Modification of metal substrates and its application to the study of redox proteins

Stability and Stabilization of Biocatalysts A. Ballesteros, F.J. Plou, J.L. Iborra and P.J. Hallir~(Editors) 9 1998 Elsevier Science B.V. All rights reserved.

697

M o d i f i c a t i o n o f m e t a l s u b s t r a t e s a n d its a p p l i c a t i o n to the s t u d y o f r e d o x proteins T. Pineda, J. M. Sevilla, A. J. Roman, R. Maduefio and M. B lazquez. Departamento de Quimica Fisica y Termodin~imica Aplicada, Facultad de Ciencias, Universidad de C6rdoba, 14004 C6rdoba, Spain.

This work deals with a comparative study of the surface modification of metal substrates (gold, mercury and platinum) by chemisorption of 6-mercaptopurine (6MP). Experimental conditions for film formation were defined and tested in ex-situ voltammetric experiments. A self-assembled monolayer (SAM) seems to be formed by judging from desorptive reduction peaks and supression of hydrogen adsorption region. The 6MP-Pt substrate allows to observe promoted quasi-reversible stable electrochemistry of metalloprotein cyt c similar to that reported on 6MP-Au and 6MP-Hg electrodes. Differences in metal-S bonding strength are evidenced but lateral interactions of 6MP-adsorbed molecules seems to play a main role on the behavior of the modified substrate.

1. INTRODUCFION The 6-mercaptopurine (6MP) molecule is able to chemisorb to the metal atoms by covalent bond through the S atom. The strength of the bond between the sulfur and a gold atom is on the order of 40-50 kcal/mol [ 1]. This high affmity of the sulfur atom for the metal together with the favorable interactions between the close-packed groups makes possible a high organization in the monolayers (SAM, self-assembled monolayers) obtained by these thioderivatives. Therefore, the high affmity of gold and other metals towards sulfur adsorption allows a diverse range of functional groups to be incorporate into the SAM or onto the exposed surface of the SAM. The electrochemical reactivity of metalloproteins at bare metal electrodes is often highly irreversible and in some cases undetectable. The rapid growth in the research related to the direct electron transfer between metalloproteins and electrodes came after the first clear demonstrations of quasi-reversible and direct electrochemistry of cyt c at a 4,4'-bipyridinemodified gold electrode [2,3]. Different promoter molecules have been studied and, it has been concluded that at least two functional groups are required to promote direct electron transfer between metalloproteins and electrodes: one of them should be able to bind to the electrode surface and the other, to show a suitable orientation to allow a favourable interaction with the electron transfer domain of the protein [4,5]. On the course of our studies on characterization of adsorbed monolayers of 6MP on gold, platinum and mercury substrates, we have tested the electrochemical response of cyt c. The 6MP molecule has proved to be a good promoter for the electrochemistry of cyt c as it has

698 been reported by Taniguchi et al., which used a 6MP-modified gold electrode [6] and in characterizing the molten globule conformation in cyclic voltammetric studies [7]. In this work we will analyze the electrochemical response of cyt c obtained at the different substrates, gold, platinum and mercury, all of them modified with 6MP.

2. EXPERIMENTAL Horse heart cyt c (type VI) and 6MP were purchased from Sigma. KOH, semiconductor grade, was purchased form Aldrich. All other reagents were of Merck p.a. grade and were used without purification. As the supporting electrolyte, buffered solutions of 0.025 M phosphoric acid at pH 7.0 in 0.1 M NaCIO4 or 0.1 M KOH were used. Milli-Q purified water was used throughout to prepare solutions. All electrochemical measurements were performed at a temperature of 25+0.1~ using thermostated Metrohm cells. The reference electrode was a saturated calomel electrode (SCE) and a platinum wire served as a counter electrode. Mercury, gold and platinum were used as working electrode. The mercury electrode was a Metrohm EA 290 hanging mercury drop electrode (HMDE) with a surface area of 0.0139 cm 2. The gold electrode was a 2 mm diameter sphere made by melting a 0.5 mm diameter gold wire in a flame at natural gas/air, resulting a few facets visible on the surface. The platinum electrode was a disk of 1.6 mm diameter from BAS (Bioanalytical Systems). Voltammetric curves were recorded on an Autolab (Ecochemie model Pgstat 20) instrument attached to a PC computer with proper software (GPES) for totally control of the experiments and data acquisition and treatment. The modified electrodes were prepared using the following procedure: the gold and platinum electrodes were sequentially polished with 0.3 and 0.05 ~tm alumina + water slurries until a shiny mirror-like finish was obtained. The electrodes were then sonicated and washed with deionized water (milli-Q). In the case of the mercury electrode, a fresh mercury drop was used in each experiment. Surface modification of the electrodes was carried out by the film transfer method of dipping the clean electrode into a solution of 100 lxM 6MP for a determined time (1 and 15 min for mercury and gold and 24 hours for platinum, respectively) following by rinsing with distilled water. For the experiments with cyt c, a homemade mierocell with an optimum volume of 500 ~tl, was coupled to the conventional cell, as a separate compartment, and connected by a built-in frit terminal.

3. RESULTS AND DISCUSSION The chemisorption of 6MP on a metal surface changes the properties of the interface. The first step in this study is to find the potencial range where the interface remains modified. At high potential values, oxidative desorption of 6MP occurs coinciding in the case of gold and platinum with the oxide monolayer formation. On the other hand, at low potential values reductive desorption could take place. It is interesting to note that the reductive desorption could be used to evaluate the surface coverage of the electrode by integration of the

699 voltammetric peak obtained under these conditions. The following reaction is thought to occur for the desorption of these molecules: M-S-R + H § + e ~ M + HS-R. Figure 1 shows the voltammograms obtained for the three substrates under the conditions where a complete monolayer is thought to exist. In order to observe the reductive desorption for the substrate, is necessary to transfer the modified electrode to a basic solution (i.e., 0.1 M KOH). At lower pH, only the desorption from mercury is observed with a good shape in the voltammogram. In the case of gold the signal is very spread compared to that in alkaline media (Fig. 2).

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-400

-800 E/mV

- 1200

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B

-

Figure 1. Cyclic voltammograms of reductive desorption of a 6MP monolayer from (A) mercury, (B) platinum and (C) gold electrodes in 0.1 M KOH solution. Scan rate 100 mV/s. Scale: (A), (B) 50 gA/cm 2, (C) 10 gA/cm 2.

0

-400 E/mV

-800

Figure 2. Cyclic voltammograms of reductive desorption of a 6MP monolayer from (A) mercury and (B) gold electrodes in 0.1 M acetic acid solution (pH 6). Scan rate 100 mV/s. Scale: (A) 50 ~tA/cm2, (B) 5 gA/cm 2.

One interesting aspect of this reductive desorption phenomena is that the potential depends strongly on the substrate nature. Then, in the case of mercury the process occurs at less negative potential and only one sharp peak is observed. This behaviour has been reported in acid medium as being due to the destruction of a condensed 2D phase [8]. Figure 1 also shows the voltammogram corresponding to the reductive desorption of the monolayer built over the gold electrode. Several peaks are observed, some of them assigned to the desorption of thio-compounds from a specific monocrystalline facet. Recent studies on reductive desorption of alkanethiolate on different gold substrates have showed that the peak potential is dependent on the surface crystallinity of the underlying gold substrate and on the differences in the binding strengths of adsorbates at terraces and step sites [9]. In the case of a platinum substrate, it seems that the reduction of the M-S bond takes place at potential close to the hydrogen evolution. A difference between the first and second sweep is observed but attempts to quantify the charge did not give any proper results. From these reduction peaks corresponding to the desorption of the 6MP molecule from the different substrates it can be concluded that the energy of the metal-sulfur bond increase in the sense Hg
700 Once the three modified substrates are characterized in term of protection by a film, we check the voltammetric response of the metalloprotein cyt c by using these substrates as electrodes. We take the advantage that in all cases, the redox signal of cyt c is able to be observed within the range of potentials where the modification is stable, since that signal always takes place at +0.2 to-0.2 V range.

Table 1 Electrochemical parameters of the electron transfer of cyt c Electrode

Epe/mV

Epa/mv

AEp(a)/mV E~

E~

103x ksh(e)/cm s~

6MP-Au

-33

44

77

5

5

4.0 (d)

6MP-Hg

-34

44

78

6

5

4.1 (d)

6MP-Pt

-50

53

103

2

3

2.3

(a) v=40 mV/s (b) The midpoint potential. (c) Obtained by digital simulation (d) From ref. 10 In Table 1 the redox parameters obtained for cyt c at the three substrates are gathered. A sample of the voltammograms obtained are plotted in Figure 3. Taking together the results on the Table 1 and comparing the voltammograrns is possible to conclude that the three substrates allow to observe direct electrochemistry of cyt c. The value of the formal redox potential, E ~ measured as the midpoint between anodic (Epa) and catodic (Epe) peak potentials of the reversible voltammogram, is very close for the three systems studied. However, the anodic and cathodic peak potentials separation from Pt and the other substrates indicate that some differences occur on the electron transfer rate constant. This is consistent with the work reported by Taniguchi et al. [ 11] on a platinum electrode in the presence of 4,4'-bipyridine, which conclude that the difference on the estimated diffusion coefficient for cyt c, is related to a less reversible electrode reaction and a lower surface activity of the promoter as compared to a gold electrode. In the case of platinum, the fact that the reductive desorption is not well observed, make difficult to establish that the 6MP monolayer is completely formed and also, to speculate about the properties of that monolayer in the sense of its ability to limit access of solution-phase molecules to the electrode surface, deffeets, etc. However, platinum is unique as it posses the property to show a definite potential region for hydrogen adsorption. It is known that when an organic monolayer is formed over this metal, the hydrogen adsorption is supressed in an extent comparable to the occupancy of the atoms at the surface for the molecules of the monolayer. This effect is observed in Figure 4, where the current density due to the hydrogen adsorption is practically absent in the voltammogram registered for the modified platinum electrode at the same potential region.

701

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E/mV Figure 3. mg/ml cyt and (w) phosphate, 40mV/s.

Cyclic voltammograms of 6 c at ('--) 6MP-Au, (---) 6MP-Hg 6MP-Pt electrodes in 25 mM 0.1 M NaCIO 4 (pH 7). Scan rate

I

-200 -400 E/mV

I

-600

Figure 4. Cyclic voltammograms of a (--) bare platinum and (---) 6MP-Pt electrodes in 25 mM phosphate, 0.1 M NaCIO4 (pH 7). Scan rate 50 mV/s

Finally, the voltammograms obtained with the three modified substrates can be analyzed by using digital simulation, after substraction of background currents, assuming n=l, cz=0.5 and the diffusion coefficient, D=7.7x10 7 cm2s~ [10]. Figure 5 shows typical background substrated cyclic voltammograms of cyt c, together with simulated data for the experiment in platinum. The results obtained from this analysis, i.e., the formal redox potential, E ~ and the electron transfer rate constant, ksh, are gathered in Table 1.

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100

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100 POTENTIAL / mV

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Figure 5. (A) Cyclic voltammogram of 6 mg/ml cyt c in 25 mM phosphate buffer (pH 7), 0.1 M NaC104 at 6MP-Pt electrode. (---) Backgound current. 03) Background substracted voltammogram of cyt c at 6MP-Pt electrode (o) and simulated data ( ~ ) . The simulation was made by using the values shown in the text.

702 It is known that the chain of self-assembled thiols monolayers on gold form closely packed, ordered monolayers with excellent non-wetting characteristics and high stability. Little is known about alkanethiol monolayers on Pt except that it is formed as judged from surface wetting. Pt shows gauche transformations that are reversibly eliminated at negative or positive potentials. It is thought that an initial disorder is what allows those conformations [ 12]. The organization of the 6MP monolayer on Pt is still unknown but, no appreciable changes are observed after succesive scans in the absence or in the presence of cyt c. Lateral interactions of the 6MP adsorbate may limit the ability of the surface transformations of the film. However, owing to the small size of the 6MP molecule as compared to the long thiol chains, the structure of the monolayer would involve deflects which leaves the metal surface in direct contact with the solution. These feactures seem not to be determinant in the achievement of the electrochemical response of the metalloprotein, leading only to a slight variation in the kinetic of the electron transfer reaction.

ACKNO~GEMENTS This work was supported by DGICYT (project PB94-0440), Junta de Andalucia and Universidad de C6rdoba.

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