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Native, denatured and reduced BSA
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Electrochimica Acta 53 (2008) 4014–4021
Native, denatured and reduced BSA Enhancement of chronopotentiometric peak H by guanidinium chloride Veronika Ostatn´a, Emil Paleˇcek ∗ Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i. Kr´alovopolsk´a 135, 612 65 Brno, Czech Republic Received 13 July 2007; received in revised form 3 October 2007; accepted 3 October 2007 Available online 23 October 2007
Abstract In proteomics and biomedicine fast techniques applicable for preliminary tests of the protein properties and structural changes are sought. Methods of electrochemical analysis have been little utilized in these fields. We show that using constant current chronopotentiometric stripping peak H, minute amounts of denatured and reduced bovine serum albumin (BSA) can be easily discriminated from native BSA. Peak H, which is due to catalytic hydrogen evolution, is greatly enhanced in the presence of non-denaturing concentrations of guanidinium chloride. The course of BSA reduction and denaturation can be followed and traces of the damaged protein can be detected in native BSA samples. © 2007 Elsevier Ltd. All rights reserved. Keywords: Constant current chronopotentiometry; Protein denaturation; Guanidinium chloride; Mercury electrodes; Catalytic hydrogen evolution
1. Introduction Proteomics and biomedicine are booming scientific fields rapidly acquiring new methods of protein analysis. Regrettably among these methods the electrochemical analysis has been so far almost missing. We believe that the recent progress in protein research, and particularly in proteomics [1] and biomedicine, represents a challenge to protein electrochemistry. In a relatively short time of its existence proteomics achieved an extensive technology development, which contributed to the fast development of this area of science. Despite the ingenuity of technologies applied in proteomics, their application in proteome studies is limited. The methods are time-consuming, exacting on sample purity and rely on powerful, expensive equipment and qualified staff. Therefore, less expensive and less time-consuming techniques, providing only restricted information but applicable to sample screening and detection of changes in protein structures and properties have been sought. Methods of electrochemical analysis have great potentialities in this respect, which have been so far little utilized. Polarog-
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Corresponding author. Tel.: +420 5 492 46 241; fax: +420 5 415 17 249. E-mail address: [email protected] (E. Paleˇcek).
0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.10.035
raphy of proteins was used in biochemistry and medicine and particularly in cancer research for several decades in the middle of the 20th century. In the recent decades attention of electrochemists turned to direct electrochemistry of redox-active center containing conjugated proteins providing fast reversible electrochemistry at solid electrodes [2–5]. This exciting field is, however, of a limited use in the fields of proteomics and biomedicine. Several years ago we asked the question whether electroactivity of usual amino acids contained in proteins can be utilized in protein research. In the beginning of the 1980s it was shown that tyrosine and tryptophan residues in proteins are oxidizable at carbon electrodes [6]. The sensitivity of the voltammetric determination was, however, rather low and the oxidation signals were not well resolved. About 10 years ago we found, in collaboration with J. Wang, that application of constant current chronopotentiometric stripping analysis (CPSA) [7], or square wave voltammetry [8] with an efficient baseline correction produced better resolved peaks and increased the sensitivity of the oxidation signals of peptides and proteins by about two orders of magnitude. Using mercury electrodes we showed that CPSA of peptides and proteins produced a well-developed peak at highly negative potentials [9,10], which was due to catalytic hydrogen evolution. By means of this peak it was possible to
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determine peptides and proteins down to nanomolar and subnanomolar concentrations. We denominated this peak as peak H, because of High sensitivity, Hydrogen evolution and Heyrovsky in whose laboratory the ability of proteins to catalyze hydrogen evolution at mercury electrodes was discovered in 1930 [11]. Peak H differs from the previously described polarographic and voltammetric electrocatalytic signals of proteins [9] (i) by its ability to detect peptides and proteins down to nanomolar and subnanomolar concentrations and (ii) by its remarkable sensitivity to local and global changes in protein structures [9,12]. We also showed that both the protein oxidation signals at carbon electrodes and peak H at mercury electrodes can be obtained not only in the conventional way, i.e., with the electrodes dipped into the analyte (in situ) but also with protein-modified electrodes (using Adsorptive Transfer Stripping, AdTS, ex situ) [13,14]. Adsorbing proteins from microliter drops made it possible to determine proteins down to femto and subfemtomole level. Peak H proved to be extremely useful in the analysis of a number of proteins, including determination of metallothioneins in tissues [15,16], determination of redox states of peptides and proteins [9,17], detection of conformational changes in mutant proteins [9], monitoring the course of protein aggregation [8,18], DNA–protein interactions [9,14], determination of point mutations in DNA [14] using double-surface method [19], etc. Recently, we have shown that native and denatured bovine serum albumin (BSA) and other proteins can be discriminated at the hanging mercury drop electrode (HMDE) using peak H [12]. BSA was denatured by urea and CPSA of both native and denatured proteins was performed at non-denaturing concentrations of urea (usually bellow 0.1 M). Under the given conditions we observed a relatively large peak H produced by 90 nM ureadenatured BSA as compared to about 50-fold smaller peak of native BSA. In this paper we show that peak H of native and denatured BSA is greatly enhanced in the presence of certain, relatively low non-denaturing concentrations of guanidinium chloride (GdmCl). We also show that peak H can be used to monitor time dependence of BSA denaturation and reduction of disulfide bonds in BSA.
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2. Experimental 2.1. Materials and methods BSA, GdmCl and urea were purchased from Sigma–Aldrich Chemical Co. All other chemicals used were of analytical grade, solutions were prepared from triply distilled water. 2.2. Apparatus and electrochemical procedures Electrochemical measurements were performed with an AUTOLAB Analyzer (EcoChemie, Utrecht, The Netherlands) in connection with VA-Stand 633 (Metrohm, Herisau, Switzerland); HMDE (area of drop 0.4 mm2 ) was used as a working electrode in the standard cell with three-electrode system. An Ag/AgCl/3M KCl electrode served as the reference electrode and a platinum wire as the auxiliary electrode. All experiments were carried out at room temperature open to air. We used constant current derivative chronopotentiometric adsorptive stripping (CP AdS) analysis (Scheme 1A). The working electrode was immersed for accumulation time, tA (usually 60 s) at accumulation potential EA −0.1 V into the electrolytic cell containing the protein solution in the background electrolyte, and the chronopotentiograms were recorded. In most experiments stirring accompanied the accumulation. Alternatively, in Adsorptive Transfer Stripping (AdTS, ex situ) (Scheme 1B and C) the protein was adsorbed at EA (−0.1 V) for tA (60 s), under stirring and the HMDE with an adsorbed protein layer was washed and transferred into a blank background electrolyte. The initial potential was −0.1 V, the potential negative limit was −1.95 V and the stripping current, Istr was −90 A, if not stated otherwise. The background electrolyte was 150 mM borate buffer pH 9.7 (if not stated otherwise). 2.3. Denaturation of proteins Denaturation was performed by incubating 14.4 M protein in 0.1 M Tris–HCl, pH 7.3 with 6 M guanidinium chloride or 8 M urea at 4 ◦ C overnight. Then the protein solution was diluted by supporting electrolyte to the final protein concentra-
Scheme 1. Comparison of AdS (in situ, conventional, in which the electrode is dipped into the analyte) (A) with AdTS (ex situ experiments, in which adsorption and electrode processes are separated). In AdTS the BSA was adsorbed either from a solution containing or not containing GdmCl (or urea), followed by peak H recording in the supporting electrolyte (not containing BSA) in presence (B) or absence of GdmCl (C).
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tion (usually about 100 nM); the final concentration of GdmCl (or urea) usually was 70 mM. Electrochemical measurement with the denatured protein was performed with the protein solutions freshly diluted from the denaturing conditions, if not stated otherwise. 3. Results and discussion In our previous paper [12] we showed that peak H of native BSA and of some other proteins is greatly increased as a result of the protein denaturation by urea. We also mentioned that similar effects can be obtained using guanidinium chloride (GdmCl) as a denaturant but we did not study the effect of non-denaturing concentrations of GdmCl on peak H. 3.1. Effect of GdmCl concentration on peak H Fig. 1A and B shows the dependence peak H area of 100 nM native and denatured BSA on non-denaturing concentrations of GdmCl in the range from 40 to 140 mM GdmCl. Starting from about 50 mM GdmCl the peak area of denatured BSA increased with GdmCl concentration up to about 130 mM GdmCl (Fig. 1B). Increasing the GdmCl concentration from 100 to 120 mM lead to a striking increase of the half-width (W1/2 ) of peak H (Fig. 1B) and formation of an additional small peak at −1.9 V. Between 120 and 130 mM GdmCl a sharp peak at −1.88 V (spike, W1/2 13 mV) appeared (Fig. 1A) upon the negative side of the original broad peak. A similar spike was observed also on voltammetric curves [20] suggesting possible surface reorganization process leading to an increase in the attractive interactions of the protein–GdmCl entities in the monolayer. Peak area of native BSA was enhanced by increasing GdmCl concentration starting from about 100 mM GdmCl.
Peak H of BSA denatured by GdmCl was always substantially higher than that of native BSA, under the same conditions (Fig. 1B). Similar results were obtained also with BSA denatured by urea (and measured in presence 11 mM urea) [12]. In Fig. 1C peaks of 100 nM native and GdmCl-denatured BSA, obtained in the presence of 70 mM GdmCl at Istr (−90 A) (i.e. under the conditions showing very large difference between the peak areas of native and denatured BSA) are compared. Under these conditions, the peak area of denatured BSA was about 20fold larger than that of native BSA (containing 70 mM GdmCl). Peak H of urea-denatured BSA (containing 70 mM urea but no GdmCl) was even smaller than that of native BSA with 70 mM GdmCl (Fig. 1C-inset). These results clearly show that GdmCl in a certain concentration range (Fig. 1) greatly enhances peak H both of denatured and native BSA. Under the above electrolyte conditions but at substantially smaller Istr (−3 A) and accumulation time tA (180 s), we were able to detect denatured BSA down to 100 pM concentration (6.9 g/L) (not shown). While the above conditions suited well for discrimination of native and denatured forms of the protein, they were less suitable for the determination of concentration of the GdmCl-denatured BSA in a wider concentration range. In the presence of 70 mM GdmCl the dependence of peak height on concentration of the GdmCl-denatured BSA was linear only in a very narrow concentration range (because of presence of the spike, Fig. 1A) and therefore less convenient for analytical purposes. To overcome this difficulty we used a different approach described in Section 3.4. Using cyclic voltammetry with HMDE and 0.2 M NaOH with 0.8 M CoCl2 and 0.2 M GdmCl as a background electrolyte Luo et al. [21] reported a BSA detection limit of 2 g/L. Their peak P was, however, not applicable for discrimination of native and denatured BSA, because of strongly alkaline medium, inducing protein denaturation and it was limited to the determi-
Fig. 1. (A) Peak H of 100 nM GdmCl-denatured BSA in presence 100 mM (dashed line), 120 mM (solid) and 140 mM (bold) GdmCl. 100 nM BSA was adsorbed on HMDE for accumulation time tA , 60 s, at accumulation potential EA , −0.1 V, from 150 mM sodium borate buffer, pH 9.7 with stirring at 1500 rpm. Chronopotentiograms were recorded (in situ, CP AdS) with stripping current Istr , −90 A. (B) dependence of peak H half-width (W1/2 ) and area of 100 nM native (- -- -) and GdmCl-denatured (–—) BSA on concentration of GdmCl. (C) peak H of native BSA with 70 mM GdmCl (dashed line) and denatured BSA with 70 mM GdmCl (solid) or with 70 mM urea (bold). Denaturation of 14.4 M BSA in 6 M GdmCl or 8 M urea was performed overnight at 4 ◦ C. Denatured BSA was always measured immediately after the removal of the denaturation conditions Inset: Detail of peak H native and urea-denatured BSA.
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nation of BSA and HSA (but not suitable for lysozyme). On the other hand, peak H was produced by all proteins so far tested [9,12,14,15,22]. 3.2. Role of GdmCl in formation of peak H To explain their peak P, Luo et al. [21] speculated that guanidine functioned not as a ligand but as a modifier of the BSA molecule, introducing a positive charge to BSA dissolved in strongly alkaline NaOH solution. Due to its positive charge, the Co(II)–BSA complex was strongly adsorbed and could accumulate at the surface of the stationary mercury electrode. On the other hand, urea associated also with BSA but it did not induce strong adsorption of the Co(II)–BSA complex. With pKa of 12.5, guanidine is protonated under physiological conditions forming guanidinium cation [CH6 N3 ]+ , with charge of +1. We were interested whether under our conditions (Fig. 1), i.e. in absence of cobalt and at less alkaline pH, the main effect of GdmCl will also consist only in an increased adsorption of BSA. To elucidate this problem we used the Adsorptive Transfer Stripping (AdTS, ex situ) method [9,13,23], in which the electrode was first immersed in the BSA solution with 70 mM GdmCl, for tA of 60 s and the electrode was removed from the BSA solution, washed and transferred to the electrolytic cell containing either a blank background electrolyte (Scheme 1C) or a background electrolyte with 70 mM GdmCl (Scheme 1B). The results were compared with the conventional AdS (in situ) measurements (Scheme 1A). When the electrode was transferred into the GdmCl-containing electrolyte (Scheme 1B) we observed a large peak (Fig. 2(b)), not too different from that obtained by means of conventional AdS (Fig. 2(a), Scheme 1A). Transfer of the electrode (with adsorbed BSA) to the electrolyte not containing any GdmCl (Fig. 2(c), Scheme 1C) produced a dramatically different result showing practically no peak H under the given conditions; when the sensitivity of the measurement was greatly increased the peak H became detectable (Fig. 2, inset). Adsorption of urea-denatured BSA (in solution containing 70 mM urea but no GdmCl) on the electrode, followed by the electrode transfer and measurement in GdmCl-containing background electrolyte, resulted in peak H similar to that produced by GdmCl-denatured BSA (Fig. 2(d), Scheme 1B). A more detailed analysis of this peak however showed that peak height was slightly lower, the peak was narrower and this area significantly smaller than that of peak of BSA adsorbed from GdmCl. These results suggested that presence or absence of GdmCl during the protein adsorption somehow affects the resulting CPS signal. This effect was, however, marginal as compared to that observed in the presence of GdmCl in the electrolyte (during the electrode process) (Figs. 1 and 2) which greatly enhanced peak H. Clearly GdmCl presence during the electrode process plays a decisive role in the enhancement of this peak. Our conclusion is thus different from that made by Luo et al. [21] who suggested that the main effect in enhancement of their peak P consisted in GdmCl influence on BSA adsorption. They believed that peak P was not due to the catalytic hydrogen evolution but rather to reduction of the BSA–GdmCl–Co(II) complex producing a remarkable adsorption current. In contrast, the peak H was, immediately
Fig. 2. Comparison of AdS (in situ, conventional) with AdTS (ex situ) experiments, in which adsorption and electrode processes are separated. In AdTS GdmCl can be present during the adsorption and absent during the electrode process or vice versa. (a) AdS peak H (bold line) of 100 nM GdmCl-denatured BSA in 150 mM sodium borate buffer, pH 9.7 with 70 mM GdmCl. (b–d) AdTS analysis: 100 nM GdmCl-denatured BSA was adsorbed from 150 mM sodium borate buffer, pH 9.7 with 70 mM GdmCl and transferred (b) to the same medium (dashed dot) or (c) to the medium not containing GdmCl (solid), (d) same as (b) but urea-denatured (dashed) BSA was adsorbed at HMDE. Adsorption was performed for tA (60 s) at EA (−0.1 V), under stirring. HMDE with adsorbed BSA layer was washed for 60 s, dried and transferred to the supporting electrolyte without or with GdmCl and peak H was recorded. Other details are as in Fig. 1. The results show that large enhancement of peak H requires presence of GdmCl in the supporting electrolyte. Inset: Detail of (c) peak H of denatured BSA.
after its discovery, assigned to the catalytic hydrogen evolution [9,10,16]. Here we tested whether the behavior of this peak may correspond to characteristics of the catalytic hydrogen evolution even in the presence of GdmCl. 3.3. Catalytic hydrogen evolution Electrochemical signals due to the catalytic hydrogen evolution are known to increase with increasing capacity of the background buffer [24,25]. We followed the dependence of peak H of 100 nM denatured BSA on concentration of sodium borate buffer (50–200 mM) with 70 mM GdmCl, pH 9.7 (Fig. 3). We observed almost linear increase of the denatured protein signal in the range of 50–150 mM sodium borate. At concentrations higher than 150 mM sodium borate in the presence of 70 mM GdmCl some precipitation of the denatured BSA frequently occurred. AdTS yielded results similar to AdS, if BSA was adsorbed from the same medium (50 mM sodium borate with 70 mM GdmCl, pH 9.7) and transferred to the background electrolytes containing different concentrations of the sodium borate
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Fig. 3. Dependence of height of peak H of denatured BSA on concentration of sodium borate buffer. Comparison of AdS and AdTS experiments. Denatured BSA was adsorbed from the electrolytic cell for tA (60 s) at open current circuit, without stirring; AdS: BSA was in 50–150 mM sodium borate buffer, pH 9.7 with 70 mM GdmCl (––); AdTS (ex situ): denatured BSA was adsorbed either (a) from 50 mM borate buffer, pH 9.7 with 70 mM GdmCl and the BSA-modified HMDE was transferred to another cell containing different concentrations of borate buffer with 70 mM GdmCl (- -- -) or (b) from different concentrations of borate buffer with 70 mM GdmCl and HMDE with BSA layer was transferred to the electrolyte with constant buffer concentration ( , 150 mM borate buffer with 70 mM GdmCl). Other details are as in Fig. 2.
buffer (Fig. 3). When BSA was adsorbed from solutions of different borate concentrations and the BSA-modified HMDE was always transferred to the same medium (150 mM sodium borate with 70 mM GdmCl, pH 9.7), much smaller changes of peak H resulted from changes in the borate concentration (Fig. 3). These results suggest that the increase of the height of peak H with increasing buffer capacity is due predominantly to its effect on the electrode process. Changes in buffer capacity during the BSA adsorption affected peak H to a lesser extent (Fig. 3). We may thus conclude that peak H of GdmCl-denatured BSA showed characteristics of the electrode processes which are due to catalytic hydrogen evolution [9]. This does not exclude smaller effects of BSA adsorption in addition to the electrocatalysis, which play the major role under the given conditions. 3.4. Dependence on BSA concentration Under the conditions described in (Fig. 1C) we observed a linear dependence of peak H on BSA concentration only in a relatively narrow concentration range because the dependence was complicated by formation of a spike on peak H (Fig. 1A). Such a narrow concentration range can be tolerated if the signals of native and denatured BSA are compared or if a structural transition or damage to the protein is traced at a known protein concentration. On the other hand if the concentration of the denatured BSA should be determined a linear dependence of
Fig. 4. Dependence of height of AdT peak H on concentration of denatured BSA in 150 mM sodium borate buffer, pH 9.7. Inset: Peak H of denatured BSA at concentrations increasing by tendency of arrow. Other details are as in Fig. 2 (c).
peak H on BSA concentration in a much wider range is necessary. In order to eliminate formation of spikes we applied AdTS method in which we transferred the GdmCl-denatured BSA into an electrolyte not containing GdmCl (Scheme 1C). Using this technique we obtained smaller peaks H but they were free of spikes, displaying a linear dependence of the height peak H on BSA concentration from about 50 to 1000 nM (Fig. 4). 3.5. BSA aggregation It is known that folded protein is in equilibrium with the denatured state and that proteins in their denatured state usually tend to aggregate [26]. Knowing this tendency, in most of our experiments we performed our measurements [12] (Figs. 1 and 2) of denatured BSA immediately after removal of the denaturation conditions. To obtain data about changes in peak H related to the BSA aggregation we measured peak H in dependence on time after removal of the denaturation conditions. BSA was denatured in 6 M GdmCl diluted to 400 nM BSA, 167 mM GdmCl, 40 mM Tris, pH 7.3. The solution was then incubated at 37 ◦ C with stirring and measured after 4-fold dilution. During the first 15 min of incubation the height of peak H decreased by about 6%. Prolonged incubation up to 180 min resulted in little changes in the peak height (Fig. 5). After 5 h of incubation a large decrease of peak H was observed and between 6 and 21 h peak H almost disappeared. Tendency to a faster aggregation was observed at pH 9.3 and in urea-denatured BSA, which aggregated much faster than GdmCl-denatured BSA. Our results show that when studying denatured BSA it is advisable to perform the measurements immediately after removal of the denaturation conditions. This is particularly critical when working with urea-denatured BSA. In studies of aggregation of denatured BSA, buffers with pH exceeding 9.7 should be used with caution.
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Fig. 5. Dependence of height of AdS peak H on time of aggregation. 400 nM GdmCl-denatured BSA with 167 mM GdmCl was incubated in 40 mM Tris–HCl, pH 7.3 with stirring in a test-tube at 37 ◦ C, samples were withdrawn in time intervals given in the figure and diluted to 100 nM BSA in 150 mM sodium borate, pH 9.7 with 70 mM GdmCl, followed by peak H recording. Other details are as in Fig. 1.
3.6. Course of BSA reduction and denaturation When denaturing disulfide bonds-containing proteins these have to be reduced to obtain fully unfolded protein. We reduced BSA using tris (2-carboxyl-ethyl) phosphine hydrochloride (phosphine), i.e. the agent frequently used for protein disulfide group reduction. 14.4 M BSA with 6 M GdmCl was denatured in presence and in absence of 10 mM phosphine in the usual way,
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i.e. at 4 ◦ C, overnight. After dilution to 100 nM BSA, the CP AdS was carried out in the presence of 70 mM GdmCl. BSA, which was simultaneously denatured and reduced (BSAden/r ), yielded a higher and wider peak H than the BSA, which was denatured in absence of the reducing agent (BSAden ) (Fig. 6A), in agreement with our previous results [12]. BSA, which was reduced with 10 mM phosphine in absence of the denaturant, produced peak H which was only slightly smaller than the peak of BSAden (Fig. 6A). These results suggest that peak H is able to reflect not only changes in the protein conformation induced by denaturing agents such as urea [12] and GdmCl (Fig. 1C) but also changes resulting from a mere reduction of the protein disulfide bonds. We were interested in whether peak H might be used also to follow kinetics of BSA denaturation and/or reduction. High concentrations of GdmCl decreased peak H of BSA making thus measurements in 6 M GdmCl (usually used for protein denaturation) difficult. We therefore used only 2 M GdmCl and increased temperature from 4 to 27 ◦ C to follow the dependence of BSA denaturation on time in the presence and in absence of phosphine. The combined effect of GdmCl and phosphine resulted in a steep increase of peak H (both the peak height and area) in the first 5 min of incubation, followed by almost no change up to 60 min (Fig. 6C). The GdmCl treatment (in absence of phosphine) produced a less steep increase of peak H after about 5 min lag period; after 20 min of incubation the peak area leveled off reaching about 50% of the area of peak H of BSAden/r in the same time intervals. Treatment of BSA by phosphine (in absence of GdmCl) resulted in a 20 min lag followed by an increase of peak H area to about the same area level as that of BSAden ; after 30 min of incubation the area of peak H of the reduced BSA did not practically change.
Fig. 6. (A) peak H of 100 nM native (dot), denatured BSA (dashed), reduced native (solid) and BSA denatured in the presence of reducing agent (bold). Denaturation and reduction of 14.4 M BSA in 6 M GdmCl with or without 10 mM phosphine was performed overnight at 4 ◦ C. (B) peak H of 100 nM native BSA alone (dashed) or after addition of 5% (solid) or 10% (bold) of reduced BSA. (C) dependence of area of peak H on time of incubation in 0.1 M Tris–HCl, pH 7.3 at 27 ◦ C. Denaturation by 2 M GdmCl (- -- -) or denaturation and reduction by 2 M GdmCl in the presence 10 mM phosphine (–䊉–) or reduction with 10 mM phosphine (in absence of GdmCl)(- -- -). Other details are as in Fig. 1A.
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We reduced BSA (in absence of GdmCl) and added small amounts of it to native BSA. Presence of 5% of the reduced BSA in the native sample was easily detected (Fig. 6B). These results are in agreement with our studies of reduced and oxidized peptides showing that peak H is an excellent indicator of their redox states [9,17]. Compared to the reduction of peptides [17], in BSA not only the reduction of disulfide groups but also the concominant protein conformational changes (resulting from the reduction of a number of disulfide groups) may strongly influence peak H of the reduced BSA. Our results suggest that peak H appears suitable for studies of BSA structural transitions. Moreover, the detection of small fractions of the reduced BSA in a large abundance of native BSA (Fig. 6B) gives a promise for detection of protein damage and of small conformational changes in mutated proteins. More work will be necessary to make conclusions about applicability of this method to different kinds of proteins and about other possible limitations of this method. On the other hand, one advantage of the method is already apparent now: the very low demand on concentration and volume (if AdTS is used) of the protein sample and very large differences in the peak heights of native and denatured proteins. 4. Conclusion Our results showed that both urea-denatured [12] and GdmCldenatured BSA (Fig. 1C) produced substantially higher peaks H than native BSA samples measured under the same conditions. In difference to the effect of urea [12], the presence of non-denaturing concentrations of GdmCl in the sample greatly enhanced peak H both in native and in denatured BSA (Fig. 1A and B). Peak H of GdmCl-denatured BSA (BSAden−G ) increased with the buffer concentration, showing characteristics of the electrode process due to catalytic hydrogen evolution [24,25]. Using AdTS technique we could estimate the effect of BSA adsorption in the electrocatalytic reaction. We showed that the composition of the background electrolyte (during the electrocatalysis) is more important for the resulting CPS signal than the composition of the adsorption medium. Under the given conditions the GdmCl–BSA associates should be positively charged, stimulating electrostatic adsorption of BSA at the negatively charged electrode. The enhancement of peak H of BSA adsorbed in the presence of GdmCl at open current circuit is not, however, due to the effect of GdmCl on BSA adsorption but to the presence of GdmCl in the electrolyte during the electrode process as shown by our AdT experiments (Fig. 2). Peak H of BSA observed in GdmCl-containing electrolyte is much wider than that obtained in absence of GdmCl (Fig. 2), suggesting not only enhancement but also qualitative changes in the involved electrode process(es). At higher concentrations of GdmCl (between 120 and 140 mM) a spike is formed suggesting presence of a BSA–GdmCl film at the electrode surface (Fig. 1A). Such film may be related to strong electrostatic interaction between positively charged BSA–GdmCl associates and negatively charged electrode, which, in addition to strong hydrophobic interactions, holds the protein firmly at the surface charged to highly negative potentials.
The differences in the heights of peak H of native and denatured BSA were surprisingly very large (regardless of presence or absence of GdmCl) and to our knowledge they cannot be compared to observations obtained with any other electrochemical method. The large difference in peak H heights of native and denatured BSA suggests that the ability of denatured BSA (adsorbed at the electrode at potentials of peak H) to catalyze hydrogen evolution is greatly enhanced as compared to native BSA. It can be expected that hydrophobic amino acid side chains (important for the protein adsorption at the hydrophobic mercury surface) are buried, and that also some potentially catalytically active proton donor groups are hidden in the structure of native BSA (stabilized by 17 disulfide bonds) in solution. In denatured BSA both types of the groups should be much more accessible. Considering these properties of native and denatured BSA one can intuitively assume that denatured BSA should produce a higher catalytic signal. On the other hand, we are aware of some observations showing that some voltammetric methods were not able to recognize native from denatured BSA [27,28]. We discuss this problem in a paper in which BSA signals of CPS and voltammetric methods are compared [20]. In this paper we show peak H produced only by native and denatured forms of a single protein. In addition to BSA we studied native and denatured forms of other proteins, such as human serum albumin, ␥-globulin, ␣-crystallin, myoglobin [12], ␣-globulin, concanavalin A and we observed large differences between the peak H heights of their native and denatured forms, in agreement with this paper. In our earlier work we showed that native double-stranded DNA and RNA (reviewed in [29–32]) yielded no or only small electrochemical responses in contrast to denatured single stranded nucleic acids which produced very large polarographic [33], voltammetric [29] and chronopotentiometric [29] signals at mercury electrodes. With natural and biosynthetic nucleic acids no exception was observed from this rule [29,32]. On the other hand in the case of proteins such a uniform electrochemical behavior can hardly be expected, because of complexity and diversity of protein structures. For example, in native unfolded proteins, such as prions or ␣-synuclein involved in Parkinson’s disease [18] the effect of denaturing agents may result in much smaller effects than in globular proteins. The chronopotentiometric responses of native and denatured proteins might thus show some surprises if a larger number of different proteins are investigated under various conditions. On the other hand, we do not expect that a significant number of proteins would be unable to produce peak H. So far we have analyzed >25 proteins and some of their mutants and about 13 different peptides and under suitable conditions all of them yielded peak H [34]. This electrocatalytic peak thus appears a promising tool for proteomics and biomedicine. More work will be, however, necessary to uncover all potentialities of peak H. Acknowledgements The authors are indebted to Dr. M. Heyrovsk´y for critical reading of the manuscript. This work was supported by grants from the Grant Agency of the Czech Republic 301/07/0490,
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202/07/P497 and from the Academy of Sciences of the Czech Republic IAA50040513 and AVOZ50040507 and Ministry of Education, Youth and Sports, CR, LC06035 and by European Community FP6 funding. This publication reflects the authors’ views and not necessarily those of the European Community. The Community is not liable for any use made of the information. References [1] S. Billova, E. Palecek, in: E. Palecek, F. Scheller, J. Wang (Eds.), Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics, Elsevier, Amsterdam, 2005, p. 751. [2] F.A. Armstrong, in: G.S. Wilson (Ed.), Bioelectrochemistry, vol. 9, WileyVCH, Weinheim, 2002, p. 11. [3] S. Borgmann, G. Hartwich, A. Schulte, W. Schuhmann, in: E. Palecek, F. Scheller, J. Wang (Eds.), Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics, Elsevier, Amsterdam, 2005, p. 599. [4] E.E. Ferapontova, S. Shleev, T. Ruzgas, L. Stoica, A. Christenson, J. Tkac, A.I. Yaropolov, L. Gordon, in: E. Palecek, F. Scheller, J. Wang (Eds.), Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics, Elsevier, Amsterdam, 2005, p. 517. [5] H. Wackerbarth, J. Zhang, M. Grubb, A.G. Hansen, B.L. Ooi, H.E.M. Christensen, J. Ulstrup, in: E. Palecek, F. Scheller, J. Wang (Eds.), Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics, Elsevier, Amsterdam, 2005, p. 485. [6] V. Brabec, V. Mornstein, Biochim. Biophys. Acta 625 (1980) 43. [7] X. Cai, G. Rivas, M.A.P. Farias, H. Shiraishi, J. Wang, E. Palecek, Anal. Chim. Acta 332 (1996) 49. [8] M. Masarik, A. Stobiecka, R. Kizek, F. Jelen, Z. Pechan, W. Hoyer, T.M. Jovin, V. Subramaniam, E. Palecek, Electroanalysis 16 (2004) 1172. [9] E. Palecek, in: E. Palecek, F. Scheller, J. Wang (Eds.), Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics, Elsevier, Amsterdam, 2005, p. 690. [10] M. Tomschik, L. Havran, M. Fojta, E. Palecek, Electroanalysis 10 (1998) 403.
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[11] J. Heyrovsky, J. Babicka, Coll. Czech. Chem. Commun. 2 (1930) 370. [12] V. Ostatna, B. Dogan, B. Uslu, S. Ozkan, E. Palecek, J. Electroanal. Chem. 593 (2006) 172. [13] E. Palecek, I. Postbieglov´a, J. Electroanal. Chem. 214 (1986) 359. [14] E. Palecek, M. Masarik, R. Kizek, D. Kuhlmeier, J. Hassmann, J. Schulein, Anal. Chem. 76 (2004) 5930. [15] R. Kizek, L. Trnkova, E. Palecek, Anal. Chem. 73 (2001) 4801. [16] M. Tomschik, L. Havran, E. Palecek, M. Heyrovsky, Electroanalysis 12 (2000) 274. [17] V. Dorcak, E. Palecek, Electroanalysis 19 (2007) 2405. [18] E. Palecek, V. Ostatna, M. Masarik, C.W. Bertoncini, T.M. Jovin, The Analyst (2007) doi:10.1039/b715815f. [19] E. Palecek, M. Fojta, F. Jelen, Bioelectrochemistry 56 (2002) 85. [20] V. Ostatna, F. Kuralay, L. Trnkova, E. Palecek, in preparation. [21] D. Luo, J. Lan, C. Zhou, C. Luo, Anal. Chem. 75 (2003) 6346. [22] L. Havran, S. Billova, E. Palecek, Electroanalysis 16 (2004) 1139. [23] E. Palecek, F. Jelen, C. Teijeiro, V. Fuc´ık, T.M. Jovin, Anal. Chim. Acta 273 (1993) 175. [24] F.G. Banica, A. Ion, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, John Wiley and Sons, Chichester, 2000, p. 1115. [25] M. Heyrovsky, in: E. Palecek, F. Scheller, J. Wang (Eds.), Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics, Elsevier, Amsterdam, 2005, p. 657. [26] R. Guerois, J. Mendes, L. Serrano, in: J. Buchner, T. Kiefhaber (Eds.), Protein Folding Handbook. Part I, Wiley-VCH, Weinheim, 2005, p. 343. [27] M.J. Honeychurch, Bioelectrochem. Bioenerg. 44 (1997) 13. [28] F. Scheller, M. J¨anchen, H.-J. Pr¨umke, Biopolymers 14 (1975) 1553. [29] E. Palecek, Electroanalysis 8 (1996) 7. [30] E. Palecek, M. Fojta, Talanta 74 (2007) 276. [31] E. Palecek, F. Jelen, Crit. Rev. Anal. Chem. 32 (2002) 261. [32] E. Palecek, F. Jelen, in: E. Palecek, F. Scheller, J. Wang (Eds.), Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics, Elsevier, Amsterdam, 2005, p. 74. [33] E. Palecek, V. Vetterl, Biopolymers 6 (1968) 917. [34] E. Palecek, V. Ostatna, Electroanalysis 19 (2007) 2383.
Electrochimica Acta 53 (2008) 4014–4021
Native, denatured and reduced BSA Enhancement of chronopotentiometric peak H by guanidinium chloride Veronika Ostatn´a, Emil Paleˇcek ∗ Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i. Kr´alovopolsk´a 135, 612 65 Brno, Czech Republic Received 13 July 2007; received in revised form 3 October 2007; accepted 3 October 2007 Available online 23 October 2007
Abstract In proteomics and biomedicine fast techniques applicable for preliminary tests of the protein properties and structural changes are sought. Methods of electrochemical analysis have been little utilized in these fields. We show that using constant current chronopotentiometric stripping peak H, minute amounts of denatured and reduced bovine serum albumin (BSA) can be easily discriminated from native BSA. Peak H, which is due to catalytic hydrogen evolution, is greatly enhanced in the presence of non-denaturing concentrations of guanidinium chloride. The course of BSA reduction and denaturation can be followed and traces of the damaged protein can be detected in native BSA samples. © 2007 Elsevier Ltd. All rights reserved. Keywords: Constant current chronopotentiometry; Protein denaturation; Guanidinium chloride; Mercury electrodes; Catalytic hydrogen evolution
1. Introduction Proteomics and biomedicine are booming scientific fields rapidly acquiring new methods of protein analysis. Regrettably among these methods the electrochemical analysis has been so far almost missing. We believe that the recent progress in protein research, and particularly in proteomics [1] and biomedicine, represents a challenge to protein electrochemistry. In a relatively short time of its existence proteomics achieved an extensive technology development, which contributed to the fast development of this area of science. Despite the ingenuity of technologies applied in proteomics, their application in proteome studies is limited. The methods are time-consuming, exacting on sample purity and rely on powerful, expensive equipment and qualified staff. Therefore, less expensive and less time-consuming techniques, providing only restricted information but applicable to sample screening and detection of changes in protein structures and properties have been sought. Methods of electrochemical analysis have great potentialities in this respect, which have been so far little utilized. Polarog-
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Corresponding author. Tel.: +420 5 492 46 241; fax: +420 5 415 17 249. E-mail address: [email protected] (E. Paleˇcek).
0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.10.035
raphy of proteins was used in biochemistry and medicine and particularly in cancer research for several decades in the middle of the 20th century. In the recent decades attention of electrochemists turned to direct electrochemistry of redox-active center containing conjugated proteins providing fast reversible electrochemistry at solid electrodes [2–5]. This exciting field is, however, of a limited use in the fields of proteomics and biomedicine. Several years ago we asked the question whether electroactivity of usual amino acids contained in proteins can be utilized in protein research. In the beginning of the 1980s it was shown that tyrosine and tryptophan residues in proteins are oxidizable at carbon electrodes [6]. The sensitivity of the voltammetric determination was, however, rather low and the oxidation signals were not well resolved. About 10 years ago we found, in collaboration with J. Wang, that application of constant current chronopotentiometric stripping analysis (CPSA) [7], or square wave voltammetry [8] with an efficient baseline correction produced better resolved peaks and increased the sensitivity of the oxidation signals of peptides and proteins by about two orders of magnitude. Using mercury electrodes we showed that CPSA of peptides and proteins produced a well-developed peak at highly negative potentials [9,10], which was due to catalytic hydrogen evolution. By means of this peak it was possible to
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determine peptides and proteins down to nanomolar and subnanomolar concentrations. We denominated this peak as peak H, because of High sensitivity, Hydrogen evolution and Heyrovsky in whose laboratory the ability of proteins to catalyze hydrogen evolution at mercury electrodes was discovered in 1930 [11]. Peak H differs from the previously described polarographic and voltammetric electrocatalytic signals of proteins [9] (i) by its ability to detect peptides and proteins down to nanomolar and subnanomolar concentrations and (ii) by its remarkable sensitivity to local and global changes in protein structures [9,12]. We also showed that both the protein oxidation signals at carbon electrodes and peak H at mercury electrodes can be obtained not only in the conventional way, i.e., with the electrodes dipped into the analyte (in situ) but also with protein-modified electrodes (using Adsorptive Transfer Stripping, AdTS, ex situ) [13,14]. Adsorbing proteins from microliter drops made it possible to determine proteins down to femto and subfemtomole level. Peak H proved to be extremely useful in the analysis of a number of proteins, including determination of metallothioneins in tissues [15,16], determination of redox states of peptides and proteins [9,17], detection of conformational changes in mutant proteins [9], monitoring the course of protein aggregation [8,18], DNA–protein interactions [9,14], determination of point mutations in DNA [14] using double-surface method [19], etc. Recently, we have shown that native and denatured bovine serum albumin (BSA) and other proteins can be discriminated at the hanging mercury drop electrode (HMDE) using peak H [12]. BSA was denatured by urea and CPSA of both native and denatured proteins was performed at non-denaturing concentrations of urea (usually bellow 0.1 M). Under the given conditions we observed a relatively large peak H produced by 90 nM ureadenatured BSA as compared to about 50-fold smaller peak of native BSA. In this paper we show that peak H of native and denatured BSA is greatly enhanced in the presence of certain, relatively low non-denaturing concentrations of guanidinium chloride (GdmCl). We also show that peak H can be used to monitor time dependence of BSA denaturation and reduction of disulfide bonds in BSA.
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2. Experimental 2.1. Materials and methods BSA, GdmCl and urea were purchased from Sigma–Aldrich Chemical Co. All other chemicals used were of analytical grade, solutions were prepared from triply distilled water. 2.2. Apparatus and electrochemical procedures Electrochemical measurements were performed with an AUTOLAB Analyzer (EcoChemie, Utrecht, The Netherlands) in connection with VA-Stand 633 (Metrohm, Herisau, Switzerland); HMDE (area of drop 0.4 mm2 ) was used as a working electrode in the standard cell with three-electrode system. An Ag/AgCl/3M KCl electrode served as the reference electrode and a platinum wire as the auxiliary electrode. All experiments were carried out at room temperature open to air. We used constant current derivative chronopotentiometric adsorptive stripping (CP AdS) analysis (Scheme 1A). The working electrode was immersed for accumulation time, tA (usually 60 s) at accumulation potential EA −0.1 V into the electrolytic cell containing the protein solution in the background electrolyte, and the chronopotentiograms were recorded. In most experiments stirring accompanied the accumulation. Alternatively, in Adsorptive Transfer Stripping (AdTS, ex situ) (Scheme 1B and C) the protein was adsorbed at EA (−0.1 V) for tA (60 s), under stirring and the HMDE with an adsorbed protein layer was washed and transferred into a blank background electrolyte. The initial potential was −0.1 V, the potential negative limit was −1.95 V and the stripping current, Istr was −90 A, if not stated otherwise. The background electrolyte was 150 mM borate buffer pH 9.7 (if not stated otherwise). 2.3. Denaturation of proteins Denaturation was performed by incubating 14.4 M protein in 0.1 M Tris–HCl, pH 7.3 with 6 M guanidinium chloride or 8 M urea at 4 ◦ C overnight. Then the protein solution was diluted by supporting electrolyte to the final protein concentra-
Scheme 1. Comparison of AdS (in situ, conventional, in which the electrode is dipped into the analyte) (A) with AdTS (ex situ experiments, in which adsorption and electrode processes are separated). In AdTS the BSA was adsorbed either from a solution containing or not containing GdmCl (or urea), followed by peak H recording in the supporting electrolyte (not containing BSA) in presence (B) or absence of GdmCl (C).
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tion (usually about 100 nM); the final concentration of GdmCl (or urea) usually was 70 mM. Electrochemical measurement with the denatured protein was performed with the protein solutions freshly diluted from the denaturing conditions, if not stated otherwise. 3. Results and discussion In our previous paper [12] we showed that peak H of native BSA and of some other proteins is greatly increased as a result of the protein denaturation by urea. We also mentioned that similar effects can be obtained using guanidinium chloride (GdmCl) as a denaturant but we did not study the effect of non-denaturing concentrations of GdmCl on peak H. 3.1. Effect of GdmCl concentration on peak H Fig. 1A and B shows the dependence peak H area of 100 nM native and denatured BSA on non-denaturing concentrations of GdmCl in the range from 40 to 140 mM GdmCl. Starting from about 50 mM GdmCl the peak area of denatured BSA increased with GdmCl concentration up to about 130 mM GdmCl (Fig. 1B). Increasing the GdmCl concentration from 100 to 120 mM lead to a striking increase of the half-width (W1/2 ) of peak H (Fig. 1B) and formation of an additional small peak at −1.9 V. Between 120 and 130 mM GdmCl a sharp peak at −1.88 V (spike, W1/2 13 mV) appeared (Fig. 1A) upon the negative side of the original broad peak. A similar spike was observed also on voltammetric curves [20] suggesting possible surface reorganization process leading to an increase in the attractive interactions of the protein–GdmCl entities in the monolayer. Peak area of native BSA was enhanced by increasing GdmCl concentration starting from about 100 mM GdmCl.
Peak H of BSA denatured by GdmCl was always substantially higher than that of native BSA, under the same conditions (Fig. 1B). Similar results were obtained also with BSA denatured by urea (and measured in presence 11 mM urea) [12]. In Fig. 1C peaks of 100 nM native and GdmCl-denatured BSA, obtained in the presence of 70 mM GdmCl at Istr (−90 A) (i.e. under the conditions showing very large difference between the peak areas of native and denatured BSA) are compared. Under these conditions, the peak area of denatured BSA was about 20fold larger than that of native BSA (containing 70 mM GdmCl). Peak H of urea-denatured BSA (containing 70 mM urea but no GdmCl) was even smaller than that of native BSA with 70 mM GdmCl (Fig. 1C-inset). These results clearly show that GdmCl in a certain concentration range (Fig. 1) greatly enhances peak H both of denatured and native BSA. Under the above electrolyte conditions but at substantially smaller Istr (−3 A) and accumulation time tA (180 s), we were able to detect denatured BSA down to 100 pM concentration (6.9 g/L) (not shown). While the above conditions suited well for discrimination of native and denatured forms of the protein, they were less suitable for the determination of concentration of the GdmCl-denatured BSA in a wider concentration range. In the presence of 70 mM GdmCl the dependence of peak height on concentration of the GdmCl-denatured BSA was linear only in a very narrow concentration range (because of presence of the spike, Fig. 1A) and therefore less convenient for analytical purposes. To overcome this difficulty we used a different approach described in Section 3.4. Using cyclic voltammetry with HMDE and 0.2 M NaOH with 0.8 M CoCl2 and 0.2 M GdmCl as a background electrolyte Luo et al. [21] reported a BSA detection limit of 2 g/L. Their peak P was, however, not applicable for discrimination of native and denatured BSA, because of strongly alkaline medium, inducing protein denaturation and it was limited to the determi-
Fig. 1. (A) Peak H of 100 nM GdmCl-denatured BSA in presence 100 mM (dashed line), 120 mM (solid) and 140 mM (bold) GdmCl. 100 nM BSA was adsorbed on HMDE for accumulation time tA , 60 s, at accumulation potential EA , −0.1 V, from 150 mM sodium borate buffer, pH 9.7 with stirring at 1500 rpm. Chronopotentiograms were recorded (in situ, CP AdS) with stripping current Istr , −90 A. (B) dependence of peak H half-width (W1/2 ) and area of 100 nM native (- -- -) and GdmCl-denatured (–—) BSA on concentration of GdmCl. (C) peak H of native BSA with 70 mM GdmCl (dashed line) and denatured BSA with 70 mM GdmCl (solid) or with 70 mM urea (bold). Denaturation of 14.4 M BSA in 6 M GdmCl or 8 M urea was performed overnight at 4 ◦ C. Denatured BSA was always measured immediately after the removal of the denaturation conditions Inset: Detail of peak H native and urea-denatured BSA.
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nation of BSA and HSA (but not suitable for lysozyme). On the other hand, peak H was produced by all proteins so far tested [9,12,14,15,22]. 3.2. Role of GdmCl in formation of peak H To explain their peak P, Luo et al. [21] speculated that guanidine functioned not as a ligand but as a modifier of the BSA molecule, introducing a positive charge to BSA dissolved in strongly alkaline NaOH solution. Due to its positive charge, the Co(II)–BSA complex was strongly adsorbed and could accumulate at the surface of the stationary mercury electrode. On the other hand, urea associated also with BSA but it did not induce strong adsorption of the Co(II)–BSA complex. With pKa of 12.5, guanidine is protonated under physiological conditions forming guanidinium cation [CH6 N3 ]+ , with charge of +1. We were interested whether under our conditions (Fig. 1), i.e. in absence of cobalt and at less alkaline pH, the main effect of GdmCl will also consist only in an increased adsorption of BSA. To elucidate this problem we used the Adsorptive Transfer Stripping (AdTS, ex situ) method [9,13,23], in which the electrode was first immersed in the BSA solution with 70 mM GdmCl, for tA of 60 s and the electrode was removed from the BSA solution, washed and transferred to the electrolytic cell containing either a blank background electrolyte (Scheme 1C) or a background electrolyte with 70 mM GdmCl (Scheme 1B). The results were compared with the conventional AdS (in situ) measurements (Scheme 1A). When the electrode was transferred into the GdmCl-containing electrolyte (Scheme 1B) we observed a large peak (Fig. 2(b)), not too different from that obtained by means of conventional AdS (Fig. 2(a), Scheme 1A). Transfer of the electrode (with adsorbed BSA) to the electrolyte not containing any GdmCl (Fig. 2(c), Scheme 1C) produced a dramatically different result showing practically no peak H under the given conditions; when the sensitivity of the measurement was greatly increased the peak H became detectable (Fig. 2, inset). Adsorption of urea-denatured BSA (in solution containing 70 mM urea but no GdmCl) on the electrode, followed by the electrode transfer and measurement in GdmCl-containing background electrolyte, resulted in peak H similar to that produced by GdmCl-denatured BSA (Fig. 2(d), Scheme 1B). A more detailed analysis of this peak however showed that peak height was slightly lower, the peak was narrower and this area significantly smaller than that of peak of BSA adsorbed from GdmCl. These results suggested that presence or absence of GdmCl during the protein adsorption somehow affects the resulting CPS signal. This effect was, however, marginal as compared to that observed in the presence of GdmCl in the electrolyte (during the electrode process) (Figs. 1 and 2) which greatly enhanced peak H. Clearly GdmCl presence during the electrode process plays a decisive role in the enhancement of this peak. Our conclusion is thus different from that made by Luo et al. [21] who suggested that the main effect in enhancement of their peak P consisted in GdmCl influence on BSA adsorption. They believed that peak P was not due to the catalytic hydrogen evolution but rather to reduction of the BSA–GdmCl–Co(II) complex producing a remarkable adsorption current. In contrast, the peak H was, immediately
Fig. 2. Comparison of AdS (in situ, conventional) with AdTS (ex situ) experiments, in which adsorption and electrode processes are separated. In AdTS GdmCl can be present during the adsorption and absent during the electrode process or vice versa. (a) AdS peak H (bold line) of 100 nM GdmCl-denatured BSA in 150 mM sodium borate buffer, pH 9.7 with 70 mM GdmCl. (b–d) AdTS analysis: 100 nM GdmCl-denatured BSA was adsorbed from 150 mM sodium borate buffer, pH 9.7 with 70 mM GdmCl and transferred (b) to the same medium (dashed dot) or (c) to the medium not containing GdmCl (solid), (d) same as (b) but urea-denatured (dashed) BSA was adsorbed at HMDE. Adsorption was performed for tA (60 s) at EA (−0.1 V), under stirring. HMDE with adsorbed BSA layer was washed for 60 s, dried and transferred to the supporting electrolyte without or with GdmCl and peak H was recorded. Other details are as in Fig. 1. The results show that large enhancement of peak H requires presence of GdmCl in the supporting electrolyte. Inset: Detail of (c) peak H of denatured BSA.
after its discovery, assigned to the catalytic hydrogen evolution [9,10,16]. Here we tested whether the behavior of this peak may correspond to characteristics of the catalytic hydrogen evolution even in the presence of GdmCl. 3.3. Catalytic hydrogen evolution Electrochemical signals due to the catalytic hydrogen evolution are known to increase with increasing capacity of the background buffer [24,25]. We followed the dependence of peak H of 100 nM denatured BSA on concentration of sodium borate buffer (50–200 mM) with 70 mM GdmCl, pH 9.7 (Fig. 3). We observed almost linear increase of the denatured protein signal in the range of 50–150 mM sodium borate. At concentrations higher than 150 mM sodium borate in the presence of 70 mM GdmCl some precipitation of the denatured BSA frequently occurred. AdTS yielded results similar to AdS, if BSA was adsorbed from the same medium (50 mM sodium borate with 70 mM GdmCl, pH 9.7) and transferred to the background electrolytes containing different concentrations of the sodium borate
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Fig. 3. Dependence of height of peak H of denatured BSA on concentration of sodium borate buffer. Comparison of AdS and AdTS experiments. Denatured BSA was adsorbed from the electrolytic cell for tA (60 s) at open current circuit, without stirring; AdS: BSA was in 50–150 mM sodium borate buffer, pH 9.7 with 70 mM GdmCl (––); AdTS (ex situ): denatured BSA was adsorbed either (a) from 50 mM borate buffer, pH 9.7 with 70 mM GdmCl and the BSA-modified HMDE was transferred to another cell containing different concentrations of borate buffer with 70 mM GdmCl (- -- -) or (b) from different concentrations of borate buffer with 70 mM GdmCl and HMDE with BSA layer was transferred to the electrolyte with constant buffer concentration ( , 150 mM borate buffer with 70 mM GdmCl). Other details are as in Fig. 2.
buffer (Fig. 3). When BSA was adsorbed from solutions of different borate concentrations and the BSA-modified HMDE was always transferred to the same medium (150 mM sodium borate with 70 mM GdmCl, pH 9.7), much smaller changes of peak H resulted from changes in the borate concentration (Fig. 3). These results suggest that the increase of the height of peak H with increasing buffer capacity is due predominantly to its effect on the electrode process. Changes in buffer capacity during the BSA adsorption affected peak H to a lesser extent (Fig. 3). We may thus conclude that peak H of GdmCl-denatured BSA showed characteristics of the electrode processes which are due to catalytic hydrogen evolution [9]. This does not exclude smaller effects of BSA adsorption in addition to the electrocatalysis, which play the major role under the given conditions. 3.4. Dependence on BSA concentration Under the conditions described in (Fig. 1C) we observed a linear dependence of peak H on BSA concentration only in a relatively narrow concentration range because the dependence was complicated by formation of a spike on peak H (Fig. 1A). Such a narrow concentration range can be tolerated if the signals of native and denatured BSA are compared or if a structural transition or damage to the protein is traced at a known protein concentration. On the other hand if the concentration of the denatured BSA should be determined a linear dependence of
Fig. 4. Dependence of height of AdT peak H on concentration of denatured BSA in 150 mM sodium borate buffer, pH 9.7. Inset: Peak H of denatured BSA at concentrations increasing by tendency of arrow. Other details are as in Fig. 2 (c).
peak H on BSA concentration in a much wider range is necessary. In order to eliminate formation of spikes we applied AdTS method in which we transferred the GdmCl-denatured BSA into an electrolyte not containing GdmCl (Scheme 1C). Using this technique we obtained smaller peaks H but they were free of spikes, displaying a linear dependence of the height peak H on BSA concentration from about 50 to 1000 nM (Fig. 4). 3.5. BSA aggregation It is known that folded protein is in equilibrium with the denatured state and that proteins in their denatured state usually tend to aggregate [26]. Knowing this tendency, in most of our experiments we performed our measurements [12] (Figs. 1 and 2) of denatured BSA immediately after removal of the denaturation conditions. To obtain data about changes in peak H related to the BSA aggregation we measured peak H in dependence on time after removal of the denaturation conditions. BSA was denatured in 6 M GdmCl diluted to 400 nM BSA, 167 mM GdmCl, 40 mM Tris, pH 7.3. The solution was then incubated at 37 ◦ C with stirring and measured after 4-fold dilution. During the first 15 min of incubation the height of peak H decreased by about 6%. Prolonged incubation up to 180 min resulted in little changes in the peak height (Fig. 5). After 5 h of incubation a large decrease of peak H was observed and between 6 and 21 h peak H almost disappeared. Tendency to a faster aggregation was observed at pH 9.3 and in urea-denatured BSA, which aggregated much faster than GdmCl-denatured BSA. Our results show that when studying denatured BSA it is advisable to perform the measurements immediately after removal of the denaturation conditions. This is particularly critical when working with urea-denatured BSA. In studies of aggregation of denatured BSA, buffers with pH exceeding 9.7 should be used with caution.
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Fig. 5. Dependence of height of AdS peak H on time of aggregation. 400 nM GdmCl-denatured BSA with 167 mM GdmCl was incubated in 40 mM Tris–HCl, pH 7.3 with stirring in a test-tube at 37 ◦ C, samples were withdrawn in time intervals given in the figure and diluted to 100 nM BSA in 150 mM sodium borate, pH 9.7 with 70 mM GdmCl, followed by peak H recording. Other details are as in Fig. 1.
3.6. Course of BSA reduction and denaturation When denaturing disulfide bonds-containing proteins these have to be reduced to obtain fully unfolded protein. We reduced BSA using tris (2-carboxyl-ethyl) phosphine hydrochloride (phosphine), i.e. the agent frequently used for protein disulfide group reduction. 14.4 M BSA with 6 M GdmCl was denatured in presence and in absence of 10 mM phosphine in the usual way,
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i.e. at 4 ◦ C, overnight. After dilution to 100 nM BSA, the CP AdS was carried out in the presence of 70 mM GdmCl. BSA, which was simultaneously denatured and reduced (BSAden/r ), yielded a higher and wider peak H than the BSA, which was denatured in absence of the reducing agent (BSAden ) (Fig. 6A), in agreement with our previous results [12]. BSA, which was reduced with 10 mM phosphine in absence of the denaturant, produced peak H which was only slightly smaller than the peak of BSAden (Fig. 6A). These results suggest that peak H is able to reflect not only changes in the protein conformation induced by denaturing agents such as urea [12] and GdmCl (Fig. 1C) but also changes resulting from a mere reduction of the protein disulfide bonds. We were interested in whether peak H might be used also to follow kinetics of BSA denaturation and/or reduction. High concentrations of GdmCl decreased peak H of BSA making thus measurements in 6 M GdmCl (usually used for protein denaturation) difficult. We therefore used only 2 M GdmCl and increased temperature from 4 to 27 ◦ C to follow the dependence of BSA denaturation on time in the presence and in absence of phosphine. The combined effect of GdmCl and phosphine resulted in a steep increase of peak H (both the peak height and area) in the first 5 min of incubation, followed by almost no change up to 60 min (Fig. 6C). The GdmCl treatment (in absence of phosphine) produced a less steep increase of peak H after about 5 min lag period; after 20 min of incubation the peak area leveled off reaching about 50% of the area of peak H of BSAden/r in the same time intervals. Treatment of BSA by phosphine (in absence of GdmCl) resulted in a 20 min lag followed by an increase of peak H area to about the same area level as that of BSAden ; after 30 min of incubation the area of peak H of the reduced BSA did not practically change.
Fig. 6. (A) peak H of 100 nM native (dot), denatured BSA (dashed), reduced native (solid) and BSA denatured in the presence of reducing agent (bold). Denaturation and reduction of 14.4 M BSA in 6 M GdmCl with or without 10 mM phosphine was performed overnight at 4 ◦ C. (B) peak H of 100 nM native BSA alone (dashed) or after addition of 5% (solid) or 10% (bold) of reduced BSA. (C) dependence of area of peak H on time of incubation in 0.1 M Tris–HCl, pH 7.3 at 27 ◦ C. Denaturation by 2 M GdmCl (- -- -) or denaturation and reduction by 2 M GdmCl in the presence 10 mM phosphine (–䊉–) or reduction with 10 mM phosphine (in absence of GdmCl)(- -- -). Other details are as in Fig. 1A.
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We reduced BSA (in absence of GdmCl) and added small amounts of it to native BSA. Presence of 5% of the reduced BSA in the native sample was easily detected (Fig. 6B). These results are in agreement with our studies of reduced and oxidized peptides showing that peak H is an excellent indicator of their redox states [9,17]. Compared to the reduction of peptides [17], in BSA not only the reduction of disulfide groups but also the concominant protein conformational changes (resulting from the reduction of a number of disulfide groups) may strongly influence peak H of the reduced BSA. Our results suggest that peak H appears suitable for studies of BSA structural transitions. Moreover, the detection of small fractions of the reduced BSA in a large abundance of native BSA (Fig. 6B) gives a promise for detection of protein damage and of small conformational changes in mutated proteins. More work will be necessary to make conclusions about applicability of this method to different kinds of proteins and about other possible limitations of this method. On the other hand, one advantage of the method is already apparent now: the very low demand on concentration and volume (if AdTS is used) of the protein sample and very large differences in the peak heights of native and denatured proteins. 4. Conclusion Our results showed that both urea-denatured [12] and GdmCldenatured BSA (Fig. 1C) produced substantially higher peaks H than native BSA samples measured under the same conditions. In difference to the effect of urea [12], the presence of non-denaturing concentrations of GdmCl in the sample greatly enhanced peak H both in native and in denatured BSA (Fig. 1A and B). Peak H of GdmCl-denatured BSA (BSAden−G ) increased with the buffer concentration, showing characteristics of the electrode process due to catalytic hydrogen evolution [24,25]. Using AdTS technique we could estimate the effect of BSA adsorption in the electrocatalytic reaction. We showed that the composition of the background electrolyte (during the electrocatalysis) is more important for the resulting CPS signal than the composition of the adsorption medium. Under the given conditions the GdmCl–BSA associates should be positively charged, stimulating electrostatic adsorption of BSA at the negatively charged electrode. The enhancement of peak H of BSA adsorbed in the presence of GdmCl at open current circuit is not, however, due to the effect of GdmCl on BSA adsorption but to the presence of GdmCl in the electrolyte during the electrode process as shown by our AdT experiments (Fig. 2). Peak H of BSA observed in GdmCl-containing electrolyte is much wider than that obtained in absence of GdmCl (Fig. 2), suggesting not only enhancement but also qualitative changes in the involved electrode process(es). At higher concentrations of GdmCl (between 120 and 140 mM) a spike is formed suggesting presence of a BSA–GdmCl film at the electrode surface (Fig. 1A). Such film may be related to strong electrostatic interaction between positively charged BSA–GdmCl associates and negatively charged electrode, which, in addition to strong hydrophobic interactions, holds the protein firmly at the surface charged to highly negative potentials.
The differences in the heights of peak H of native and denatured BSA were surprisingly very large (regardless of presence or absence of GdmCl) and to our knowledge they cannot be compared to observations obtained with any other electrochemical method. The large difference in peak H heights of native and denatured BSA suggests that the ability of denatured BSA (adsorbed at the electrode at potentials of peak H) to catalyze hydrogen evolution is greatly enhanced as compared to native BSA. It can be expected that hydrophobic amino acid side chains (important for the protein adsorption at the hydrophobic mercury surface) are buried, and that also some potentially catalytically active proton donor groups are hidden in the structure of native BSA (stabilized by 17 disulfide bonds) in solution. In denatured BSA both types of the groups should be much more accessible. Considering these properties of native and denatured BSA one can intuitively assume that denatured BSA should produce a higher catalytic signal. On the other hand, we are aware of some observations showing that some voltammetric methods were not able to recognize native from denatured BSA [27,28]. We discuss this problem in a paper in which BSA signals of CPS and voltammetric methods are compared [20]. In this paper we show peak H produced only by native and denatured forms of a single protein. In addition to BSA we studied native and denatured forms of other proteins, such as human serum albumin, ␥-globulin, ␣-crystallin, myoglobin [12], ␣-globulin, concanavalin A and we observed large differences between the peak H heights of their native and denatured forms, in agreement with this paper. In our earlier work we showed that native double-stranded DNA and RNA (reviewed in [29–32]) yielded no or only small electrochemical responses in contrast to denatured single stranded nucleic acids which produced very large polarographic [33], voltammetric [29] and chronopotentiometric [29] signals at mercury electrodes. With natural and biosynthetic nucleic acids no exception was observed from this rule [29,32]. On the other hand in the case of proteins such a uniform electrochemical behavior can hardly be expected, because of complexity and diversity of protein structures. For example, in native unfolded proteins, such as prions or ␣-synuclein involved in Parkinson’s disease [18] the effect of denaturing agents may result in much smaller effects than in globular proteins. The chronopotentiometric responses of native and denatured proteins might thus show some surprises if a larger number of different proteins are investigated under various conditions. On the other hand, we do not expect that a significant number of proteins would be unable to produce peak H. So far we have analyzed >25 proteins and some of their mutants and about 13 different peptides and under suitable conditions all of them yielded peak H [34]. This electrocatalytic peak thus appears a promising tool for proteomics and biomedicine. More work will be, however, necessary to uncover all potentialities of peak H. Acknowledgements The authors are indebted to Dr. M. Heyrovsk´y for critical reading of the manuscript. This work was supported by grants from the Grant Agency of the Czech Republic 301/07/0490,
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