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1 (This is a sample cover image for this issue. The actual cover is not yet available at this time.) This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. ther uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier s archiving and manuscript policies are encouraged to visit:

2 Electrochemistry Communications 23 (2012) Contents lists available at SciVerse ScienceDirect Electrochemistry Communications journal homepage: Protein reducing agents dithiothreitol and tris(2-carboxyethyl)phosphine anodic oxidation Inês Barreira Santarino, Severino Carlos B. liveira, Ana Maria liveira-brett Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Coimbra, Portugal article info abstract Article history: Received 23 May 2012 Received in revised form 15 June 2012 Accepted 19 June 2012 Available online 29 June 2012 Keywords: Dithiothreitol Tris(2-carboxyethyl)phosphine Proteins xidation Glassy carbon electrode Protein reducing agents dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) are crucial to disrupt disulfide bonds for qualitative and quantitative analysis in proteomics. Protein bond disruption is very important for analyzing proteins as single subunits. The electrochemical oxidation mechanism of DTT and TCEP was investigated using cyclic and differential pulse voltammetry over a wide p range on a glassy carbon electrode. The oxidation mechanism of both compounds is irreversible, p-dependent and proceeds in a single step. The anodic oxidation of DTT is associated with the oxidation of the sulfhydryl group with lowest pk a, while the anodic oxidation of TCEP is associated with the oxidation of the phosphine group in the molecule. The diffusion coefficients of DTT and TCEP were calculated, and the redox mechanisms are proposed Published by Elsevier B.V. 1. Introduction The interest in proteomics as a tool for drug development and innumerable other applications continues to expand at a fast rate. Proteomic analysis has recently been conducted on tissues, biofluids, subcellular components and enzymatic pathways related to various disease and toxicological states, in both animal and human models. Several recent studies have attempted to integrate proteomics data with genomics and/or metabolomics data in a biologic system approach. Among the methods which contributed to proteomic data analysis are X-ray diffraction analysis, nuclear magnetic resonance, electron and scanning force microscopies, electrophoresis, MALDI-TF mass spectrometry and chromatography. Two-dimensional polyacrilamide gel electrophoresis (2D-electrophoresis) is capable of resolving over 1800 proteins in a single gel, being important as the primary tool in proteomics research where multiple proteins must be separated for parallel analysis [1]. Due to protein structural complexity, the use of the protein reducing agents dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), Fig. 1, are crucial to disrupt disulfide bonds for qualitative and quantitative analysis in proteomics [1 5]. DTT is a thiol reducing agent that is often used in high concentration to force equilibrium towards reducing cysteines. owever, disulfide reduction by thiols has some disadvantages because, when reacting protein sulfhydryls react with extrinsic probes, the S groups of the reductant compete directly with those of the protein, for attachment of thiol-reactive labels. Therefore, they have to be removed from the Corresponding author. address: brett@ci.uc.pt (A.M. liveira-brett). sample before the protein is labeled. Also, DTT is not stable for a long time in the reduced form, and solvent-inaccessible disulfide bonds are difficult to be reduced by DTT, so reduction of disulfide bonds is sometimes carried out under denaturing conditions, at high temperatures, or in the presence of other denaturing agents, such as urea or sodium dodecyl sulfate (SDS) [3,4]. DTT is used in all techniques which involve electrophoresis, such as Western blot, and it has recently been applied in zymography to determine protein activity [6]. Thiol groups usually immobilize DNA on gold surfaces in biosensors. A common use of DTT is as a deprotecting thiolated DNA. The terminal sulfurs of thiolated DNA have a tendency to oxidize and form dimers in solution, especially in the presence of oxygen. Normally DTT is mixed with a DNA solution and allowed to react, and is then removed by filtration (solid catalyst) or by chromatography (liquid form). TCEP, compared with DTT, has been shown to be a significantly more suitable and selective reductant in acid media, since it does not react with other functional groups found in proteins and also because its removal is not necessary before labeling the protein sample. TCEP is generally impermeable to the hydrophobic protein core, allowing its use for the selective reduction of disulfides that have aqueous exposure [2,4,5]. owever, TCEP is two times more expensive than DTT. Thus, although TCEP has advantages over DTT, the choice of reductant depends on the application specificity [4]. Research on the redox behavior of thiols and phosphines using electrochemical techniques has the potential to provide valuable insights into the redox reaction mechanisms of these molecules that are very relevant for evaluating their reductant activity at sulfhydryl and phosphine groups. Voltammetric methods, due to their high sensitivity, have been used to study the redox behavior of thiols [7] and /$ see front matter 2012 Published by Elsevier B.V. doi: /j.elecom

3 I.B. Santarino et al. / Electrochemistry Communications 23 (2012) A 1 a B DTT 40 na TCEP 1 a 10 na p E / V (vs. Ag/AgCl) p E / V (vs. Ag/AgCl) C 1.4 E pa / V (vs. Ag/AgCl) P TCEP P DTT p D E S - S -2e P -2e P S S DTT TCEP Fig. 1. 3D plot of DP voltammograms base line corrected in 50 μm: (A) DTT and (B) TCEP as a function of p; (C) plot of DTT peak 1 a ( ) E pa and of TCEP peak 1 a ( ) E pa vs. p; proposed oxidation mechanisms: (D) DTT and (E) TCEP. phosphines [8] in solution. owever, concerning DTT and TCEP, their redox properties have only been studied by cyclic voltammetry, in order to characterize the redox behavior of proteins, using a mercury electrode and in specific conditions [9,10]. The oxidation processes of DTT and TCEP, owing to their importance as reducing agents in biochemistry and numerous other applications in biotechnology, need clarification. The objective of this paper is the investigation of the anodic behavior of DTT and TCEP, Fig. 1,forawiderangeof solution conditions using cyclic and differential pulse voltammetry at a glassy carbon electrode. DTT and TCEP are oxidizable and clarification of their oxidation pathways can provide a model for the mechanistic study of electron transfer between DTT-protein and TCEP-protein and also with solid surfaces, and establish the support for fabrication of new biosensors and biomedical devices. 2. Experimental Dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) were obtained from Sigma and used without further purification. A stock solution of 0.20 M of DTT was prepared in deionized water and stored at room temperature. TCEP solution of the desired concentration was prepared directly in the buffer electrolyte solution prior to use. The supporting electrolyte solutions prepared with analytical grade reagents and purified water from a Millipore Milli-Q system

4 116 I.B. Santarino et al. / Electrochemistry Communications 23 (2012) (conductivity 0.1 μs cm 1 ), were: 0.1 M acetate buffer 3.5bpb5.5, 0.1 M phosphate buffer 7.0bpb8.0, 0.1 M borate buffer 7.0bpb1 for TCEP, 0.1 M sodium hydroxide 9.0bpb1 for DTT. The p measurements were carried out with a Crison microp 2001 p-meter with an Ingold combined glass electrode. All experiments were done at room temperature (25±1 C). Voltammetric experiments were carried out using a μautolab potentiostat with GPES 4.9 software, Metrohm/Autolab, Utrecht, The Netherlands. Measurements were carried out using a glassy carbon working electrode (GCE) (d=1.5 mm), a Pt wire counter electrode, and an Ag/AgCl (3 M KCl) as reference electrode, in a 2 ml onecompartment electrochemical cell. The experimental conditions for differential pulse (DP) voltammetry were: pulse amplitude 50 mv, pulse width70ms,andscanrate5mvs 1. The GCE was polished using 1 μm diamond spray (Kemet, Kent, UK) before every electrochemical assay. After polishing, the electrode was rinsed thoroughly with Milli-Q water. Following this mechanical treatment, the GCE was placed in buffer supporting electrolyte and various DP voltammograms were recorded until a steady state baseline voltammogram was obtained. 3. Results and discussion The oxidation behavior of DTT, Fig. 1D, and TCEP, Fig. 1E, at a GCE was studied by cyclic voltammetry (CV) in electrolytes with different ps. CVs of 500 μm DTT in acid media, 0.1 M acetate buffer p=4.0, showed, at a high positive potential, one irreversible oxidation peak 1 a, at E p1a =+1.33 V, corresponding to the oxidation of the S group. CVs of 500 μm DTT in neutral media, 0.1 M phosphate buffer p=7.0, showed the anodic peak 1 a,ate p1a =+4 V, and the peak current decreased in successive scans. Compared with acid media, the oxidation potential of the peak 1 a was shifted to a less negative value with increasing p and the peak current was higher. DTT is only oxidized at high positive potentials, near the potential of oxygen evolution. CVs of 500 μm TCEP in acid media, 0.1 M acetate buffer p=4.0, showed one irreversible anodic peak 1 a,ate p1a =+1 V, associated with the oxidation of the phosphine group in the molecule and the peak currents decreased in the successive scans. CVs were also obtained at different scan rates in 500 μm DTT, in 0.1 M phosphate buffer p=7.0, and in 500 μm TCEP, in 0.1 M acetate buffer p=4.0, and both molecules presented a very similar behaviour. The peak currents for scan rates 5 mv s 1 ν 900 mv s 1 were: for DTT I p1a (μa)= ν 1/2,andforTCEPI p1a (μa)= ν 1/2, showing a diffusion controlled process, in agreement with the hydrophilic character of DTT and TCEP at these ps [2 5]. The effect of p for low concentrations of DTT and TCEP on the electrochemical oxidation was studied using DP voltammetry in supporting electrolytes 2.0 p 12.0, in order to evaluate the number of electrons and protons involved in the oxidation of DTT and TCEP. DP voltammograms in 50 μm DTT in 3 p 10 showed that the oxidation occurs in one single step, peak 1 a, and the potential shifted to more negative values with increasing p, Fig. 1A, indicating the existence of an acid base equilibrium close to the electrochemical reaction and that protons are involved in the oxidation process. The thiol group with the lowest pk a =8.3 is deprotonated and oxidized first [11]. Considering the E pa vs. p plot, Fig. 1C, the slope of the dotted line is 30 mv per p unit, and half-height width of peak 1 a, the oxidation reaction involves two electrons and only one proton. The peak 1 a current versus p, Fig. 1A, shows that the peak currents are much higher for 2.0bpb8.0, due to the effect of p on ionization of the S group with the lowest pk a =8.3. Since that at alkaline p the S group is fully ionized (deprotonated), increasing DTT hydrophilicity and consequently decreasing DTT adsorption on the GCE surface was observed. A mechanism for the oxidation of DTT is proposed, where DTT peak 1 a corresponds to the oxidation of the sulfhydryl group with the lowest pk a =8.3, involving two electrons and followed by deprotonation and formation of a radical that, after direct nucleophilic attack by water, produces the final oxidation product, based on the CV and DP voltammetry results and in agreement with the literature [7], Fig. 1D. DP voltammograms in 50 μm TCEP in different electrolytes showed one p-dependent oxidation peak, peak 1 a, the potential shifted to more negative values with increasing the p and the highest peak current was for p=3.0, Fig. 1B and C. For 3 p 9 the p-dependence was linear with a slope of 30 mv per p unit, Fig. 1C, and considering the half-height width of peak 1 a, the oxidation involves two electrons and only one proton. The effect of p on the peak currents was also examined, and in 50 μm TCEP the most sensitive response was observed in acidic media, Fig. 1B, and in neutral media the TCEP peak currents were much smaller. Successive DP voltammograms in 0.1 M borate buffer p=7.0 clearly demonstrated that the electrode surface is rapidly blocked by the polymerization of the TCEP oxidation product. To remove the polymeric film adsorbed on the GCE surface, it was necessary to place the GCE in ethanol for 15 min in the ultrasonic bath and, prior to polishing, 10 CVs were performed in acid supporting electrolyte. A mechanism for the oxidation of TCEP is proposed, Fig. 1E, where peak 1 a corresponds to the oxidation of phosphine in the molecule with the transfer of two electrons and one proton, involving the formation of a radical that after direct nucleophilic attack by water produces the final oxidation product. In another pathway, the radical can react with another radical starting the polymerization process, leading to the adsorbed products on the electrode surface. It was found that the relative reaction rates of these two pathways depend on the p of TCEP solution. CVs for different scan rates were performed and considering the number of electrons transferred n=2 determined by DP voltammetry, the diffusion coefficients of DTT in p=7.0 D DTT = cm 2 s 1,and TCEP in p 4.0 D TCEP = cm 2 s 1, were determined. For this calculation, the GCE electroactive area was determinated by CV from a plot of I pa vs. ν 1/2 using a solution of 2.5 mm hexacyanoferrate and the diffusion coefficient of hexacyanoferrate in phosphate buffer was D = cm 2 s 1 [12]. 4. Conclusions The electrochemical oxidation study showed that DTT and TCEP undergo oxidation at a GCE and the oxidation mechanism of both compounds is irreversible, p-dependent, and proceeds in a single step. The anodic oxidation process of DTT occurs at the sulfhydryl group with lowest pk a while TCEP occurs at the phosphine group in the molecule following a similar mechanism, involving two electrons followed by deprotonation and formation of a radical that after direct nucleophilic attack by water produces the final oxidation product. The diffusion coefficients D DTT = cm 2 s 1 and D TCEP = cm 2 s 1, were determined and the oxidation mechanisms proposed. The research on the redox mechanisms of reducing agents DTT or TCEP is crucial to understand the disruption of disulfide bonds for qualitative and quantitative analysis in proteomics. Acknowledgments Financial support from Fundação para a Ciência e Tecnologia (FCT), Portugal, BTID grant (I.B. Santarino), Post-Doctoral grant SFR/BPD/71965/2010 (S.C.B. liveira), project PTDC/QUI/098562/ 2008, PP (co-financed by the European Community Funds FSE e FEDER/CMPETE), CEMUC-R (Research Unit 285), are gratefully acknowledged. References [1] W. Reiner, T. Naven, Proteomics in Practice, Wiley-VC Verlag Gmb, Berlin, [2] J.A. Burns, J.C. Butler, J. Moran, G.M. Whitesides, Journal of rganic Chemistry 56 (1991)

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