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Department of Geology and Geophysics, University of Wyoming, Laramie, Wyoming 82071, USA
* E-mail address of corresponding author: Anidhi{at}uwyo.edu
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Abstract |
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 TOP Abstract INTRODUCTIONEXPERIMENTAL METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: Cytochrome c • Fe Oxide • Hematite • Protein • Sorption
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INTRODUCTION |
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TOPAbstractINTRODUCTIONEXPERIMENTAL METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Direct electron transfer between redox-active proteins (e.g. cytochromes) and electrodes is affected by sorption (Lojou and Bianco, 2000). Hence, characterizing sorption of cytochromes on mineral surfaces is useful for understanding electrochemical behavior essential to their application in biosensor and bioanalytical systems (Chen et al., 2002). For example, it has been shown that c-type cytochromes change conformation upon adsorption so as to affect electron transfer to mineral (kaolinite, mont-morillonite, goethite) -modified electrode surfaces (Sallez et al., 2000). Slab optical wave guide spectroscopy (Santos et al., 2003), quartz microbalance techniques (Lojou and Bianco, 2003) and electrochemical surface-enhanced resonance Raman spectroscopy (Dick et al., 2000) have been used to study sorption and related conformation changes of cytochromes on surfaces. In addition, UV-visible spectroscopy was used in situ to indicate conformational alteration of myoglobin adsorbed on polymethylsiloxane (Anderson and Robertson, 1995). We elected to use wet chemical techniques (with UV-visible spectroscopy as a conformational probe) to characterize sorption of Hcc to hematite. Our objectives were (1) to characterize sorption of Hcc on hematite as a function of pH, time of sorption, and ionic strength; (2) to investigate the direct electrochemistry of Hcc on hematite; and (3) to monitor for possible conformation change using UV-visible spectroscopy and electrochemistry. Hematite was chosen because it is the only nominally ferric oxide that can be a sufficiently good semiconductor to use as an electrode in electrochemical experiments with cytochromes. Hcc has been extensively studied and characterized, which provides us with as complete a comparative background for a cytochrome as exists; for example, conformational states and structural changes have been extensively correlated with UV-visible absorption bands, circular dichroism bands, and redox potentials so that changes in these properties can be readily interpreted in terms of protein folding. This degree of structural information is not available for most cytochromes (such as those utilized by dissimilatory Fe-reducing bacteria), and Hcc thus provides us with a very useful and indeed necessary first step in the characterization of other proteins interacting with oxide mineral surfaces.
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EXPERIMENTAL METHODS |
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TOPAbstractINTRODUCTIONEXPERIMENTAL METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Aqueous experiments
Sorption experiments for hematite suspensions were conducted in 30 mL polycarbonate centrifuge tubes following a procedure described by Khare et al.(2004). Protein retention to these tubes is minimal as per manufacturer’s recommendation. All samples had a suspended solids concentration of 1.50 g kg–1, constant ionic strength of 0.01 M KCl and total sample mass of 30 ± 0.01 g. Aqueous solutions for sorption experiments (KCl, HCl, KOH all at 0.01 M and Hcc at 0.0001 M) were prepared using analytical-grade reagents and degassed (heated and N2 purged) deionized water. Stock hematite suspension was shaken on a reciprocating shaker at 1 Hz for at least 30 min before use. 0.478 g of hematite was weighed while vigorously stirring a stock suspension on a magnetic stirrer, and brought to ~30% of the final mass with 0.01 M KCl. 5000 µL of 0.0001 M Hcc solution were added slowly to each vigorously stirred sample. The pH was adjusted using 0.01 M HCl or 0.01 M KOH, and each sample was brought to its final mass. The sample headspace was flushed with N2 gas. To characterize the sorption kinetics, samples were shaken for 1, 5, 20, 40, 60, 90 and 120 h on a reciprocating floor shaker at 0.5 Hz and 21°C. The pH varied by an average of 0.2 pH units and was not adjusted during equilibration. After equilibration, samples were centrifuged at ~6000 g for 10 min, and the supernatant solutions were decanted. The pH was measured in a portion of the supernatant solution before filtering and the remaining solutions were filtered using 0.2 µm polycarbonate filter membranes. Dissolved Hcc was measured in the supernatant solutions using UV-visible absorbance of the Soret band at 408 nm. The concentration of Hcc sorbed on hematite was determined as the difference between total added Hcc and Hcc measured in supernatant solutions. To determinine the pH dependence of Hcc sorption, the pH was adjusted from 1.7 to 12.3. A 0.1 M HCl or 0.1 M KOH solution was used in addition to 0.01 M HCl or 0.01 M KOH to adjust the pH to 1.7, 2.0 or to 11.0, 12.0 or 12.3, respectively.
To determine the ionic strength dependence of Hcc sorption, the background KCl concentration was varied from 0.1 to 0.5 M and the supernatant liquids were analyzed against standards of similar molarity. In addition, HCl and KOH of similar molarity were used for pH adjustment to achieve the desired ionic strength. For both the pH and ionic strength sorption dependence, the samples were equilibrated for 20 h and the pH was not adjusted during equilibration.
Model calculations
Hcc charge as a function of pH was calculated using an algorithm developed by Tabb (http://fields.scripps.edu/DTASelect/20010710-pI-Algorithm.pdf) that predicts the isoelectric point at pH 10, in agreement with Theorell and Akesson (1941). The surface charge of hematite as a function of pH was calculated using MICROQL and data from Stumm and Morgan (1996). As an approximation for van der Waals attraction, a Hamaker constant of 10–20 J was used with a simple block model (references in the caption of Figure 1b
), giving an attractive force of ~0.05 nN. Hamaker constants for virus-hematite interaction through water (Vilker et al., 1981), if used as an approximation for protein-hematite interaction, give slightly smaller attractive forces. To estimate electrostatic forces between a hematite surface and Hcc, we used a point charge corresponding to the charge of an Hcc molecule positioned 1.0 nm above a 100 nm2 area with a surface charge density calculated for the hematite surface using MICROQL. Hcc and hematite surface charge density for each pH were used in the pH-dependent force calculation; a dielectric constant of 10 was used as an approximation for interfacial structured water. Hexagonal close packing of 3.4 nm spheres (the size of Hcc in native globular conformation) was used to calculate the number of monolayers of Hcc sorbed on the surface of hematite. If Hcc unfolds and changes shape upon adsorption, however, this assumption may no longer apply. We offer the number of monolayers as one way of assessing the relative amount of adsorbed protein; it should be thought of as a monolayer equivalent for hexagonally close-packed native globular protein based simply on the amount of available surface area and the amount of sorbed protein.
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Electrochemical characterization of Hcc using hematite electrodes
Cyclic voltammograms (CV) were obtained using a hematite (single crystal) working electrode, Pt wire counter electrode and an oxidized Ag wire pseudo- reference calibrated against an Ag/AgCl reference electrode. Clean hematite crystals with electron donor impurity (Sn and Ti) concentration of 2 x 10–3 atom.% were suspended in 0.1 M KCl with or without added Hcc. The CVs were collected using a EG&G 263 A potentiostat controlled using a PC.
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RESULTS AND DISCUSSION |
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TOPAbstractINTRODUCTIONEXPERIMENTAL METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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. Sorption was minimal at pH 4, increased slightly at pH 6.8, and showed a pronounced increase at pH 9.5 (Figure 1a). These isotherms differ from those of other proteins (human plasma albumin, HPA, and bovine pancreas ribonuclease, Rnase) sorbing to hematite in that they do not show well expressed plateau values (Koutsoukos et al., 1983). Our adsorption envelope results showed that Hcc sorption depended strongly on pH, with most sorption occurring between pH 8 and 10 (Figure 1b). The isoelectric point of Hcc is ~10 (e.g. Theorell and Akesson, 1941) while the point of zero charge for hematite is ~pH 8.5 (e.g. Parks and deBruyn, 1962); hence, the narrow pH range of sorption (at pH values for which we would expect opposite charge on the Hcc and hematite) suggests a primarily electrostatic attraction. We estimated electrostatic and van der Waals forces (see Methods) between hematite and Hcc, and the resulting pH-dependent net forces are plotted in Figure 1b. The peak in Hcc sorption coincides well with the calculated region of net attractive force (Figure 1b).
Hcc is a folded protein that changes conformation with pH (Boffi et al., 2001), affecting optical absorption. At pH<3.0, intramolecular electrostatic repulsion leads to unfolding and the exposure of hydrophobic portions of the molecule to solution (which may affect adsorption) and is reflected in a blue shift in the Soret band position (Figure 2a). In the pH 3–7 range there is no shift in Soret band position, but from pH 7 to 12 there is a small but systematic blue shift of 3 nm (Figure 2a). Thus, there may be conformational changes taking place in the pH range of peak adsorption (pH 8 to 10) that could affect the adsorption reaction. Nevertheless, the clear correspondence between peak adsorption and electro- static attraction (Figure 1b) suggests that possible conformational changes (for example) in the hydrophobic vs. hydrophilic nature of Hcc are not contributing in an obvious way to adsorption.
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Adsorption of Hcc on quartz and other mineral oxide surfaces has previously been attributed mainly to electrostatic interactions (Santos et al., 2003; Sallez et al., 2000). The operation of Hcc in its physiological setting (interaction with biological membranes and with cytochrome c oxidase) is thought to be primarily electrostatically controlled (e.g. Pinheiro 1994; Millett and Durham, 1996). Pinheiro (1994) showed that two different conformational states are induced in Hcc upon complexation with negatively charged surfaces. The conformation of state I is similar to that of Hcc in solution where native low-spin Fe configuration of heme is preserved, but in state II the heme groove opens leading to thermal and potential dependent equilibrium between a 5-coordinated high-spin and a 6-coordinated low-spin configuration. The equilibrium between states I and II as well as the conformation equilibrium in state II appear to be controlled by the electrostatic interaction between the positively charged lysine residues surrounding the heme groove and a negatively charged surface (Pinheiro 1994), similar to the situation expected for adsorption to hematite at pH >pzc.
Conformational changes upon sorption that might affect the ability of Hcc to desorb, and hence also its overall sorption behavior needs to be considered. Our Hcc supernatant solutions, in the context of microscopic reversibility, can be considered to be a mixture of ‘native’ Hcc that has never been sorbed to hematite with Hcc that has been sorbed and then desorbed. The UV- visible spectra for supernatant solutions at pH 7–12 were red-shifted, while those for Hcc solutions in the same range showed a systematic blue shift (Figure 2a, b). These differences suggest a conformational change, caused by sorption, that is carried into solution upon subsequent desorption. This in turn suggests that 20 h of reaction time is insufficient to equilibrate with respect to a slow exchange process between surface and solution.
The peak sorption occurs with a surface coverage equal to ~7 monolayers of Hcc (Figure 1b). This high surface coverage is probably caused by a combination of factors, including (1) van der Waals attraction between Hcc molecules near the Hcc isoelectric point, leading to agglomeration, (2) alignment of the Hcc dipoles negative-to-positive as a result of orientation in the weak electric field between pH 8.5 and 10, and (3) possible conformational change of Hcc in the sorbed state such that the assumption of 3.4 nm spheres used to calculate monolayer coverage no longer holds.
Kinetics of Hcc sorption on hematite
The rate of initial sorption was rapid and essentially constant over the pH range, 3–10 with Hcc reaching 85–90% of its maximum sorption capacity within an hour (Figure 3a). Sorption kinetics and amounts appear similar at pH 3 and 7 (Figure 3a). At pH 9.7, after ~60 h, the total amount of sorbed Hcc appears to decrease slowly. Because the amount of sorption is extremely sensitive to pH in this range (Figure 2a), differences in pH as shown in the error bars could result in apparent reduction in sorption. In addition, the blue shift in the position of the Soret band with time (at pH 9.66; Figure 3b) suggests that Hcc of altered conformation might be slowly building up in solution. As indicated in the UV-visible results, such shifts can also result in higher optical absorptivities than native protein (which could cause an artifactual apparent decrease in sorption). Hence, because longer-term variations in apparent sorption density may be related to slow conformation change rather than to absolute amounts of adsorption, an operational equilibration time of 20 h for pH and ionic strength-dependent experiments was chosen.
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). Ionic strength has little effect on the amount of Hcc sorbed, which hovers within error of zero (Figure 4a). At pH 9.5, between the IEP of hematite and Hcc, there is electrostatic attraction between hematite and Hcc; if Hcc is treated as a diffuse layer ion attracted to the vicinity of a charged surface, we would expect to see the total amount sorbed to decrease with ionic strength at pH 9.5. This seems to be the case, with the exception of sorption at 300 mM KCl. At pH 9.5, there is a relatively high molar absorptivity at 100 mM KCl, but a relatively low molar absorptivity at 300 mM KCl; this appears to correlate inversely with the adsorption results at these KCl concentrations (Figure 4b). In contrast, there is essentially no variability for the results at pH 3.0 (Figure 4b). We emphasize that we have calibrated the concentration measurement at each ionic strength value, so that an unusually small molar absorptivity at 300 mM does not lead to an artificially low concentration measurement at 300 mM and thus to an artificially high sorption density for Hcc. Instead, the changes in molar absorptivity may reflect conformation changes in the Hcc that affect its ability to sorb. In addition, the full width at half maximum height (fwhm) for the Soret band in our absorption spectra does not increase with increasing ionic strength, suggesting that Hcc aggregation did not occur in our systems (Whitford et al., 1991; Peng et al., 2000).
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). In later scans, a reduction wave at positive potential grows into the voltammograms and the oxidation broadens on the high-potential side. The average of the initial reduction and oxidation wave potentials gives a measured midpoint potential of ~10 mV positive (relative to the Ag/AgCl reference), which is slightly below that expected for native Hcc at 50 mV (Yeh and Kuwana, 1977; Bowden et al., 1984; Chen et al., 2002). The higher-potential waves have been observed previously and attributed to hemes experiencing a positive charge environment (Chen et al., 2002). The slightly negative reduction potential compared to native cytochromes suggest that at pH 9.5 slight unfolding of Hcc may have taken place due to sorption on hematite consistent with the results from UV-visible spectroscopy. Voltammograms taken without Hcc (control) show no peaks (data not shown).
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CONCLUSIONS |
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TOPAbstractINTRODUCTIONEXPERIMENTAL METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Footnotes |
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(Received 17 September 2004; revised 16 May 2005)
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