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Effect of Ionic Strength on the Electrochemical Behavior of Glutathione on a Phospholipid Self-Assembled Monolayer on Mercury R. Herrero,† J. L. Barriada,† J. M. Lo´pez-Fonseca,‡ M. R. Moncelli,§ and M. E. Sastre de Vicente*,† Departamento de Quı´mica Fundamental e Industrial, University of La Corun˜ a, 15071 A Corun˜ a, Spain, Departamento de Quı´mica Fı´sica y Analı´tica, University of Oviedo, 33006 Oviedo, Spain, and Dipartimento di Chimica, University of Florence, 50121 Florence, Italy Received April 29, 1999. In Final Form: September 16, 1999
A voltammetric study of the permeability of redox couple oxidized glutathione/reduced glutathione (GSSG/GSH) through a membrane biomimetic model has been carried out. The biomimetic model has been obtained by deposition of a self-assembled monolayer of phospholipid on a mercury electrode. Experimental results show that the oxidized form of GSSG is able to penetrate through the phospholipid monolayer to produce an irreversible adsorption peak on the mercury surface. The dependence of current and peak potential on the ionic strength has been studied. No voltammetric signal has been obtained for the reduced form GSH. A simple model of the double layer is employed to analyze the experimental curves. Results obtained are briefly compared with some studies of transport carried out in cell cultures.
Introduction The redox couple reduced glutathione (GSH)/oxidized glutathione (GSSG) is involved in many cellular functions, especially in antioxidant defense. Furthermore, the glutathione redox ratio (i.e., GSH/GSSG) is correlated with oxidative stress, which may occur under some physiological and pathological conditions.1-4 Such a biochemical relevance of the transport of the GSH/GSSG couple aroused our interest in studying their redox behavior on a membrane model system constituted by a phospholipid monolayer adsorbed on mercury at different values of ionic strength. Previous work on this membrane model using voltammetric techniques on a mercury electrode has been carried out by Miller,5 who performed permeability experiments involving molecules of high molecular weight and biochemical interest. Nelson6,7 has examined the characteristics of some monolayers and the penetration of various * To whom correspondence may be addressed: telephone number, 34 981 167000 (ext. 2198); fax number, 34 981 167065; e-mail:
[email protected]. † University of La Corun ˜ a. ‡ University of Oviedo. § University of Florence. (1) Hwang, C.; Sinskey, A. J.; Lodish, H. F. Science 1992, 257, 14961502. (2) Hwang, C.; Lodish, H. F.; Sinskey, A. J. Measurement of glutathione redox state in cytosol and secretory pathway of cultured cells. In Methods in Enzymology; Packer, L., Ed.; Academic Press: San Diego, CA, 1995; Vol. 251, pp 212-221. (3) Akerboom, T. P. M.; Sies, H. Transport of glutathione, glutathione disulfide and glutathione conjugates across the hepatocyte plasma membrane. In Methods in Enzymology; Academic Press: San Diego, CA, 1989; Vol. 173, pp 523-534. (4) Meister, A. Glutathione Metabolism. In Methods in Enzymology; Packer, L., Ed.; Academic Press: San Diego, CA, 1995; Vol. 251, pp 3-7. (5) Miller, I. R. Structural and energetics aspects of charge transport in lipid layers and in biological membranes. In Topics in Bioelectrochemistry and Bioenergetics; Milazzo, G., Ed.; John Wiley: Bristol, 1981; Vol. 4, pp 161-224. (6) Nelson, A.; Benton, A. J. Electroanal. Chem. 1986, 202, 253-270.
chemical species through them. Moncelli et al. have also used this model in double-layer studies.8,9 Polarography of glutathione on a mercury electrode was initially studied by Kolthoff.10 Moreover, references to previous works on glutathione redox couple on a mercury electrode can be found in a recent work devoted to find the best analytical conditions for the determination of GSSG, among other substances, by means of cathodic stripping voltammetry.11 A recent review of Heyrovsky et al. about the voltammetry of the cystine12 on mercury electrode is also of interest due to the analogies between the voltammetric behavior of both molecules. In this work, the ability of GSSG and GSH to penetrate through a monolayer of dioleoylphosphatidylcholine (DOPC) deposited on mercury is studied by voltammetry. Some results are compared with studies of transport on real cells, and they seem to support the fact that a preferential transport of GSSG over GSH from the cytosol into the endoplasmatic reticulum exists. Experimental Section The chemicals used in this work were mercury (Merck Suprapur), KCl (Merck p.a., heated at 500 °C in a muffle furnace to remove organic impurities), KH2PO4 and Na2HPO4 (Merck pro-analysis), and L-glutathione, reduced 98%, and L-glutathione, oxidized 90%, both from Aldrich. Dioleoylphosphatidylcholine (purity grade 1) was supplied by Lipid Products (South Nutfield, Surrey, U.K.) as solutions containing 100 mg of phospholipid in (7) Nelson, A.; Auffret, N.; Borlakoglu, J. Biochim. Biophys. Acta 1990, 1021, 205-216. (8) Moncelli, M. R.; Guidelli, R. J. Electroanal. Chem. 1992, 326, 331-338. (9) Moncelli, M. R.; Becucci, L.; Guidelli, R. Biophys. J. 1994, 66, 1969-1980. (10) Stricks, W.; Kolthoff, I. M. J. Am. Chem. Soc. 1952, 74, 46464653. (11) Banica, F. G.; Fogg, A. G.; Moreira, J. C. Talanta 1995, 42, 227234. (12) Heyrovsky´, M.; Mader, P.; Vesela´, V.; Fedurco, M. J. Electroanal. Chem. 1994, 369, 53-70.
10.1021/la990527t CCC: $19.00 © 2000 American Chemical Society Published on Web 04/21/2000
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5 mL of chloroform plus methanol. The working solutions for spreading at the air-water interface were prepared every day by dilution of 50 µL of the stock in 1 mL of pentane and storing the solution at -20 °C. All measurements were carried out at 25 ( 0.1 °C in aqueous solutions containing different amounts of 4 M KCl stock solution to keep constant the ionic strength. Glutathione concentration was studied from 6.6 × 10-6 to 1.3 × 10-4 M at ionic strength range from 0.005 to 0.200 M. The pH was controlled with a HPO42-/H2PO4- buffer. The overall concentration of the acidic and basic components of the buffer is referred to as the buffer concentration. The homemade hanging mercury drop electrode employed in the measurements, the cell, and the detailed procedure to produce self-assembled lipid monolayers deposited on mercury are described elsewhere.9,13 All potentials were measured versus a saturated calomel electrode (SCE). Differential capacitance measurements were made by means of Metrohm Herisau Polarecord E 506 polarograph using phasesensitive ac voltammetry. The baseline potential was overlapped with a sinusoidal signal of 10 mV amplitude and a 75 Hz frequency, using a lag angle of 90°. Differential capacity of the lipid monolayer was constantly measured against the applied potential to check the stability and reproducibility of the film. All experiments were conducted on a fully covered electrode where the minimum capacity was 1.7 ( 0.1 µF‚cm-2. Cyclic voltammetry measurements were made with a Metrohm E 506 polarograph connected to a Metrohm VA-Scanner E 612 triangular signal generator. Voltammograms were recorded on a Linseis LX 1600 recorder. The pH was measured before and after each experiment by means of a Crison micro pH 2000 pH-meter fitted to a radiometer combined glass electrode with an Ag/AgCl reference electrode. The electrode performance was periodically checked with solutions of accurately known pH.
Results Oxidized glutathione. The effect of experimental parameters such as stirring time and initial potential has been analyzed in order to find the best conditions to incorporate GSSG in the phospholipid monolayer and observe the subsequent reduction on the electrode. The recording after stirring solution with no potential applied reveals that GSSG is able to penetrate through the phospholipid monolayer (Figure 1). On the other hand, agitation with potential applied resulted in an inhibition effect on the GSSG penetration, and so, no voltammetric signal is observed in this situation. A change in the initial potential confirms that GSSG penetrates while the phospholipid monolayer is perfectly stable and not as a consequence of monolayer disruption at more positive potentials than -0.2 V (Figure 1c,d). The bulk solution was first subjected to mild stirring for different times, t, and the corresponding charge Q associated to the reduction peak was then measured. This quantity was found to increase progressively with t, without attaining a maximum limiting value (Figure 2). The curve of the differential capacity vs E obtained at t ) 0 in the presence of GSSG practically coincides with that obtained with pure DOPC (Figure 3a); in particular, the differential capacity along the flat minimum, namely, from -0.2 to -0.8 V, assumes the same value of about 1.7 µF‚cm-2 in both cases. Conversely, at any stirring time different from 0, a plot of the pseudocapacity vs E shows a hump due to GSSG electroreduction which develops on the positive side of this potential region and then returns to the value of pure DOPC. A depresion in the first reorientation peak of the DOPC is also observed (Figure 3b). Repeated cycling on the same drop cancels the effects (13) Moncelli, M. R.; Becucci, L. J. Electroanal. Chem. 1995, 385, 183-189.
Figure 1. Cyclic voltammograms of oxidized glutathione at a DOPC-coated Hg electrode in 0.1 mol‚dm-3 KCl, 0.01 mol‚dm-3 phosphate buffer, pH 7.3, and scan rate 0.2 V s-1 at different experimental conditions: (a) [GSSG] ) 0, Ei ) -0.05 V, Ef ) -1.10 V; (b) [GSSG] ) 1.33 × 10-5 mol‚dm-3, Ei ) -0.20 V, Ef ) -1.10 V, 5 min stirring time with E ) -0.20 V; (c) [GSSG] ) 1.04 × 10-4 mol‚dm-3, Ei ) -0.20 V, Ef ) -1.10 V, 5 min stirring time without applied potential; (d) [GSSG] ) 1.04 × 10-4 mol‚dm-3, Ei ) -0.40 V, Ef ) -1.10 V, 5 min stirring time without applied potential. Electrode area was 13.6 × 10-3 cm2.
Figure 2. Plot of the charge for oxidized glutathione against stirring time as obtained from cyclic voltammograms of 6.60 × 10-6 mol‚dm-3 GSSG at a DOPC coated Hg electrode in 0.005 mol‚dm-3 KCl, 1 × 10-3 mol‚dm-3 phosphate buffer, pH 7.3, scan rate 0.2 V s-1. Electrode area was 13.6 × 10-3 cm2.
due to the accumulation of GSSG, and the differential capacity vs E curve of pure DOPC is restored. Cyclic voltammograms show a reduction peak in the negative potential scan while no oxidation peak is found
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Figure 5. Plots of ln(v) vs FEp/(RT) as obtained from cyclic voltammograms of 6.60 × 10-6 mol‚dm-3 oxidized glutathione at a DOPC-coated Hg electrode in KCl and 1.0 × 10-3 mol‚dm-3 phosphate buffer at ionic strengths 0.005 ([), 0.011 (9), and 0.052 (2) mol‚dm-3. The inset shows a plot of the charge-transfer coefficient, R, obtained from the slope of the straight lines vs ionic strength.
Figure 3. C vs E curves for a DOPC-coated Hg electrode (a) in the absence and (b) in the presence of 1.30 × 10-4 mol‚dm-3 oxidized glutathione after 5 min of stirring time without applied potential in 0.1 mol‚dm-3 KCl, 0.01 mol‚dm-3 phosphate buffer, pH 7.3, scan rate 7.5 mV s-1.
Figure 4. Plots of log(Ip) vs log(v) as obtained from cyclic voltammograms of oxidized glutathione at a DOPC-coated Hg electrode in 0.1 mol‚dm-3 KCl, 0.01 mol‚dm-3 phosphate buffer, pH 7.3, at different values of [GSSG] ) 6.60 × 10-6 (O), 2.65 × 10-5 (4), 5.28 × 10-5 (3), 1.04 × 10-4 (]) mol‚dm-3. Electrode area was 13.6 × 10-3 cm2. The straight lines have unit slope.
in the subsequent positive potential scan. The reduction peak is observed to vanish when multiple cycling is performed on the same drop, and so all analysis stated below are referred to the reduction peak obtained in the first negative potential scan after agitation during 5 min. Figure 4 shows the plot of log(Ipeak) vs log(v), where v is the scan rate, at different concentrations of GSSG and ionic strength fixed at 0.100 M. All the straight lines in the figure have unit slope. Plotting ln(v) against the dimensionless quantity FEp/(RT) at constant pH and
Figure 6. Plots of -FEp(2.3RT) vs pH as obtained from cyclic voltammograms of 2.62 × 10-5 mol‚dm-3 oxidized glutathione at a DOPC-coated Hg electrode after 5 min of stirring time without applied potential in KCl + phosphate buffer solutions of different pH values and ionic strength: 0.010 ([); 0.100 (9); 0.200 (2) mol‚dm-3. Scan rate was 0.2 V s-1. The straight lines have unit slope.
GSSG concentration yields straight lines over the whole ionic strength investigated, as shown in Figure 5. The slope from each straight line is equal to the apparent values (R*) that the charge-transfer coefficient exhibits due to the effect of the double layer, and their dependence on the ionic strength can be seen in the inset of Figure 5. Plots of -FEp/(2.3RT) against pH at constant scan rate are also linear with a slope equal to unity at any ionic strength (Figure 6). Over the whole pH range investigated, a change in the buffer concentration from 5 × 10-4 to 1 × 10-2 M at constant pH has no appreciable effect on GSSG reduction. The effect of ionic strength on the reduction peak has been studied attending to the modification of peak current and peak potential. A well-defined trend is constantly observed; a moderate peak current decreasing and a peak potential shifting to more positive values are observed as the ionic strength is increased up to a concentration about 0.1 M. Further increase in the ionic strength has no effect on both parameters (Figure 7). Reduced Glutathione. Similar voltammetric analyses have been carried out on the reduced form of glutathione GSH. In this case no voltammetric signal is found due to the oxidation of GSH on mercury. No change in differential capacity measurements has been found, which is evidence of the absence of GSH in the monolayer. Cyclic voltammograms of GSH have been recorded both on a bare mercury electrode and in the presence of phospholipid, at potentials where the phospholipid monolayer is stable but in the absence of which two reduction peaks and the corresponding oxidation peaks are obtained.
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evidence of an adsorptive reduction process. Moreover, the high values of the charge associated to the reduction peak (Figure 2) and the distortion observed in the pseudocapacity-applied potential plot (Figure 3b), as a consequence of GSSG accumulation, seem to indicate that GSSG is adsorbed on the mercury surface directly instead of the phospholipid polar heads region. Figure 3b shows a decrease of the first reorientation peak of the phospholipid which is indicative of a partial replacement of DOPC molecules initially adsorbed on mercury by GSSG molecules. Both effects observed due to repetitive scans on the same drop, namely, the restoration of the differential capacity vs E curve as that of the pure lipid and the progressive decrease of the reduction peak until complete vanishment, are indicative that the reduced form does not remain adsorbed on the electrode surface; hence, reaction product is eliminated from the lipidic region. Voltammetric measurements carried out on the reduced form, GSH, show evidence of the absence of faradaic current, and an invariable differential capacity vs E curve confirms the absence of GSH in the monolayer. To provide an interpretation of the experimental behavior shown in Figure 7, the following approach will be adopted. Let the charge-transfer step have the general form
Ox(ads) + ne- f Products
(1)
According to Hubbard,14,15 the current-potential equation can be expressed in a convenient form by the expression
[
i ) nFA*koγoxΓox exp -
R*F (E - Eo) RT
]
(2)
where γox expresses the relation of the activity of chemisorbed reactant aox with coverage Γox (aj ) γjΓj), and R* and *ko are the apparent values of the rate parameters, which are expected to depend markedly upon double layer composition. Their relation to the true values R and ko is given by
C (z - R) Cd
R* ) R +
(3)
F φ *ko ) ko exp (R - z) RT 2
[
Figure 7. Plots of (a) reduction peak current (Ip) vs ionic strength (I), (b) Ep vs I, and (c) Ep vs log(I) as obtained from cyclic voltammograms of 6.60 × 10-6 mol‚dm-3 oxidized glutathione at a DOPC-coated Hg electrode in KCl + phosphate buffer of pH 7.3. Scan rates were 0.100 ([), 0.200 (9), and 0.400 (2) V s-1. Electrode area was 13.6 × 10-3 cm2.
]
(4)
where
φ2 )
C ∆V Cd
(5)
Discussion
φ2 is the potential at the plane of electron transfer of the species j, z is the charge of the electroactive molecule, C is the electrode capacitance, Cd is the capacitance of diffuse layer, and ∆V represents the potential applied to the electrode relative to the potential of zero charge (∆V ) E - Ez), Ez is the potential of zero charge. Combining the above equations16,17 yields the following expressions of voltammetric peak current and peak
Differential capacity (Figure 3) and cyclic voltammetry (Figure 1) measurements indicate that GSSG penetrates through the lipid monolayer in the absence of any applied potential, and subsequently, it can be reduced on the mercury surface when a potential scan is imposed. A bellshaped reduction peak, together with the unit slope obtained in the plots of log(Ipeak) vs log(v) (Figure 4), gives
(14) Lane, R. F.; Hubbard, A. T. J. Phys. Chem. 1973, 77, 14011410. (15) Lane, R. F.; Hubbard, A. T. J. Phys. Chem. 1973, 77, 14111421. (16) Laviron, E. Electroanal. Chem. Interface Electrochem. 1974, 52, 355-393. (17) Srinivasan, S.; Gileadi, E. Electrochim. Acta 1966, 11, 321335.
These signals disappear in the presence of the lipid monolayer, which suggests that the lipid monolayer inhibits penetration of GSH, despite GSH being electroactive on mercury.
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potential as a function of R*, *ko, and φ2
ip )
R*nF2AvΓox RTe
(6) o
RTγox*k RT ln Ep ) E + R*F R*Fv o
(7)
The parameter φ2 includes the influence of the double layer through the relation C/Cd the dependence of which is in turn related to the nature and concentration of the supporting electrolyte. To calculate the dependence of differential capacity on the concentration of the electrolyte, let the potential drop along the whole interface E be the sum of two contributions, namely, one being the potential drop of the diffuse layer ∆φd and the other involving all the remaining potential drops related to the monolayer ∑Vi*d.
E)
∑i Vi*d + ∆φd
(8)
Now, if the electrolyte solution is very dilute, ∆φd . ΣiVi*d and then the change of potential drop E, with electrolyte solution c is given by ∂E/∂c ) ∂∆φd/∂c. Clearly, a modification of the double layer structure, such as a change in the ionic strength, affects the kinetics of the electrode process. Consequently, a variation of peak current or peak potential is expected. On the other hand, when the solution is very concentrated ∑iVi*d . ∆φd and ∂E/∂c ≈ 0. No effect of the ionic strength on the kinetics should be then observed. Depending on the relative value of each of the contributions ∑Vi*d and ∆φd, the slope of the E vs c plot may be expected to vary from 0 to the maximum value allowed by the Gouy-Chapman theory (∂φd/∂c). The diffuse layer potential φd can be calculated from modeling the double layer of the lipid monolayer-coated electrode as two capacitors in series. Thus, the total capacitance per cm2 of the double layer C is made up of that due to the phospholipid monolayer Cm and that due to the diffuse double layer Cd.
1 1 1 ) + C Cm Cd
(9)
The phospholipid monolayer is assumed to behave as an ideal capacitor, whereas the capacitance of the diffuse layer is calculated from Gouy-Chapman theory, which proposes the following equation at 25 °C18
Cd ) 228zc1/2 cosh(19.5zφd) (µF cm-2)
(10)
where z is the absolute value of the charge of each ion in a z:z electrolyte and c is the electrolyte concentration in mol/L. This model has been recently employed by Becka and Miller19 to study electron-transfer processes on alkanethiol monolayer coated gold electrodes. The two algebraic relationships of eqs 9 and 10 can be combined with eq 5 to yield the expresion
φd )
∆V Cm 1/2
228zc
cosh(19.5zφd) + Cm
(11)
(18) Bard, A. J.; Faulkner, L. R. Electrochemical methods: fundamentals and applications; Wiley: New York, 1980. (19) Becka, A. M.; Miller, C. J. J. Phys. Chem. 1993, 97, 6233-6239.
The value of φd for a given electrode potential and electrolyte concentration can be obtained from an iterative resolution of this equation by, for example, a NewtonRaphson procedure.20 Not even for this simple model is it possible to obtain an analytical solution of φd vs c, except in particular cases as, for example, those described by Mairanovskii21 who has obtained simple linear φd vs log(c) relationships, but only for potential values very far from the zero charge potential. The eq 11 predicts that the higher the value of ∆V, the larger the effect of the double layer. Similarly, a gradual decrease in the potential of the diffuse layer is also predicted as ionic strength is progressively increased, and so, the potential peak is expected to be shifted to more positive values with increasing ionic strength. As it is shown in Figure 7, GSSG exhibits a linear logaritmic dependence of its peak potential on the ionic strength (Figure 7c). Moreover, peak current yields an exponential decrease as ionic strength increases (Figure 7a). Such behavior is probably caused by an electrostatic repulsion on the mercury surface, which leads to the depression of the anionic form of the sulfur mercury derivative. According to the values of the dissociation constants reported in the bibliography,22 the anionic form is supposed to be predominant at pH ≈ 7. A straight line with intercept equal to 2 is obtained when the values of R*, taken from the inset of Figure 5, are plotted against C/Cd data, calculated according to the model described above. Such value of intercept can be justified on the basis of eq 3, taking the double layer independent charge-transfer coefficient to R ) 2. The same result is obtained if double layer effects are considered to be negligible for the highest ionic strengths, then R is directly obtained from experimental data, as shown in the inset of Figure 5. For an electrode reaction which consists of a series of consecutive elementary steps, some of which are necessarily electron-transfer steps, while some others are chemical in nature, a value of R ) 2 implies that the ratedetermining step is a chemical step following the reversible uptake of two electrons.23,24 When dealing with organic compounds, the most common chemical steps are, by far, those involving the uptake or release of one proton. The slope of the -FEp/(2.3RT) vs pH plot (Figure 6) being approximately equal to unity allows one to conclude that the rate chemical step is the protonation of the reduced form.24 The reduction of GSSG on a DOPC monolayer does not depend on the buffer concentration at constant pH and, hence, does not satisfy the principles of general acidbase catalysis. This behavior can be justified by assuming that the protonation takes place inside the DOPC monolayer which is practically impermeable to the H2PO4- ions on the time scale of our measurements whereas it may be permeated by the proton itself. In this case, the role of the buffer is exclusively that of mantaining the pH just outside (20) Press, W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling, W. T. Numerical recipes in FORTRAN: the art of scientific computing, 2nd ed.; Cambridge University Press: Cambridge (England) and New York, 1992. (21) Mairanovskii, S. G. Catalytic and kinetic waves in polarography; Plenum Press: New York, 1968. (22) Rabenstein, D. L.; Guevremont, R.; Evans, C. A. Glutathione and its metal complexes. In Metal ions in biological systems. Amino acids and derivatives as ambivalent ligands; Sigel, H., Ed.; Marcel Dekker: New York, 1979; Vol. 9; pp 103-141. (23) Bockris, J. O. M.; Reddy, A. K. N. Modern electrochemistry; an introduction to an interdisciplinary area; Plenum Press: New York, 1970. (24) Moncelli, M. R.; Herrero, R.; Becucci, L.; Guidelli, R. Biochim. Biophys. Acta 1998, 1364, 373-384.
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the lipid layer constant. Hence, the rate-determining protonation step is affected by a change in pH but not by a change in the buffer concentration at constant pH. Conclusions Voltammetric measurements of the glutathione redox couple GSSG/GSH show that the oxidized form GSSG is able to penetrate a self-assembled phospholipid monolayer deposited on mercury and then reduce on the mercury surface. The analysis of the irreversible adsorption peak obtained for the reduction process leads to the conclusion that the rate-determining step is a protonation step involving the proton as the only effective proton donor following the reversible uptake of two electrons. A simple two-capacitor model corresponding to the phospholipid monolayer and the diffuse double layer was employed to study the dependence of the peak current and peak potential on the ionic strength. On the other hand, the reduced form of glutathione GSH has been found to be not able to penetrate the phospholipid monolayer. The reason for such different behavior of reduced and oxidized form of glutathione is not clear.
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The greater ability of GSSG to penetrate through the monolayer of DOPC agrees well with the results of different recent biological research on the redox behavior of glutathione in the endoplasmatic reticulum.1 These experiments demostrate the preferential transport of oxidized glutathione compared to reduced form. Results obtained in this work, i.e., easy penetration of GSSG relative to GSH through DOPC, suggest that the phospholipid monolayer coated mercury electrode works actually as a model for the cellular membrane which separates the cytosol from the endoplasmatic reticulum. However, the authors are aware that making such an extrapolation of this kind should be taken with caution. Acknowledgment. M.E.S.V. thanks Xunta de Galicia for financial support received through Project XUGA 10310B97. J.L.B. thanks the Ministerio de Educacio´n y Cultura for the fellowship granted. Thanks are due to T. Vilarin˜o for her helpful comments. LA990527T
