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Electrochemical sensing of concanavalin A and ovalbumin interaction in solution
Accepted Manuscript Electrochemical sensing of concanavalin A and ovalbumin interaction in solution Veronika Vargová, Robert Helma, Emil Paleček, RNDr. Veronika Ostatná, Ph.D. PII:
S0003-2670(16)30818-2
DOI:
10.1016/j.aca.2016.06.055
Reference:
ACA 234679
To appear in:
Analytica Chimica Acta
Received Date: 9 March 2016 Revised Date:
14 June 2016
Accepted Date: 29 June 2016
Please cite this article as: V. Vargová, R. Helma, E. Paleček, V. Ostatná, Electrochemical sensing of concanavalin A and ovalbumin interaction in solution, Analytica Chimica Acta (2016), doi: 10.1016/ j.aca.2016.06.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Electrochemical sensing of concanavalin A and ovalbumin interaction in solution
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Veronika Vargová, Robert Helma, Emil Paleček, Veronika Ostatná 1
Institute of Biophysics, The Czech Academy of Science, v.v.i., Královopolská 135, 61265 Brno, Czech
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Republic
1 Corresponding Author:
RNDr. Veronika Ostatná, Ph.D.
Institute of Biophysics CAS, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic Phone: +420 541 517 162; Fax: +420 541 517 249; E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract In an attempt to develop a label- and reagent- free electrochemical method for the detection of lectinglycoprotein interactions, we tested lectin-concanavalin A (ConA), glycoprotein-ovalbumin (Ova) and their complex using chronopotentiometric stripping (CPS) analysis and a hanging mercury drop
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electrode. Incubation of ConA with Ova resulted in an increase of the CPS peak H of the complex as compared to the CPS peaks of individual Ova and ConA proteins. Qualitatively similar results were obtained with other glycoprotein-lectin couples (ConA-RNase B and lectin from Sambucus nigra-
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fetuin). Using the CPS method, we were able to follow the course of complex formation in solution. Comparable responses of Ova, ConA and ConA-Ova complex were obtained not only at the mercury
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electrode but also with solid amalgam electrodes, which are more suitable for parallel analysis. It can be anticipated that electrochemical methods, namely CPS, will find application in glycomics and proteomics. Keywords
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Protein-protein interactions; lectin-glycoprotein interactions; ovalbumin; concanavalin A; constant current chronopotentiometric stripping; mercury electrodes;
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Introduction
Protein-protein association is at the center of diverse biological processes ranging from molecular
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motors to cell signaling. Proteins catalyze reactions, transport, form the building blocks of viral capsids, traverse the membranes to form regulated channels, and are involved in the transmission of information from DNA to RNA [1]. Most current methods study biomacromolecules and their complexes in diluted solutions or in crystals, greatly differing from cellular environments in highly concentrated liquids and at various structures carrying electric charges [2,3]. In electrochemical methods using small polarizable electrodes, the interactions of surface-confined biomacromolecules, such as nucleic acids, proteins and carbohydrates (including mutual biomacromolecule interactions, 2
ACCEPTED electric field effects, etc.) can be studied [4].MANUSCRIPT For almost 15 years we used constant current chronopotentiometric stripping (CPS) analysis and Hg-containing electrodes, which allowed observation of the well-developed electrocatalytic peak of peptides and proteins. This peak, termed peak H, was due to the catalytic hydrogen evolution, displaying sensitivity to local and global changes in protein structure [4-8] even for poorly soluble membrane proteins [9,10]. This peak can
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be useful in protein analysis for monitoring protein aggregation [7], denaturation [4,5], glycation [11] and for analysis of protein redox states [4]. New methods utilizing peak H were developed for DNAprotein interactions [12]. This method, using protein signals, is based on new principles, which have
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so far not been used in DNA-protein interaction analysis. It was possible to discriminate between the
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stabilities of tumor suppressor p53 core domain (p53CD) complexes with different DNAs on the basis of their susceptibility to the electric field effects at the electrode by controlling the temperature and/or current density. In this paper we attempted to apply CPS analysis and peak H to investigate lectin-glycoprotein interactions, representing a special case of protein-protein interactions.
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Lectin from Canavalia ensiformis, (Concanavalin A, ConA) is a legume protein, which was extracted from jack bean by the method of Sumner and Howell in 1936 [13]. The unusual biological properties of ConA are directly related to its saccharide binding activity, which in turn, is regulated
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by metal ion binding [14]. ConA has a binding specificity for α-D-glucose and α-D-mannose sugars,
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with weak binding to monosaccharides and stronger binding to oligosaccharides such as those found on the cell surface of glycoproteins and glycolipids [15]. ConA is a metalloprotein, requiring the presence of metal ions for proper binding function; each subunit of ConA contains one manganese (Mn2+) and one calcium (Ca2+) ion [16]. These biological properties of ConA have led to its utilization in various biotechnological applications [17,18].
Ovalbumin (Ova) is a glycoprotein which has a single N-glycosylation site (asparagine 292) occupied by a heterogeneous population of attached neutral glycans. The high-mannose 3
ACCEPTED MANUSCRIPT (predominantly mannopentaose-di-(N-acetyl-D-glucosamine)-Man5GlcNAc2 and mannohexaose-di(N-acetyl-D-glucosamine)-Man6GlcNAc2, (Fig. 1)) and hybrid type are the most abundant [19]. More than 25 distinct, neutral glycans have been reported [20], however, over the 12 glycoforms were observed in very low abundance [21]. Mandal et al. showed that the α(1,6) core arm of Man5 which contains the trimannosyl moiety, is the primary binding epitope for lectin ConA [22]. The
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interaction of ConA-Ova was previously studied by different methods, such as calorimetry, nuclear magnetic resonance dispersion measurements as well as electrochemistry [4,22-25]. Voltammetry was used for Ova detection using ConA labeled by daunomycin [24,25]. Also electrochemical
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impedance spectroscopy was used as label-free method for Ova detection using ConA attached at
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electrode surface [26].
In this paper we attempted to apply CPS analysis to lectin-glycoprotein interaction studies. We found that, using peak H, free lectins and free glycoproteins can be distinguished from the lectinglycoprotein complexes and the time course of the complex formation can be followed. The
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interaction of ConA with Ova was studied in detail; free ConA did not yield any peak H. Formation of the ConA-Ova complex resulted in an increase in peak H, which was tentatively explained by a conformational change in Ova. Similar behavior was also observed in studies of other lectin-
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glycoprotein complexes.
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2. Materials and Methods
2.1. Reagents and Solutions.
Concanavalin A, from Canavalia ensiformis (ConA, 25.6 kDa, 6 arginine (Arg), 12 lysine (Lys), 6 histidine (His) but no cysteine (Cys) residues in the protein monomer; ConA forms a tetramer at neutral pH), Albumin from chicken egg white (Ova, 42.8 kDa, 15 Arg, 20 Lys, 7 His Cys 6), Bovine serum albumin (BSA), RNase B, fetuin, lectin from Ulex europaneus (UEA) and lectin from Sambucus nigra (SNA) were purchased from Sigma-Aldrich. Stock solutions were prepared in triply 4
ACCEPTED MANUSCRIPT distilled water. The final concentration of the solutions was determined spectrophotometrically. All other chemicals were of analytical grade. 2.2. Apparatus. The experiments were performed with a three electrode system connected to a µAutolab III
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potentiostat (Metrohm-Autolab). The working electrode was a hanging mercury drop electrode (area 0.4 mm2) controlled by a VA-stand 663 (Metrohm). Ag|AgCl|3 M KCl was used as the reference electrode and platinum wire as a counter electrode. All experiments were carried out at a temperature
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of 26 °C open to air with constant current chronopotentiometric stripping.
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2.3. Procedures.
Analytes were adsorbed on the HMDE for accumulation time tA at accumulation potential EA in the background electrolyte (50 mM phosphate, pH 7). In Adsorptive Stripping (in situ) experiments: HMDE were immersed into the electrolytic cell (with the stirred solution containing the protein in the background electrolyte). After sample accumulation at EA –0.1 V, for tA 240 s (if not stated
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otherwise), chronopotentiograms or voltammograms were then recorded from the initial potential, Ei of –0.1 V to the final potential, Ef of –2 V. CPS: stripping current Istr of –30 µA (if not stated
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otherwise). Alternating current (ac) voltammetry: step of 4 mV; frequency of 66.21 Hz; scan rate of 15.7 mV/s. Adsorptive Transfer (AdT, ex situ) stripping: Sample was adsorbed at open current
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potential for 60 s (if not stated otherwise) from 5 µL drop. Then the protein-modified electrode was transfer to the electrolytic cell with blank background electrolyte (not containing any protein) to perform CPS.
2.3.1. Complex preparation. Complexes were prepared in 2 different ways. Preparation in solution: Stock solutions of albumin (Ova, RNaseB, fetuin or BSA) and lectin (ConA, lectin UEA or lectin SNA) were mixed in 50 mM phosphate, pH 7 to a final protein 5
ACCEPTED MANUSCRIPT concentration of 6 µM. This mixture in the ratio 1:1 was incubated in a thermomixer for 1 hour at 25 °C and 300 rpm. A special case of complex preparation in solution was preparation directly in the electrolytic cell: Stock solutions of Ova and ConA were mixed in 50 mM phosphate, pH 7 in an electrolytic cell to a final 1 µM concentration. The mixture was stirred and incubated at room
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temperature. After varying periods of incubation chronopotentiograms were recorded. 2.3.2. Solid amalgam electrode preparation
The meniscus solid amalgam electrode (SAE) was prepared as previously described [27,28]. Briefly,
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the silver solid amalgam electrode was dipped into the mercury and a meniscus of liquid mercury was formed at the electrode surface. The SAE was activated by applying a constant potential of –2.2
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V to the electrode in 0.2 M KCl without deaeration for 5 min. A regeneration of the SAE was then carried out by repeated cyclic voltammograms at a scan rate of 10 V/s from 0.1 V to –1.9 V in 50 mM Na-phosphate, pH 7, without deaeration. Scans were repeated 100-times. The activation and regeneration of the SAE preceded each measurement. Further manipulation with protein-modified
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SAE was the same as with HMDE. 2.3.3. Denaturation of Ova and ConA.
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40 µM ConA and 45 µM Ova were denatured in 8 M urea at 4 ºC overnight. The protein solutions were then diluted by Na-phosphate, pH 7 to the final protein concentration. Electrochemical
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measurement with the denatured protein was performed with freshly diluted protein solution. 1 µM ConA was measured in the presence of 200 mM urea and Ova in 177 mM urea (non-denaturing concentration).
2.4. Native agarose gel electrophoresis Fluorescein isothiocyanate labeled ConA (ConA-FITC) was incubated with increasing amount (1-4 µg) of Ova and bovine serum albumin (BSA) in 50 mM Na-phosphate, pH 7. Proteins were mixed with 6x loading buffer (20% sucrose) and were loaded on native agarose gel (1% in 0.33x TBE). The 6
samples were running on the gel ACCEPTED for 2 hours at MANUSCRIPT 70 V in a cold room. Fluorescence of ConA-FITC was detected by scanning of gel on TyphoonFLA 9000 imager (GE Healthcare). 3. Results and discussion Considering the ability of CPS to recognize and detect p53 sequence-specific binding to DNA [12]
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we decided to apply this method for protein-protein interaction studies. For our studies we have chosen the lectin-glycoprotein system [29]. Lectins are proteins well suited to the recognition of glycans on glycoproteins in their natural, intact form [4,17]. Lectin molecules are capable of
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producing a peak H similar to other proteins (not shown), but we found that under conditions when Ova, serum albumins and other proteins (Fig. 2A) [8] produce a peak H, in native lectin ConA this
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peak was absent. Using Ova as a glycoprotein and ConA as a lectin we obtained a system in which we could follow the effect of ConA-Ova binding on the Ova CPS responses. 3.1. Lectin-glycoprotein formation in solutions
A complex of lectin ConA with the glycoprotein Ova was formed in solution. Some conditions of
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complex preparation was taken from ref. [24]. To be sure that the lectin-glycoprotein complex was formed, we tested its formation using ConA conjugated with fluorescein isothiocyanate (ConA-
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FITC) and native agarose gel. Fig. SI-1 shows that ConA migrated in two different fractions in agreement with previous findings [30]. We titrated ConA-FITC with Ova and BSA (BSA was taken
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as negative control). With increasing of Ova concentration, new band appeared indicating complex formation (Fig. SI-1, line 8-10). Addition of BSA as control resulted in no changes of the bands.
Sample of Ova, ConA and ConA-Ova complex was adsorbed at a HMDE at an accumulation potential, EA of ˗0.1 V during an accumulation time, tA of 240 s in 50 mM phosphate, pH 7, followed by chronopotentiogram recording at a stripping current, Istr of ˗30 µA. 1 µM ConA-Ova complex yielded a single well-developed peak H (at a potential, Ep of ˗1.92 V), while a 6-times smaller peak H with two maxima (peak H1 at Ep of -1.93 V and peak H2 at Ep of -1.97) was observed for free 1 7
MANUSCRIPT µM Ova. The difference betweenACCEPTED the peak H of free Ova and ConA-Ova complex was detected across the entire studied concentration range (Fig. 2B, C). With increasing Ova concentration, the peak H area gradually increased up to 1 µM and reached saturation. The peak H of ConA-Ova also increased up to 1 µM concentration, but more steeply. Similar results were obtained by the measuring of dependence of 1 µM Ova and ConA on accumulation time (not shown). In both cases,
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free ConA did not yield any peak H in the given potential window (Fig. 2), probably due to the lower content of electroactive amino acid residues (6 Arg, 12 Lys, 6 His, 0 Cys) in the protein monomer (MW 25 615 Da) as compared to the content of electroactive residues of Ova (15 Arg, 20 Lys, 7 His
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and 6 Cys, MW 42 750 Da) according to ExPASy [31]. In addition to the study of Ova in complex
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with ConA, we tested a mixture of Ova with lectin from Ulex europaeus (UEA), binding fucose residues [17], which does not specifically bind to Ova. The mixture of Ova with UEA yielded a peak H, which was almost the same as the peak H of free Ova (Fig. 2A) across the entire concentration range (Fig. 2C). In contrast to free ConA, free UEA yielded single peak H, which was smaller than peak, of Ova (Fig. 2). Our data are thus in agreement with the literature [4,22-25] showing specific
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interaction of Ova with ConA but not with UEA.
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3.1.1. ConA-Ova complex formation in an electrolytic cell. Using CPS peak H, we investigated ConA-Ova complex formation in solution. In contrast to the
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previous approach, where the complex was formed at a concentration of 6 µM in a test tube, ConA was incubated with 1 µM Ova directly in the electrolytic cell. Stirring accompanied accumulation, followed by CPS recording. The obtained CPS peak H in t = 0 min, i.e. without incubation, yielded a peak H with 2 maxima (peak H1 EP −1.93 V; peak H2 EP −1.97 V) almost the same as the peak of Ova alone. With prolonged incubation time, peak H1 and peak H2 shifted to more negative potentials and peak H1 increased (Fig. 3). After 10 min, the mixture of ConA-Ova yielded only a single peak, which slowly increased with increasing incubation time. After incubation for longer than 60 min, the
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MANUSCRIPT CPS peak H of ConA-Ova did ACCEPTED not change. The increase in peak H after ConA-Ova complex formation could be due to (i) increased accessibility of catalytically active amino acid residues (particularly Arg, Lys, His and Cys) [4], (ii) conformational changes in Ova and/or ConA after complexation and (iii) structural changes after their contact with the electrode. We cannot exclude the possibility that the ConA-Ova complex was adsorbed at the electrode surface in a different way
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than free Ova, and therefore other electro-catalytically active residues could participate in the catalysis. According to the literature, specific binding of ConA to certain oligo- and polysaccharides may involve extended interaction of the protein with several carbohydrate residues [14]. A partial
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change in the circular dichroism spectrum of ConA was obtained after interaction with high mannose
interaction with glycopeptide. 3.1.2. Phase sensitive ac voltammetry.
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type glycopeptides cleaved from Ova [32], suggesting structural changes in ConA resulting from the
Using ac voltammetry in phase-out mode, we studied the adsorption of Ova, ConA and their complex
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at a HMDE. Both voltammograms of the free proteins were similar in potential ranges from -0.1V to -0.4 V and from -0.75 V to -1.9 V, respectively (Fig. 4). In contrast to ConA, Ova produced a peak at about -0.6 V, probably due to the reduction Cys-Hg bonds (a peak at this potential was also obtained
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in phase-in mode, not shown) and consecutive reorientation of the Ova molecule at the electrode surface. We observed similar voltammograms for the ConA-Ova complex to that of free Ova, with
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about a 40% smaller peak at -0.7 V. We can assume that the cysteine residues in Ova interacted with the electrode even after the complex formation with ConA. 3.2. Dependence on Istr
The rate of the overall process in CPS is imposed by the value of the applied current (Fig. SI-2). It is important that the electrode potential shifts slower during an electrode process than in its absence, when potential changes can reach extremely high rates [4,5]. This feature is critical in protein structure analysis [4]. To obtain peak H at highly negative potentials, such as −1.9 V the surface9
ACCEPTED attached protein is exposed to the electricMANUSCRIPT field effects at negative potentials causing unfolding/denaturation of the native protein [4,5] or dissociation/disintegration of the DNA-protein complex [12]. These processes depend on the time of exposure at negative potentials, which is related to stripping current (Istr) intensity [4,8]. By applying high negative current intensities to a relatively small working electrode, exposure to negative potential can be reduced to milliseconds [8].
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Firstly, we looked at the dependence of the signals of native (natOva) and urea-denatured Ova (denOva) on the Istr intensities at a constant temperature (26 °C, Fig. 5A). Between −10 µA and −20 µA the peak areas of natOva and denOva were almost the same, suggesting electric field driven
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denaturation of natOva due to prolonged exposure to the electric field. On the other hand at Istr
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intensities more negative than −24 µA, the peak H area of natOva was several times smaller than that of denOva and the ratio of denOva/natOva increased at more negative Istr intensities (not shown). Between −20 µA and −24 µA, structural transition due to the destabilization of natOva by electric field effects was observed. The presented data were in good agreement with the data obtained from BSA and other proteins [8,33]. Subsequently, we compared the Istr signal dependences of the ConA-
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Ova complex prepared in solution to those of free natOva and denOva (Fig. 5A). At Istr intensities, where the peak H areas of natOva and denOva were almost the same, the peak H area of the complex was about 20 % smaller than those of free Ova´s. The peak H of the ConA-Ova complex was shifted
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by about 30 mV to more negative potentials as compared to the peaks of Ova, suggesting a more
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difficult reduction process. A similar difference between the peak H of the complex and free denOva was observed for all studied Istr intensities (Fig. 5). We can speculate that the smaller peak H of the ConA-Ova complex than the peak H of denOva could be caused by changes in the accessibility of electro-active residues (Lys, Arg, His and Cys), as well as different adsorption of free Ova and its complex with ConA at the charged interface. With the shift of Istr intensities to values more negative than −20 µA, the difference between the peak H of natOva and ConA-Ova increased (Fig. 5A, right axis). At Istr −30 µA the peak area of ConA-Ova was about 6-times higher than the peak area of free natOva. The mixture of natOva and lectin UEA, which did not interact specifically with Ova, yielded 10
almost the same Istr dependence ACCEPTED as that of free MANUSCRIPT natOva, confirming our suggestion that using our method, specific Ova binding to ConA can be detected at a wide range of Istr values close to room temperature. We denatured ConA in order to better understand its CPS behavior. We found that denatured ConA (denConA) produces a peak H under conditions (Fig. 5B) in which, this peak was absent in native ConA (Fig. 1A). The peak H of denConA appeared at potentials by 100 mV more
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negative than the peak of the ConA-Ova complex (Fig. 5B) at less negative Istr intensities. The appearance of the peak H of denConA can be explained by the increased accessibility of the electroactive amino acid residues as compared to native tetrameric ConA in which many of these
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residues can be buried in its structure. To obtain more data on the behavior of native and denatured
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ConA we measured the dependence of their CPS responses on Istr. Between −10 µA and −16 µA, the peak H area of denConA was almost the same as the peak area of denOva. At more negative Istr intensities, the peak H of denConA gradually decreased and at Istr intensities, under which the peak H of the complex was higher than the peak H of Ova, the peak H of denConA was smaller than that of free natOva. The appearance of the peak for denConA indicates that ConA might also contribute to
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the increase in the peak H of the complex. Changes in ConA structure after glycan binding have been previously described [32]. To our knowledge, conformational changes in Ova after ConA-Ova complex formation have not yet been described. Our results suggest that Ova also underwent
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structural changes, leading to the peak H increase, after complex formation. At the Istr intensities
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where the difference between natOva and the ConA-Ova complex was observed, denOva yielded a higher peak than the peak of the ConA-Ova complex while denConA produced a peak H smaller than that of natOva (Fig. 5A), suggesting changes in Ova binding in the ConA-Ova complex. Further work should be done to elucidate the adsorption behavior of the ConA-Ova complex at charged interfaces in more detail.
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3.3. Solid amalgam electrode
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No doubt, HMDE offers better sensitivity and reproducibility (less than ±5 %, Fig. 2A) than solid electrodes but it is less convenient for parallel analysis of biomacromolecules. On the other hand SAE yielded similar results to those obtained with HMDE, with the reproducibility of solid
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electrodes (about ±10 %, Fig. 6B). Here we used a SAE to test its suitability for the study of lectinglycoprotein interactions under the same conditions as used for HMDE. Firstly we followed dependence of peak H area on accumulation time, tA for 1 µM Ova and ConA-Ova complex (Fig. 6A). With prolonging tA, peak H area of Ova gradually increased up 240 s and leveled off. The peak
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H area of ConA-Ova complex steeply increased up to tA 180 s and did not change at longer tA. For
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further experiments with SAE, tA of 240 s were chosen. In contrast to the previous experiments at SAE, an adsorptive transfer (ex situ) method was used; 1 µM sample was adsorbed for 240 s at open current potential from 5 µL drop at the electrode; modified SAE was transferred to background electrolyte followed by chronopotentiogram recording. Almost the same results were observed for SAE by both methods (ex situ and in situ method; Fig. 6B, C) but much smaller quantities of proteins
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could be used for the ex situ method. At the SAE, ConA, Ova and the complex of Ova with ConA and UEA yielded results very similar (Fig. 6C) to those obtained with the HMDE (Fig. 1A). As
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described earlier [5,28,34], SAEs offer equally reproducible results to other solid electrodes and can be easily miniaturized and applied in protein arrays [34].
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3.4. Other lectin-glycoprotein complexes In addition to the ConA-Ova complex, we studied a complex of ConA with another glycoprotein belonging to the high mannose type, namely RNase B (Fig. 7A) containing a single N-linked glycosylation site [21]. Similarly to the Con-Ova complex, the ConA-RNase B complex yielded a higher peak H shifted to more negative potentials than the peak H of free RNase B within a wide concentration (Fig. 7A, SI-4A). On the other hand RNase A, the nonglycosylated form of RNase B, yielded almost the same signal when in a mixture with ConA as that of the free protein (Fig. 7B, SI12
ACCEPTED 4A), in good agreement with previous findingsMANUSCRIPT [35]. Similar results were observed also for the combination of the lectin Sambucus nigra (SNA) and the glycoprotein fetuin (Fig. 7C). In contrast to ConA, the lectin SNA, containing Cys residues, yielded a peak H which appeared at more positive potentials than the peak H of fetuin (Fig. 7C). The complex of SNA with fetuin again produced a peak H higher than the individual peaks H of the free lectin and glycoprotein between 1 µM and 2.5
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µM (Fig. SI-4B). In contrast to the ConA-Ova and ConA-RNase B complexes, the peak potential of the SNA-fetuin complex was more positive than the peak of the glycoprotein, which could be due to
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the contribution of an intrinsic signal of the lectin SNA.
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4. Conclusion
The investigation of protein-protein interactions belongs to one of the most important fields in current molecular biology, which affects various areas of biomedicine. It is predicted that 50-80% of human proteins are posttranslationally modified by glycans, i.e. glycoproteins [36-38]. Two
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molecules of the same protein from the same tissue may have different glycans [39]. Changes in glycan identity in a glycoprotein can have a dramatic effect in pathological processes [4,17]. Despite newly emerging and powerful methods such as biochips/arrays, high throughput mass spectroscopy,
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liquid spectroscopy and nuclear magnetic resonance [36,40,41], new methods for the study of protein-protein, as well as lectin-glycoprotein interactions are still sought, representing a challenge
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for electrochemists. CPS in combination with mercury-containing electrodes was utilized here for the first time in the analysis of glycoprotein-lectin interactions. We show that lectin ConA, specifically bound to the glycoprotein Ova, yielded peaks differing from the peaks H of free Ova and ConA at concentrations starting from about 100 nM (Figs. 1-5) at the given experimental conditions. Lower concentrations could be reached by further optimization of the method (Fig. SI-3). Also, other lectinglycoprotein couples, namely ConA-RNase B and lectin SNA-fetuin were tested and similar results were obtained after their interactions. The combination of CPS with solid amalgam electrodes
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ACCEPTED MANUSCRIPT [28,34], amenable for parallel analysis, yields instant results and does not require any labeling or modification of the protein under study. Moreover, µL volumes can be used for the adsorptive transfer method, which allows determination of the ConA-Ova complex in picomole amounts (Fig. 6, SI-3). Protein-protein interactions have been little studied by electrochemical methods. Here, we introduce
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new principles in this analysis of lectin-glycoprotein interactions. The novelty of our method can be seen in measuring the protein response resulting from the lectin-glycoprotein binding. So far, voltammetry was used for Ova detection using ConA labeled by daunomycin [24,25]. Also
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electrochemical impedance spectroscopy (EIS) was used as label-free method for Ova detection
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using ConA attached at electrode surface [26] (see Table 1 in SI for more details). In EIS dielectric and electric properties of the surface-attached lectin resulting from its interaction with glycoprotein were measured. Our CPS method is highly sensitive to changes in protein structure [4,5,10,33] and in addition to a mere detection of the lectin-glycoprotein binding it can, after its further development, contribute to the elucidation of the conformational changes resulting from the lectin-glycoprotein
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interactions. Our previous work showed that CPS is protein structure-sensitive method detecting various kind of conformational changes in proteins. For example, (i) early changes preceding aggregation of α-synuclein (involved in Parkinson’s disease) were detected by peak H in accordance
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with the dynamic light scattering results. These changes were, however, not detectable by
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fluorescence measurements [7]; (ii) changes of BSA damaged by singlet oxygen detected by CPS were in agreement with mass spectrometric and optical spectroscopic data [33,42]; (iii) the CPS peaks H of wild-type and mutant of tumor suppressor p53 showed excellent correlation with structural and stability data and provided additional insights into the differential dynamic behavior of the proteins [6]. These results suggested that CPS peak H has a number of advantages [4]. Compared to established methods of protein analysis, such as mass spectrometry, optical spectroscopy and nuclear magnetic resonance, etc, electrochemical methods are inexpensive, fast, simple and more convenient for 14
MANUSCRIPT diagnostics, because they do not ACCEPTED require expensive instrumentation. In addition advantages of CPS analysis include (i) high sensitivity of protein determination and small volume requirements (few µL of nanomolar protein concentration are usually sufficient for ex situ method) (ii) high sensitivity to changes in protein structures, (iii) easy miniaturization, and development of chips for parallel analysis. We can conclude that the proposed CPS method can after its further development
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complement the established methods. Acknowledgements
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This study was supported by project No. 13-00956S to VO from the Czech Science Foundation.
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dimensional structure of concanavalin-A. 3. Structure of monomer and its interactions with metals
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ACCEPTED MANUSCRIPT desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography, Anal. Biochem., 250 (1997) 82-101. [20] D.J. Harvey, D.R. Wing, B. Kuster, I.B.H. Wilson, Composition of N-linked carbohydrates from ovalbumin and Co-purified glycoproteins, J. Am. Soc. Mass Spectrom., 11 (2000) 564-571. [21] M. Thaysen-Andersen, S. Mysling, P. Hojrup, Site-specific glycoprofiling of N-linked
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ACCEPTED MANUSCRIPT [30] G.H. McKenzie, W.H. Sawyer, Binding properties of dimeric and tetrameric concanavalin-A binding of ligands to noninteracting macromolecular acceptors, J. Biol. Chem., 248 (1973) 549-556. [31] S.I.o. Bioinformatics, Bioinformatics resource portal. [32] N.M. Young, K.J. Neurohr, R.E. Williams, Unique effects of glycopeptides on the circular dichroism of concanavalin A, peanut agglutinin and the pea lectin, Biochim.Biophys. Acta-Protein
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Struct. M., 701 (1982) 142-145.
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Electrochim. Acta, 187 (2016) 662-669.
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[39] R.M. Schmaltz, S.R. Hanson, C.H. Wong, Enzymes in the synthesis of glycoconjugates, Chem. Rev., 111 (2011) 4259-4307.
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bovine serum albumin and the roleACCEPTED of the protein MANUSCRIPT nano-compartmentalization, Free Radical Biol.
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Med., 94 (2016) 99-109.
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ACCEPTED MANUSCRIPT Figures legends Fig. 1 Structure of high mannose glycans A. mannopentaose-di-(N-acetyl-D-glucosamine) and B.
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mannohexaose-di-(N-acetyl-D-glucosamine)
Fig. 2 A. CPS peak H of 1 µM ovalbumin (Ova, black), concanavalin A (ConA, cyan), lectin from Ulex europeus (UEA, blue), ConA-Ova complex (red) and mixture of Ova and lectin UEA (magenta)
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at a hanging mercury drop electrode (HMDE) in 50 mM Na-phosphate, pH 7. Dependence of B. peak H potential, EP and C. peak H area on concentration of native () and denatured () Ova (black),
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ConA (▼, cyan), lectin UEA (◊, blue), ConA-Ova complex (●, red) and mixture of Ova with lectin UEA (▲, magenta). D. CPS peaks H of 100 nM Ova (solid black line), ConA-Ova complex (solid red line) and background electrolyte (dashed line). Protein was adsorbed at an accumulation potential, EA of −0.1 V for an accumulation time, tA of 240 s, followed by chronopotentiogram recording with
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Istr of −30 µA at 26 °C. Complex preparation in solution: 6 µM ConA (or UEA) was incubated with 6 µM Ova in 50 mM Na-phosphate, pH 7 for 1 h at 25 °C. Agitation at 300 rpm accompanied
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incubation.
Fig. 3 Dependence of A. EP and B. peak area on the incubation time of 1 µM ConA with 1 µM Ova
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incubated in 50 mM phosphate, pH 7 in an electrolytic cell. C. CPS peak H of 1 µM ConA incubated with 1 µM Ova for the given time. Other details as in Fig. 2.
Fig. 4 Ac voltammogram of 1 µM Ova (black), ConA (cyan) and ConA-Ova complexes (red) and background electrolyte (dashed line).
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ACCEPTED MANUSCRIPT Fig. 5 A. Dependence of peak H area on Istr for natOva (, solid line, black), denOva (, dashed line, black), denConA (∆, blue), a mixture of Ova with UEA (▲, magenta) and ConA-Ova complex (●, red). Right axis: Dependence of peak area ratio of ConA-Ova complex to Ova (green) on Istr. B. CPS peak H of 1 µM denatured Ova (dashed line, black), denConA (blue) and ConA-Ova complex (red)
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at Istr of −30 µA. Other details as in Fig. 2
Fig. 6 A. Dependence of peak H area on tA for 1 µM natOva (, black) and ConA-Ova complex (●, red) at SAE. B. CPS peak H of 1 µM Ova (black) and ConA-Ova complex (red) at tA of 240 s at SAE.
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Other details as in Fig. 1 C. Adsorptive transfer CPS peak H of 1 µM Ova (black), 1 µM ConA
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(cyan), lectin UEA (blue), ConA-Ova complex (red) and mixture Ova with 1 µM UEA (magenta) at a meniscus amalgam electrode in 50 mM Na-phosphate, pH 7. Istr of ˗40 µA. Protein was adsorbed at open current potential for tA of 240 s from 5 µL drop at electrode. Modified SAE was transferred in background electrolyte for CPS recording.
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Fig. 7 Adsorptive transfer CPS peak H of 1 µM A. RNase B (black), B. RNase A (gray), ConA (cyan) and their complexes with ConA (orange, yellow) and of C. 1 µM fetuin (black), lectin
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Sambucus nigra (SNA, blue) and SNA-fetuin complex (magenta). Istr of A., B. -50 µA, C. -20 µA
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ACCEPTED MANUSCRIPT Highlights Label-free electrochemical methods for detection of glycoprotein-lectin interactions
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Chronopotentiometric detection of ovalbumin-concanavalin A complex formation
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Conformational changes in lectin-glycoprotein complex detected by peak H
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S0003-2670(16)30818-2
DOI:
10.1016/j.aca.2016.06.055
Reference:
ACA 234679
To appear in:
Analytica Chimica Acta
Received Date: 9 March 2016 Revised Date:
14 June 2016
Accepted Date: 29 June 2016
Please cite this article as: V. Vargová, R. Helma, E. Paleček, V. Ostatná, Electrochemical sensing of concanavalin A and ovalbumin interaction in solution, Analytica Chimica Acta (2016), doi: 10.1016/ j.aca.2016.06.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Electrochemical sensing of concanavalin A and ovalbumin interaction in solution
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Veronika Vargová, Robert Helma, Emil Paleček, Veronika Ostatná 1
Institute of Biophysics, The Czech Academy of Science, v.v.i., Královopolská 135, 61265 Brno, Czech
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Republic
1 Corresponding Author:
RNDr. Veronika Ostatná, Ph.D.
Institute of Biophysics CAS, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic Phone: +420 541 517 162; Fax: +420 541 517 249; E-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abstract In an attempt to develop a label- and reagent- free electrochemical method for the detection of lectinglycoprotein interactions, we tested lectin-concanavalin A (ConA), glycoprotein-ovalbumin (Ova) and their complex using chronopotentiometric stripping (CPS) analysis and a hanging mercury drop
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electrode. Incubation of ConA with Ova resulted in an increase of the CPS peak H of the complex as compared to the CPS peaks of individual Ova and ConA proteins. Qualitatively similar results were obtained with other glycoprotein-lectin couples (ConA-RNase B and lectin from Sambucus nigra-
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fetuin). Using the CPS method, we were able to follow the course of complex formation in solution. Comparable responses of Ova, ConA and ConA-Ova complex were obtained not only at the mercury
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electrode but also with solid amalgam electrodes, which are more suitable for parallel analysis. It can be anticipated that electrochemical methods, namely CPS, will find application in glycomics and proteomics. Keywords
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Protein-protein interactions; lectin-glycoprotein interactions; ovalbumin; concanavalin A; constant current chronopotentiometric stripping; mercury electrodes;
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Introduction
Protein-protein association is at the center of diverse biological processes ranging from molecular
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motors to cell signaling. Proteins catalyze reactions, transport, form the building blocks of viral capsids, traverse the membranes to form regulated channels, and are involved in the transmission of information from DNA to RNA [1]. Most current methods study biomacromolecules and their complexes in diluted solutions or in crystals, greatly differing from cellular environments in highly concentrated liquids and at various structures carrying electric charges [2,3]. In electrochemical methods using small polarizable electrodes, the interactions of surface-confined biomacromolecules, such as nucleic acids, proteins and carbohydrates (including mutual biomacromolecule interactions, 2
ACCEPTED electric field effects, etc.) can be studied [4].MANUSCRIPT For almost 15 years we used constant current chronopotentiometric stripping (CPS) analysis and Hg-containing electrodes, which allowed observation of the well-developed electrocatalytic peak of peptides and proteins. This peak, termed peak H, was due to the catalytic hydrogen evolution, displaying sensitivity to local and global changes in protein structure [4-8] even for poorly soluble membrane proteins [9,10]. This peak can
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be useful in protein analysis for monitoring protein aggregation [7], denaturation [4,5], glycation [11] and for analysis of protein redox states [4]. New methods utilizing peak H were developed for DNAprotein interactions [12]. This method, using protein signals, is based on new principles, which have
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so far not been used in DNA-protein interaction analysis. It was possible to discriminate between the
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stabilities of tumor suppressor p53 core domain (p53CD) complexes with different DNAs on the basis of their susceptibility to the electric field effects at the electrode by controlling the temperature and/or current density. In this paper we attempted to apply CPS analysis and peak H to investigate lectin-glycoprotein interactions, representing a special case of protein-protein interactions.
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Lectin from Canavalia ensiformis, (Concanavalin A, ConA) is a legume protein, which was extracted from jack bean by the method of Sumner and Howell in 1936 [13]. The unusual biological properties of ConA are directly related to its saccharide binding activity, which in turn, is regulated
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by metal ion binding [14]. ConA has a binding specificity for α-D-glucose and α-D-mannose sugars,
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with weak binding to monosaccharides and stronger binding to oligosaccharides such as those found on the cell surface of glycoproteins and glycolipids [15]. ConA is a metalloprotein, requiring the presence of metal ions for proper binding function; each subunit of ConA contains one manganese (Mn2+) and one calcium (Ca2+) ion [16]. These biological properties of ConA have led to its utilization in various biotechnological applications [17,18].
Ovalbumin (Ova) is a glycoprotein which has a single N-glycosylation site (asparagine 292) occupied by a heterogeneous population of attached neutral glycans. The high-mannose 3
ACCEPTED MANUSCRIPT (predominantly mannopentaose-di-(N-acetyl-D-glucosamine)-Man5GlcNAc2 and mannohexaose-di(N-acetyl-D-glucosamine)-Man6GlcNAc2, (Fig. 1)) and hybrid type are the most abundant [19]. More than 25 distinct, neutral glycans have been reported [20], however, over the 12 glycoforms were observed in very low abundance [21]. Mandal et al. showed that the α(1,6) core arm of Man5 which contains the trimannosyl moiety, is the primary binding epitope for lectin ConA [22]. The
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interaction of ConA-Ova was previously studied by different methods, such as calorimetry, nuclear magnetic resonance dispersion measurements as well as electrochemistry [4,22-25]. Voltammetry was used for Ova detection using ConA labeled by daunomycin [24,25]. Also electrochemical
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impedance spectroscopy was used as label-free method for Ova detection using ConA attached at
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electrode surface [26].
In this paper we attempted to apply CPS analysis to lectin-glycoprotein interaction studies. We found that, using peak H, free lectins and free glycoproteins can be distinguished from the lectinglycoprotein complexes and the time course of the complex formation can be followed. The
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interaction of ConA with Ova was studied in detail; free ConA did not yield any peak H. Formation of the ConA-Ova complex resulted in an increase in peak H, which was tentatively explained by a conformational change in Ova. Similar behavior was also observed in studies of other lectin-
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glycoprotein complexes.
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2. Materials and Methods
2.1. Reagents and Solutions.
Concanavalin A, from Canavalia ensiformis (ConA, 25.6 kDa, 6 arginine (Arg), 12 lysine (Lys), 6 histidine (His) but no cysteine (Cys) residues in the protein monomer; ConA forms a tetramer at neutral pH), Albumin from chicken egg white (Ova, 42.8 kDa, 15 Arg, 20 Lys, 7 His Cys 6), Bovine serum albumin (BSA), RNase B, fetuin, lectin from Ulex europaneus (UEA) and lectin from Sambucus nigra (SNA) were purchased from Sigma-Aldrich. Stock solutions were prepared in triply 4
ACCEPTED MANUSCRIPT distilled water. The final concentration of the solutions was determined spectrophotometrically. All other chemicals were of analytical grade. 2.2. Apparatus. The experiments were performed with a three electrode system connected to a µAutolab III
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potentiostat (Metrohm-Autolab). The working electrode was a hanging mercury drop electrode (area 0.4 mm2) controlled by a VA-stand 663 (Metrohm). Ag|AgCl|3 M KCl was used as the reference electrode and platinum wire as a counter electrode. All experiments were carried out at a temperature
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of 26 °C open to air with constant current chronopotentiometric stripping.
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2.3. Procedures.
Analytes were adsorbed on the HMDE for accumulation time tA at accumulation potential EA in the background electrolyte (50 mM phosphate, pH 7). In Adsorptive Stripping (in situ) experiments: HMDE were immersed into the electrolytic cell (with the stirred solution containing the protein in the background electrolyte). After sample accumulation at EA –0.1 V, for tA 240 s (if not stated
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otherwise), chronopotentiograms or voltammograms were then recorded from the initial potential, Ei of –0.1 V to the final potential, Ef of –2 V. CPS: stripping current Istr of –30 µA (if not stated
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otherwise). Alternating current (ac) voltammetry: step of 4 mV; frequency of 66.21 Hz; scan rate of 15.7 mV/s. Adsorptive Transfer (AdT, ex situ) stripping: Sample was adsorbed at open current
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potential for 60 s (if not stated otherwise) from 5 µL drop. Then the protein-modified electrode was transfer to the electrolytic cell with blank background electrolyte (not containing any protein) to perform CPS.
2.3.1. Complex preparation. Complexes were prepared in 2 different ways. Preparation in solution: Stock solutions of albumin (Ova, RNaseB, fetuin or BSA) and lectin (ConA, lectin UEA or lectin SNA) were mixed in 50 mM phosphate, pH 7 to a final protein 5
ACCEPTED MANUSCRIPT concentration of 6 µM. This mixture in the ratio 1:1 was incubated in a thermomixer for 1 hour at 25 °C and 300 rpm. A special case of complex preparation in solution was preparation directly in the electrolytic cell: Stock solutions of Ova and ConA were mixed in 50 mM phosphate, pH 7 in an electrolytic cell to a final 1 µM concentration. The mixture was stirred and incubated at room
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temperature. After varying periods of incubation chronopotentiograms were recorded. 2.3.2. Solid amalgam electrode preparation
The meniscus solid amalgam electrode (SAE) was prepared as previously described [27,28]. Briefly,
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the silver solid amalgam electrode was dipped into the mercury and a meniscus of liquid mercury was formed at the electrode surface. The SAE was activated by applying a constant potential of –2.2
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V to the electrode in 0.2 M KCl without deaeration for 5 min. A regeneration of the SAE was then carried out by repeated cyclic voltammograms at a scan rate of 10 V/s from 0.1 V to –1.9 V in 50 mM Na-phosphate, pH 7, without deaeration. Scans were repeated 100-times. The activation and regeneration of the SAE preceded each measurement. Further manipulation with protein-modified
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SAE was the same as with HMDE. 2.3.3. Denaturation of Ova and ConA.
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40 µM ConA and 45 µM Ova were denatured in 8 M urea at 4 ºC overnight. The protein solutions were then diluted by Na-phosphate, pH 7 to the final protein concentration. Electrochemical
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measurement with the denatured protein was performed with freshly diluted protein solution. 1 µM ConA was measured in the presence of 200 mM urea and Ova in 177 mM urea (non-denaturing concentration).
2.4. Native agarose gel electrophoresis Fluorescein isothiocyanate labeled ConA (ConA-FITC) was incubated with increasing amount (1-4 µg) of Ova and bovine serum albumin (BSA) in 50 mM Na-phosphate, pH 7. Proteins were mixed with 6x loading buffer (20% sucrose) and were loaded on native agarose gel (1% in 0.33x TBE). The 6
samples were running on the gel ACCEPTED for 2 hours at MANUSCRIPT 70 V in a cold room. Fluorescence of ConA-FITC was detected by scanning of gel on TyphoonFLA 9000 imager (GE Healthcare). 3. Results and discussion Considering the ability of CPS to recognize and detect p53 sequence-specific binding to DNA [12]
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we decided to apply this method for protein-protein interaction studies. For our studies we have chosen the lectin-glycoprotein system [29]. Lectins are proteins well suited to the recognition of glycans on glycoproteins in their natural, intact form [4,17]. Lectin molecules are capable of
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producing a peak H similar to other proteins (not shown), but we found that under conditions when Ova, serum albumins and other proteins (Fig. 2A) [8] produce a peak H, in native lectin ConA this
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peak was absent. Using Ova as a glycoprotein and ConA as a lectin we obtained a system in which we could follow the effect of ConA-Ova binding on the Ova CPS responses. 3.1. Lectin-glycoprotein formation in solutions
A complex of lectin ConA with the glycoprotein Ova was formed in solution. Some conditions of
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complex preparation was taken from ref. [24]. To be sure that the lectin-glycoprotein complex was formed, we tested its formation using ConA conjugated with fluorescein isothiocyanate (ConA-
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FITC) and native agarose gel. Fig. SI-1 shows that ConA migrated in two different fractions in agreement with previous findings [30]. We titrated ConA-FITC with Ova and BSA (BSA was taken
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as negative control). With increasing of Ova concentration, new band appeared indicating complex formation (Fig. SI-1, line 8-10). Addition of BSA as control resulted in no changes of the bands.
Sample of Ova, ConA and ConA-Ova complex was adsorbed at a HMDE at an accumulation potential, EA of ˗0.1 V during an accumulation time, tA of 240 s in 50 mM phosphate, pH 7, followed by chronopotentiogram recording at a stripping current, Istr of ˗30 µA. 1 µM ConA-Ova complex yielded a single well-developed peak H (at a potential, Ep of ˗1.92 V), while a 6-times smaller peak H with two maxima (peak H1 at Ep of -1.93 V and peak H2 at Ep of -1.97) was observed for free 1 7
MANUSCRIPT µM Ova. The difference betweenACCEPTED the peak H of free Ova and ConA-Ova complex was detected across the entire studied concentration range (Fig. 2B, C). With increasing Ova concentration, the peak H area gradually increased up to 1 µM and reached saturation. The peak H of ConA-Ova also increased up to 1 µM concentration, but more steeply. Similar results were obtained by the measuring of dependence of 1 µM Ova and ConA on accumulation time (not shown). In both cases,
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free ConA did not yield any peak H in the given potential window (Fig. 2), probably due to the lower content of electroactive amino acid residues (6 Arg, 12 Lys, 6 His, 0 Cys) in the protein monomer (MW 25 615 Da) as compared to the content of electroactive residues of Ova (15 Arg, 20 Lys, 7 His
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and 6 Cys, MW 42 750 Da) according to ExPASy [31]. In addition to the study of Ova in complex
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with ConA, we tested a mixture of Ova with lectin from Ulex europaeus (UEA), binding fucose residues [17], which does not specifically bind to Ova. The mixture of Ova with UEA yielded a peak H, which was almost the same as the peak H of free Ova (Fig. 2A) across the entire concentration range (Fig. 2C). In contrast to free ConA, free UEA yielded single peak H, which was smaller than peak, of Ova (Fig. 2). Our data are thus in agreement with the literature [4,22-25] showing specific
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interaction of Ova with ConA but not with UEA.
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3.1.1. ConA-Ova complex formation in an electrolytic cell. Using CPS peak H, we investigated ConA-Ova complex formation in solution. In contrast to the
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previous approach, where the complex was formed at a concentration of 6 µM in a test tube, ConA was incubated with 1 µM Ova directly in the electrolytic cell. Stirring accompanied accumulation, followed by CPS recording. The obtained CPS peak H in t = 0 min, i.e. without incubation, yielded a peak H with 2 maxima (peak H1 EP −1.93 V; peak H2 EP −1.97 V) almost the same as the peak of Ova alone. With prolonged incubation time, peak H1 and peak H2 shifted to more negative potentials and peak H1 increased (Fig. 3). After 10 min, the mixture of ConA-Ova yielded only a single peak, which slowly increased with increasing incubation time. After incubation for longer than 60 min, the
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MANUSCRIPT CPS peak H of ConA-Ova did ACCEPTED not change. The increase in peak H after ConA-Ova complex formation could be due to (i) increased accessibility of catalytically active amino acid residues (particularly Arg, Lys, His and Cys) [4], (ii) conformational changes in Ova and/or ConA after complexation and (iii) structural changes after their contact with the electrode. We cannot exclude the possibility that the ConA-Ova complex was adsorbed at the electrode surface in a different way
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than free Ova, and therefore other electro-catalytically active residues could participate in the catalysis. According to the literature, specific binding of ConA to certain oligo- and polysaccharides may involve extended interaction of the protein with several carbohydrate residues [14]. A partial
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change in the circular dichroism spectrum of ConA was obtained after interaction with high mannose
interaction with glycopeptide. 3.1.2. Phase sensitive ac voltammetry.
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type glycopeptides cleaved from Ova [32], suggesting structural changes in ConA resulting from the
Using ac voltammetry in phase-out mode, we studied the adsorption of Ova, ConA and their complex
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at a HMDE. Both voltammograms of the free proteins were similar in potential ranges from -0.1V to -0.4 V and from -0.75 V to -1.9 V, respectively (Fig. 4). In contrast to ConA, Ova produced a peak at about -0.6 V, probably due to the reduction Cys-Hg bonds (a peak at this potential was also obtained
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in phase-in mode, not shown) and consecutive reorientation of the Ova molecule at the electrode surface. We observed similar voltammograms for the ConA-Ova complex to that of free Ova, with
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about a 40% smaller peak at -0.7 V. We can assume that the cysteine residues in Ova interacted with the electrode even after the complex formation with ConA. 3.2. Dependence on Istr
The rate of the overall process in CPS is imposed by the value of the applied current (Fig. SI-2). It is important that the electrode potential shifts slower during an electrode process than in its absence, when potential changes can reach extremely high rates [4,5]. This feature is critical in protein structure analysis [4]. To obtain peak H at highly negative potentials, such as −1.9 V the surface9
ACCEPTED attached protein is exposed to the electricMANUSCRIPT field effects at negative potentials causing unfolding/denaturation of the native protein [4,5] or dissociation/disintegration of the DNA-protein complex [12]. These processes depend on the time of exposure at negative potentials, which is related to stripping current (Istr) intensity [4,8]. By applying high negative current intensities to a relatively small working electrode, exposure to negative potential can be reduced to milliseconds [8].
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Firstly, we looked at the dependence of the signals of native (natOva) and urea-denatured Ova (denOva) on the Istr intensities at a constant temperature (26 °C, Fig. 5A). Between −10 µA and −20 µA the peak areas of natOva and denOva were almost the same, suggesting electric field driven
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denaturation of natOva due to prolonged exposure to the electric field. On the other hand at Istr
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intensities more negative than −24 µA, the peak H area of natOva was several times smaller than that of denOva and the ratio of denOva/natOva increased at more negative Istr intensities (not shown). Between −20 µA and −24 µA, structural transition due to the destabilization of natOva by electric field effects was observed. The presented data were in good agreement with the data obtained from BSA and other proteins [8,33]. Subsequently, we compared the Istr signal dependences of the ConA-
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Ova complex prepared in solution to those of free natOva and denOva (Fig. 5A). At Istr intensities, where the peak H areas of natOva and denOva were almost the same, the peak H area of the complex was about 20 % smaller than those of free Ova´s. The peak H of the ConA-Ova complex was shifted
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by about 30 mV to more negative potentials as compared to the peaks of Ova, suggesting a more
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difficult reduction process. A similar difference between the peak H of the complex and free denOva was observed for all studied Istr intensities (Fig. 5). We can speculate that the smaller peak H of the ConA-Ova complex than the peak H of denOva could be caused by changes in the accessibility of electro-active residues (Lys, Arg, His and Cys), as well as different adsorption of free Ova and its complex with ConA at the charged interface. With the shift of Istr intensities to values more negative than −20 µA, the difference between the peak H of natOva and ConA-Ova increased (Fig. 5A, right axis). At Istr −30 µA the peak area of ConA-Ova was about 6-times higher than the peak area of free natOva. The mixture of natOva and lectin UEA, which did not interact specifically with Ova, yielded 10
almost the same Istr dependence ACCEPTED as that of free MANUSCRIPT natOva, confirming our suggestion that using our method, specific Ova binding to ConA can be detected at a wide range of Istr values close to room temperature. We denatured ConA in order to better understand its CPS behavior. We found that denatured ConA (denConA) produces a peak H under conditions (Fig. 5B) in which, this peak was absent in native ConA (Fig. 1A). The peak H of denConA appeared at potentials by 100 mV more
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negative than the peak of the ConA-Ova complex (Fig. 5B) at less negative Istr intensities. The appearance of the peak H of denConA can be explained by the increased accessibility of the electroactive amino acid residues as compared to native tetrameric ConA in which many of these
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residues can be buried in its structure. To obtain more data on the behavior of native and denatured
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ConA we measured the dependence of their CPS responses on Istr. Between −10 µA and −16 µA, the peak H area of denConA was almost the same as the peak area of denOva. At more negative Istr intensities, the peak H of denConA gradually decreased and at Istr intensities, under which the peak H of the complex was higher than the peak H of Ova, the peak H of denConA was smaller than that of free natOva. The appearance of the peak for denConA indicates that ConA might also contribute to
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the increase in the peak H of the complex. Changes in ConA structure after glycan binding have been previously described [32]. To our knowledge, conformational changes in Ova after ConA-Ova complex formation have not yet been described. Our results suggest that Ova also underwent
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structural changes, leading to the peak H increase, after complex formation. At the Istr intensities
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where the difference between natOva and the ConA-Ova complex was observed, denOva yielded a higher peak than the peak of the ConA-Ova complex while denConA produced a peak H smaller than that of natOva (Fig. 5A), suggesting changes in Ova binding in the ConA-Ova complex. Further work should be done to elucidate the adsorption behavior of the ConA-Ova complex at charged interfaces in more detail.
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3.3. Solid amalgam electrode
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No doubt, HMDE offers better sensitivity and reproducibility (less than ±5 %, Fig. 2A) than solid electrodes but it is less convenient for parallel analysis of biomacromolecules. On the other hand SAE yielded similar results to those obtained with HMDE, with the reproducibility of solid
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electrodes (about ±10 %, Fig. 6B). Here we used a SAE to test its suitability for the study of lectinglycoprotein interactions under the same conditions as used for HMDE. Firstly we followed dependence of peak H area on accumulation time, tA for 1 µM Ova and ConA-Ova complex (Fig. 6A). With prolonging tA, peak H area of Ova gradually increased up 240 s and leveled off. The peak
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H area of ConA-Ova complex steeply increased up to tA 180 s and did not change at longer tA. For
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further experiments with SAE, tA of 240 s were chosen. In contrast to the previous experiments at SAE, an adsorptive transfer (ex situ) method was used; 1 µM sample was adsorbed for 240 s at open current potential from 5 µL drop at the electrode; modified SAE was transferred to background electrolyte followed by chronopotentiogram recording. Almost the same results were observed for SAE by both methods (ex situ and in situ method; Fig. 6B, C) but much smaller quantities of proteins
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could be used for the ex situ method. At the SAE, ConA, Ova and the complex of Ova with ConA and UEA yielded results very similar (Fig. 6C) to those obtained with the HMDE (Fig. 1A). As
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described earlier [5,28,34], SAEs offer equally reproducible results to other solid electrodes and can be easily miniaturized and applied in protein arrays [34].
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3.4. Other lectin-glycoprotein complexes In addition to the ConA-Ova complex, we studied a complex of ConA with another glycoprotein belonging to the high mannose type, namely RNase B (Fig. 7A) containing a single N-linked glycosylation site [21]. Similarly to the Con-Ova complex, the ConA-RNase B complex yielded a higher peak H shifted to more negative potentials than the peak H of free RNase B within a wide concentration (Fig. 7A, SI-4A). On the other hand RNase A, the nonglycosylated form of RNase B, yielded almost the same signal when in a mixture with ConA as that of the free protein (Fig. 7B, SI12
ACCEPTED 4A), in good agreement with previous findingsMANUSCRIPT [35]. Similar results were observed also for the combination of the lectin Sambucus nigra (SNA) and the glycoprotein fetuin (Fig. 7C). In contrast to ConA, the lectin SNA, containing Cys residues, yielded a peak H which appeared at more positive potentials than the peak H of fetuin (Fig. 7C). The complex of SNA with fetuin again produced a peak H higher than the individual peaks H of the free lectin and glycoprotein between 1 µM and 2.5
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µM (Fig. SI-4B). In contrast to the ConA-Ova and ConA-RNase B complexes, the peak potential of the SNA-fetuin complex was more positive than the peak of the glycoprotein, which could be due to
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the contribution of an intrinsic signal of the lectin SNA.
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4. Conclusion
The investigation of protein-protein interactions belongs to one of the most important fields in current molecular biology, which affects various areas of biomedicine. It is predicted that 50-80% of human proteins are posttranslationally modified by glycans, i.e. glycoproteins [36-38]. Two
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molecules of the same protein from the same tissue may have different glycans [39]. Changes in glycan identity in a glycoprotein can have a dramatic effect in pathological processes [4,17]. Despite newly emerging and powerful methods such as biochips/arrays, high throughput mass spectroscopy,
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liquid spectroscopy and nuclear magnetic resonance [36,40,41], new methods for the study of protein-protein, as well as lectin-glycoprotein interactions are still sought, representing a challenge
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for electrochemists. CPS in combination with mercury-containing electrodes was utilized here for the first time in the analysis of glycoprotein-lectin interactions. We show that lectin ConA, specifically bound to the glycoprotein Ova, yielded peaks differing from the peaks H of free Ova and ConA at concentrations starting from about 100 nM (Figs. 1-5) at the given experimental conditions. Lower concentrations could be reached by further optimization of the method (Fig. SI-3). Also, other lectinglycoprotein couples, namely ConA-RNase B and lectin SNA-fetuin were tested and similar results were obtained after their interactions. The combination of CPS with solid amalgam electrodes
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ACCEPTED MANUSCRIPT [28,34], amenable for parallel analysis, yields instant results and does not require any labeling or modification of the protein under study. Moreover, µL volumes can be used for the adsorptive transfer method, which allows determination of the ConA-Ova complex in picomole amounts (Fig. 6, SI-3). Protein-protein interactions have been little studied by electrochemical methods. Here, we introduce
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new principles in this analysis of lectin-glycoprotein interactions. The novelty of our method can be seen in measuring the protein response resulting from the lectin-glycoprotein binding. So far, voltammetry was used for Ova detection using ConA labeled by daunomycin [24,25]. Also
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electrochemical impedance spectroscopy (EIS) was used as label-free method for Ova detection
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using ConA attached at electrode surface [26] (see Table 1 in SI for more details). In EIS dielectric and electric properties of the surface-attached lectin resulting from its interaction with glycoprotein were measured. Our CPS method is highly sensitive to changes in protein structure [4,5,10,33] and in addition to a mere detection of the lectin-glycoprotein binding it can, after its further development, contribute to the elucidation of the conformational changes resulting from the lectin-glycoprotein
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interactions. Our previous work showed that CPS is protein structure-sensitive method detecting various kind of conformational changes in proteins. For example, (i) early changes preceding aggregation of α-synuclein (involved in Parkinson’s disease) were detected by peak H in accordance
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with the dynamic light scattering results. These changes were, however, not detectable by
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fluorescence measurements [7]; (ii) changes of BSA damaged by singlet oxygen detected by CPS were in agreement with mass spectrometric and optical spectroscopic data [33,42]; (iii) the CPS peaks H of wild-type and mutant of tumor suppressor p53 showed excellent correlation with structural and stability data and provided additional insights into the differential dynamic behavior of the proteins [6]. These results suggested that CPS peak H has a number of advantages [4]. Compared to established methods of protein analysis, such as mass spectrometry, optical spectroscopy and nuclear magnetic resonance, etc, electrochemical methods are inexpensive, fast, simple and more convenient for 14
MANUSCRIPT diagnostics, because they do not ACCEPTED require expensive instrumentation. In addition advantages of CPS analysis include (i) high sensitivity of protein determination and small volume requirements (few µL of nanomolar protein concentration are usually sufficient for ex situ method) (ii) high sensitivity to changes in protein structures, (iii) easy miniaturization, and development of chips for parallel analysis. We can conclude that the proposed CPS method can after its further development
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complement the established methods. Acknowledgements
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This study was supported by project No. 13-00956S to VO from the Czech Science Foundation.
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References
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ACCEPTED MANUSCRIPT Figures legends Fig. 1 Structure of high mannose glycans A. mannopentaose-di-(N-acetyl-D-glucosamine) and B.
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mannohexaose-di-(N-acetyl-D-glucosamine)
Fig. 2 A. CPS peak H of 1 µM ovalbumin (Ova, black), concanavalin A (ConA, cyan), lectin from Ulex europeus (UEA, blue), ConA-Ova complex (red) and mixture of Ova and lectin UEA (magenta)
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at a hanging mercury drop electrode (HMDE) in 50 mM Na-phosphate, pH 7. Dependence of B. peak H potential, EP and C. peak H area on concentration of native () and denatured () Ova (black),
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ConA (▼, cyan), lectin UEA (◊, blue), ConA-Ova complex (●, red) and mixture of Ova with lectin UEA (▲, magenta). D. CPS peaks H of 100 nM Ova (solid black line), ConA-Ova complex (solid red line) and background electrolyte (dashed line). Protein was adsorbed at an accumulation potential, EA of −0.1 V for an accumulation time, tA of 240 s, followed by chronopotentiogram recording with
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Istr of −30 µA at 26 °C. Complex preparation in solution: 6 µM ConA (or UEA) was incubated with 6 µM Ova in 50 mM Na-phosphate, pH 7 for 1 h at 25 °C. Agitation at 300 rpm accompanied
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incubation.
Fig. 3 Dependence of A. EP and B. peak area on the incubation time of 1 µM ConA with 1 µM Ova
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incubated in 50 mM phosphate, pH 7 in an electrolytic cell. C. CPS peak H of 1 µM ConA incubated with 1 µM Ova for the given time. Other details as in Fig. 2.
Fig. 4 Ac voltammogram of 1 µM Ova (black), ConA (cyan) and ConA-Ova complexes (red) and background electrolyte (dashed line).
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ACCEPTED MANUSCRIPT Fig. 5 A. Dependence of peak H area on Istr for natOva (, solid line, black), denOva (, dashed line, black), denConA (∆, blue), a mixture of Ova with UEA (▲, magenta) and ConA-Ova complex (●, red). Right axis: Dependence of peak area ratio of ConA-Ova complex to Ova (green) on Istr. B. CPS peak H of 1 µM denatured Ova (dashed line, black), denConA (blue) and ConA-Ova complex (red)
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at Istr of −30 µA. Other details as in Fig. 2
Fig. 6 A. Dependence of peak H area on tA for 1 µM natOva (, black) and ConA-Ova complex (●, red) at SAE. B. CPS peak H of 1 µM Ova (black) and ConA-Ova complex (red) at tA of 240 s at SAE.
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Other details as in Fig. 1 C. Adsorptive transfer CPS peak H of 1 µM Ova (black), 1 µM ConA
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(cyan), lectin UEA (blue), ConA-Ova complex (red) and mixture Ova with 1 µM UEA (magenta) at a meniscus amalgam electrode in 50 mM Na-phosphate, pH 7. Istr of ˗40 µA. Protein was adsorbed at open current potential for tA of 240 s from 5 µL drop at electrode. Modified SAE was transferred in background electrolyte for CPS recording.
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Fig. 7 Adsorptive transfer CPS peak H of 1 µM A. RNase B (black), B. RNase A (gray), ConA (cyan) and their complexes with ConA (orange, yellow) and of C. 1 µM fetuin (black), lectin
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Sambucus nigra (SNA, blue) and SNA-fetuin complex (magenta). Istr of A., B. -50 µA, C. -20 µA
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ACCEPTED MANUSCRIPT Highlights Label-free electrochemical methods for detection of glycoprotein-lectin interactions
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Chronopotentiometric detection of ovalbumin-concanavalin A complex formation
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Conformational changes in lectin-glycoprotein complex detected by peak H
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