak frequencies. The percentage area of the deconvoluted peaks gives the relative amount of the components. In the present work, the maximum number of the components which can be identified in the deconvoluted amide-I band is 5 for meaningful fitting. Unless using less or a larger number of peaks, the fitting gives neither satisfactory results nor improves R2 values, and the deconvoluted peaks do not have definite positions. Since the secondary structures are stabilized by hydrogen bonds between amide CdO and N-H groups, the position of the components depends on the patterns and the strength of the hydrogen bonds. For stronger hydrogen bonding, the vibration is observed at higher wavenumber. The β-sheet structures have the strongest hydrogen bonds and exhibit an amide-I maximum at much lower frequency than R-helices.86 In the case of inter- (A1 component) and intramolecular (A2 component) aggregates, the hydrogen bonds formed between CdO and N-H groups of any polypeptide strands with which they came into contact. The consequence is that many hydrogen bonds are formed between polypeptide chains in a neighboring protein molecule, forming an aggregate stabilized by very strong intermolecular hydrogen bonds.80 The
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Figure 7. FT-IR spectra of Hb amide bands: (A) Hb pure (CHb ) 0.1 mg/mL), (B) normalized amide-I band of pure Hb (CHb ) 0.1 mg/mL), (C) Hb (CHb ) 0.1 mg/mL) with silver (CAg ) 5 mM), and (D) normalized amide-I band of Hb (CHb ) 0.1 mg/mL) with silver (CAg ) 5 mM), respectively.
1666 cm-1 component can be ascribed to the vibration modes originated by β-turns in the structures (T-component).86–88 The panels A and C of Figure 7 show the original FTIR spectra of native Hb and its bioconjugate at pH ) 6.8, where an intense band in the amide-I region centered at 1653 cm-1 could be observed indicating that Hb is in an R-helix-rich conformation. As compared with that for the native protein, this band in the bioconjugate showed obvious changes in both shape and peak position (1653-1649.8 cm-1), which suggested the occurrence of changes in the secondary structure of the Hb in the conjugates. Detailed information about this conformational change was then obtained by deconvoluting its superposed peaks. The panels B and D of Figure 7 represent the fitting curves of the normalized amide-I peak of Hb pure and Hb conjugated, respectively. The fitting results are represented in Table 3. For all the fittings, the square of the correlation coefficient R2 is found to be 0.999. The summary of the fitting result is represented by a bar diagram in Figure 8. The β/R ratio for different films is presented in the inset of Figure 8. Figure 8 shows that the Hb-silver conjugate is composed of a large amount of β-component (49.33%) and of a small amount of R-component (7.57%). Since the β/R ratio (6.50) is much greater than unity, some R-helix obviously can be converted into the β-sheet when Hb is conjugated with silver (which is
TABLE 3: Fitting Parameters Using Multipeak Fitting Technique of the Amide-I Band for Hb and Hb-Silver Conjugatea area (%)
position (cm-1)
fwhm (cm-1)
conformers
A
B
A
B
A
B
A1 β R T A2
13.50 24.87 43.95 14.50 03.18
08.54 49.33 07.57 28.20 06.36
1622.5 1644.6 1657.5 1669.4 1689.1
1618.7 1642.6 1656.0 1670.5 1687.5
22.26 22.79 33.93 19.59 14.52
18.42 24.11 11.31 18.21 12.18
a A represents the Hb-pure; B represents Hb-silver conjugate. Area (%) ) 100 represents the total area under curve; fwhm represents the full width at half-maximum of a peak.
also seen in solution phase CD spectra). In this context, it is to be stated that there is a discrepancy in the result of secondary structure calculation from CD and FTIR spectra. However, the trend of their change is the same in both cases. The amount of R-helix is larger as obtained from CD spectra than the FTIR results. This is due to the aqueous medium in solution phase CD spectra, whereas the aqueous medium is absent in thinfilm FTIR spectra; rather, the hydrophobic air phase is present there. Also FTIR results in, for pure Hb, the amount of intermolecular aggregate A1 ) 13.50% and intramolecular
Hb-Ag Interaction and Bioconjugate Formation
J. Phys. Chem. B, Vol. 114, No. 20, 2010 7069 Acknowledgment. We thank DST, Government of India (Project No.-SR/S2/CMP-0051/2006), for partial financial support. Mrityunjoy Mahato thanks CSIR, Government of India, for providing the CSIR-NET fellowship. References and Notes
Figure 8. Bar diagrams of different conformations (obtained from fitted data of FTIR deconvolution) of pure Hb and Hb-silver conjugate. Inset shows the variation of β/R for conjugate formation.
aggregate A2 ) 3.18%, but in the case of conjugate, A2 ) 6.36% and A1 ) 8.54%. From these data, it is seen that A1 is decreasing and A2 is increasing, which indicates that intramolecular aggregates dominate over intermolecular aggregates leading to greater unfolding and greater binding. 4. Conclusion In conclusion, in this work we have studied the conformational changes of Hb before and after conjugation with silver by a combination of spectroscopic techniques. It shows that Hb in the bioconjugate underwent conformational changes on both the secondary and the tertiary structure levels. The UV-vis absorption study shows that broadening and shifting of the Soret band, which demonstrates the generation of the conformational heterogeneity of heme in porphyrin moieties of Hb in a bioconjugate. The red shift of Trp fluorescence indicates the internal stark effect occurred within Hb, and the change in intensity of Trp emission indicates the microenvironment around Trp residues has a drastic change in polarity as well as hydrophobicity. Moreover, the fluorescence quenching after the formation of a Hb-Ag bioconjugate follows the modified Stern-Volmer (S-V) plot. The S-V plot along with the timeresolved fluorescence study indicates the presence of both static and dynamic types of quenching. In addition, the reduction potential values of the entities (Hb-heme, Ag+, and Trp) indicate that the possible electron transfer may account for the spectral change in absorption, emission, and CD spectroscopy. The FE-SEM study shows the bioconjugate structures. The secondary structure calculation from both CD and FTIR spectra indicates the conformational changes in Hb. Also, the deconvolution of a normalized amide-I band indicates that R-helix to β-sheet conversion is larger in conjugate than in pure Hb, and intramolecular aggregates (arising from larger hydrogen bonding between the carbonyl bonds) dominate over intermolecular aggregates, leading to a greater unfolding of Hb in bioconjugate. On the other hand, the FTIR data coupled with CD and UV-vis absorption data show the change in secondary structure as well as tertiary structure. Finally, these studies suggest that the interaction of silver with Hb causes side effects due to the R-helix to β-sheet transition and protein aggregation. The biological and pharmacological significance of this work is evident since the Hb molecule uses manufacturing in multiple drugs and medicines.
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