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Fixed charge and transport numbers in isolated pepper fruit cuticles from membrane potential measurements: Donnan and diffusion potential contributions
Colloids and Surfaces A: Physicochemical and Engineering Aspects 159 (1999) 423 – 430 www.elsevier.nl/locate/colsurfa
Fixed charge and transport numbers in isolated pepper fruit cuticles from membrane potential measurements: Donnan and diffusion potential contributions J. Benavente a,*, A. Mun˜oz b, A. Heredia b, A. Can˜as a b
a Departamento de Fı´sica Aplicada, Facultad de Ciencias, Uni6ersidad de Ma´laga, E-29071 Ma´laga, Spain Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias, Uni6ersidad de Ma´laga, E-29071 Ma´laga, Spain
Abstract Membrane potential of native isolated pepper cuticles were measured with NaCl and CaCl2 solutions for a wide range of concentrations (10 − 3 BC(M)B10 − 1). From membrane potential results the fixed charge concentration, X, and the transport number of the ions in the cuticular membrane were obtained. Taking into account the X value, an estimation of the Donnan potential contribution to the membrane potential was carried out; it was found that the Donnan exclusion of co-ions only occurs at low concentrations, while at concentrations higher than 10 − 2 M the membrane potential is mainly due to the different mobility of the ions in the cuticular membrane (diffusion potential). Concentration dependence for both fixed charge concentration and transport numbers was also considered. The influence of chemical and thermal treatments on membrane potential and electrical resistance was also studied. A decrease in the experimental values of both parameters as a consequence of chemical treatment was found; results obtained with an annealed pepper membrane also show a reduction in the electrical resistance and in the contribution of the Donnan potential to the measured membrane potential. These facts indicate a more open structure for the pepper cuticles due to both different treatments. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Chemical and thermal treatments; Electrokinetic parameters; Fruit cuticles
1. Introduction Plant cuticles are biological polymer membranes of heterogeneous composition [1] which cover the aerial organs of terrestrial plants. They * Corresponding author. Tel. + 34-5-2131929; fax: +34-52132000. E-mail address: j – [email protected] (J. Benavente)
constitute the interface between the environment and the plant cells and, although their main function is to minimise water loss from plants when stomata are closed, the cuticles are not impermeable to water (or electrolyte solutions) and under some conditions, they cannot prevent wilting. In fact, their transport properties are of great biological and practical interest as can be seen from different studies dealing with cuticular transpira-
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tion, uptake of nutrients, or herbicides, fungicides and xenobiotics applied to the foliage [2,3]. The cuticular membrane model generally adopted is a porous membrane with the pore size depending mainly upon swelling in the presence of water [4]. Scho¨nherr and Bukovac [5] showed that cuticular pores have polar regions composed by the following components: (i) cutin, which is a weakly polar compound due to hydroxyl and unesterified carboxyl groups; (ii) polyuronic acids associated with cellulose and pectin in the secondary cuticle; and (iii) some cuticular waxes with polar substitutents. It was reported that at a pH\ 3 the cuticles have a net negative charge, which may affect the sorption and transport of electrolytes [5,6]. These results also indicate an asymmetrical behavior of the two morphological surfaces of the cuticular membrane [7,8]: the inner surface, contacting the cell wall, appears with less hydrophobic character that the highly hydrophobic outer surface, covered by waxes. In this paper, the membrane potential for isolated fruit cuticular membranes (pepper cuticles) with NaCl and CaCl2 solutions at different concentrations was studied, which allows one to get information about characteristic membrane parameters such as membrane effective fixed charge concentrations and ion transport numbers. Contributions of both, Donnan and diffusion potentials to the measured membrane potential, are also estimated. Since fruit cuticles in their native stage contain different cations sorbed in their matrix (exchangeable cations) [5], which could interact with the electrolyte solutions during transport mechanism masking the results, membrane potential and electrical resistance for a pepper fruit isolated cuticle in sodium-form (Na+ ions should only be present in the cuticular matrix) were also measured. The comparison of the results obtained with native and sodium-form cuticles shows how changes in the cuticular matrix can affect the electrokinetic parameters. On the other hand, temperature effects on the structure of native cuticles were also studied from comparison of membrane potential and electrical resistance results for native and annealed isolates cuticles.
2. Experimental
2.1. Material Different samples of astomatous cuticles from pepper (Capsicum anuum, L.) fruits were used. The cuticles were enzymatically isolated following the procedure described by Shafer and Bukovac [9]. The cuticles will hereafter be called PC(Na+). Exchangeable cations were removed from a cuticle sample by shaking in a 1 M HCl solution for 15 min (three changes) followed by washing with deionized water to remove sorbed HCl. The cuticle in the sodium form, PC(Na+), was obtained after equilibration with 10 − 2 M NaCl in Tris buffer solution at pH= 7.0 in a bath shaker for 24 h at room temperature. Thereafter the cuticles were washed five times in 10 ml of deionized water (10 min each) to remove sorbed Na+ ions. In order to determine the effect of temperature on electrokinetic parameters, a native isolated cuticle was annealed at 60°C for 2 h. Experiments were carried out using NaCl and CaCl2 (Merck™, 99.5% purity) aqueous solutions at different concentrations at constant temperature t= 25.09 0.3°C and pH 6.7 9 0.3. Before use, the cuticles were kept for at least 48 h in distilled water, and later they were immersed for 12 h in a solution of the appropriate concentration.
2.2. Membrane potential and electrical resistance measurements The measuring cell is similar to that indicated in Ref. [10], and it basically consists of two halfcells, of 10 cm3 each, with a membrane area of 0.7 cm2. To minimize the concentration-polarization at the membrane/ solution interfaces, a magnetic stirrer with a stirrer rate of 500 rpm was placed at the bottom of each half-cell. Membrane potential measurements were carried out keeping constant the concentration of the solution at one side of the membrane, C2, and increasing gradually the concentration of the solution at the other side, C1 . Six different values for the constant concentration were considered (C2 = 10 − 3, 2 × 10 − 3, 5 × 10 − 3, 10 − 2, 5 × 10 − 2 and
J. Bena6ente et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 159 (1999) 423–430
10 − 1 M), while C1 ranged between 5 × 10 − 4 and 5× 10 − 1 M. In order to see if the membrane asymmetry can be detected from membrane potential measurements, the constant concentration was placed in contact with both the inner, C1, and the outer, C o, membrane surfaces; in all measurements, the electrode placed into the solution in contact with the outer membrane surface was grounded, this means: Df = f(C i) −f(C o). The electrical resistance measurements were carried out using an alternating current bridge (Wayne Kerr, Automatic Precision Bridge, model B905) at a frequency of 1 KHz, which was connected to the cell via Ag/AgCl electrodes. Resistance values were determined for different NaCl and CaCl2 solutions, for concentration ranging between 10 − 3 and 5× 10 − 2 M, having the solution at both sides of the membranes the same concentration. Resistance measurements were made with the cuticular membranes placed in the membrane holder (Rmd) and without them (Rd), the difference between both values was taken as the membrane resistance. In both experiments three series of measurements were carried out with each native membrane sample for each constant concentration, and the results presented in this paper correspond to the average of those values.
where u + and u − are the cationic and anionic mobilities, z + and z − their valencies, respectively; R and F are the gas and Faraday constant, and T the temperature of the system. t + and t − are the cation and anion transport numbers in the membrane, and they are related to the mobilities by ti = zi ui /i zi ui ; since water transport is not considered, t + and t − represents the apparent transport numbers in the membrane.- a Donnan potential difference at each membrane/solution interface, which is due to the different concentration of co-ions (ions with the same charge as the membrane) in the electrolyte solution and in the membrane (CB and C m B , respectively). Assuming diluted solutions (ai : Ci), and taking into account the conditions of electroneutrality in the solution and in the membrane, the following relation between the distribution of co-ions in the membrane and the solution can be derived as a function of the concentration of fixed charge in the membrane, X, [13,14] ( zB /zA) m rB = (C m B /CB)= [ ZB CB/( zB C B + zX X)]
(2) where zA, zB and zX are the counter-ions, co-ions and membrane charge valencies, respectively. The expression for the Donnan potential is DfDon = f m − f=(RT/zBF)ln(rB)
(3)
In general, the potential difference measured at both sides of a membrane or ‘membrane potential’, Dfm, can be represented by
3. Theory
Dfm = DfDoni + Dfdif + DfDono
According to the Teorell – Meyer – Sievers theory [11,12], the electrical potential difference measured at both sides of a charged membrane when it is separating an electrolyte solution of different concentrations (C1 and C2) basically consists of two terms:- a diffusion potential across the membrane, which is due to the different mobility of the ions in the membrane and, for diluted solutions (concentrations are used instead of activities) it can be expressed as [13] Dfdiff =(RT/F)([u + / z + ]− [u − / z − ])ln(C1/C2) = (RT/F)([t + / z + ]− [t − / z − ])ln(C1/C2)
425
(1)
a diffusion potential across the membrane, Dfdif, and two Donnan potentials, DfDoni and DfDono, one at each membrane/solution interface. Taking into account Eqs. (1) and (3), the following expression for the membrane potential can be written [13]: Dfm = (RT/wzF){ln(C1[(1+4y 21)1/2 + 1]/C2 [(1+4y 22)1/2 + 1]+wU ln[(1+4y 21)1/2 − wU] /(1+4y 22)1/2 − wU)}
(4)
w is +1 or −1, for anionic or cationic membranes, respectively, and yj = zjksCj /wX, ks being
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the salt partition coefficient (ks :1); the parameter U is related to the transport number of the ions in the membrane (U =[t + / z + ]− [t − / z − ]). The fitting of the membrane potential values measured at different salt concentrations allow us to determine the effective fixed charge and the parameter U. Because of the ion transport number represents the amount of current transported for one ion with respect to the total current crossing the membrane, tj =Ii/IT, this means t + + t − = 1, cation and anion transport numbers (apparent transport numbers) can also be obtained from membrane potential measurements as a function of the external salt concentration.
Average values of the fixed charge concentration and cation transport numbers in native pepNa + Ca2 + and t + , are shown per cuticles, X, t + in Table 1 for the whole range of concentrations. The negative fixed charge must be due to the dissociation of COOH- groups from the fatty acids in the polyesters and from -OH phenolic groups [6], depending on the electrolyte, no significant differences were found. However, important differences in the values of cation transport numbers for both electrolytes were obtained, that Na + is the value for t + was much higher than that
4. Results and discussion Fig. 1(a and b) shows the experimental values for the membrane potential, Dfm versus ln(C i/C o) at different values of the constant concentration, with NaCl and CaCl2 solutions, respectively. As can be seen from these pictures, the experimental values correspond to different parabolas, with the maximum/minimum shifting to the left, when the constant concentration increases. A comparison of the Dfm values obtained at a given concentration (0.05 NaCl) for both reverse external situations is shown in Fig. 2. From this picture, some differences in Dfm values depending on the membrane surface in contact with the constant concentration can be observed, being this effect more evident at low concentrations. From Eq. (4), and taking into account the external condition for the curve, the fixed charge concentration in the membrane can be obtained [15]: X=2UCext/(1 − U 2)1/2
(5)
where Cext is the concentration at the maximum/ minimum of the curve [16 – 18]. The parameter U can be obtained by the slope of the experimental points measured at high external concentrations (Cext X), when the Donnan potential contribution can be neglected; from this value, the transport numbers of the ions in the cuticular membranes were also obtained.
Fig. 1. Membrane potential, Dfm, versus ln(C i/C o) for native pepper cuticles at different NaCl concentrations for both external reverse situations. ( +) 0.001 M, () 0.005M, () 0.01 M, (X) 0.02M and ( ) 0.05M. (a) C i =cte; (b) Co = cte.
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427
the formation of highly hydrated environments due to the calcium solvation potential. Membrane permselectivity, Ps( + ), which is a measure of the selectivity of the counter ions over the co-ions, can be obtained from transport numbers [13]: o o )/(1 − t + ) Ps( + ) = (t + − t +
(6)
o +
where t represents the transport number of the cations in solution. Ps( + ) values for both electrolytes are also indicated in Table 1, where the higher selectivity of pepper cuticles for Na+ ions is clearly shown. Values in Table 1 are the average of at least 18 measurements (three with each membrane sample) and the interval of errors account for changes in both the variability of the native pepper cuticles and the measurements themselves, for this reason small differences in these parameters due to the membrane asymmetry or concentration dependence can be masked. In order to verify these two points, concentration dependence for the average values of the cation transport numbers obtained with a given membrane sample for both external reverse situations (C o = constant or C i =constant) are shown in Table 2. An important decrease of t + when the concentration increases was obtained (around 20 and 40% for NaCl and CaCl2 solutions, respectively); however, no clear influence of membrane asymmetry on t + values was obtained, since the differences found for both external reverse situations are always included in the error interval. Influence of Donnal potential on the experimental membrane potential results can be determined by means of Eq. (2), once the value of the fixed charge in the membrane was obtained. Fig. 3 shows a comparison of the experimental values
Fig. 2. Membrane potential, Dfm, versus In(C i/C o) for native pepper cuticles at different CaCl2 concentrations for both external reverse situations. ( +) 0.001 M, () 0.005 M, () 0.01 M, (X) 0.02M and ( ) 0.05 M. (a) C i = cte; (b) C o =cte. Ca2 + , which does not differ very much for t + from its value in solution. Differences must be due to the high affinity of Ca2 + for some polysaccharides (mainly polyuronic acids) present as components of the cuticular matrix [19] as well as
Table 1 Na+ Ca2+ and t+ , and cation permselectivity, Averages values of the fixed charge concentration, X, cation transport numbers, t+ + Ps(+) for native and Na -form pepper cuticles Ion type
NaCl CaCl2
Na+-form cuticle
Native cuticle X×103 M
t+
Ps(+)
X×103 M
t+
Ps(+)
−2.09 0.6 −1.69 0.5
0.719 0.06 0.4590.06
52% 9%
−(0.690.2)
(0.63 90.04)
40%
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Table 2 Concentration dependence of cation transport numbers for a pepper fruitcuticle CaCl2
NaCl C
C o = cte t+
C i =ctet+
C o =cte t+
C i =cte t+
0.0055 0.0060 0.0075 0.015 0.030 0.045 0.055
(0.83 90.04) (0.76 90.03) (0.75 90.04) (0.74 90.03) (0.71 90.02) (0.69 90.02) (0.67 90.02)
(0.87 9 0.04) (0.78 9 0.04) (0.77 9 0.03) (0.76 90.04) (0.72 9 0.02) (0.71 9 0.02) (0.70 90.02)
(0.56 90.03) (0.53 9 0.04) (0.52 9 0.04) (0.46 9 0.03) (0.43 9 0.02) (0.41 9 0.02) (0.39 9 0.02)
(0.59 90.04) (0.57 90.04) (0.55 90.04) (0.40 90.03) (0.39 90.02) (0.36 90.02) (0.36 90.02)
and those obtained by subtraction of the Donnan potential at both membrane – solution interfaces. Almost linear relationships between Dfm and ln(C1/C2) were obtained for the calculated values, which agrees with the assumption that, in this case, the membrane potential is due to the diffusion potential. The possible influence of chemical treatment on membrane structure and electrokinetic parameters was also studied from membrane potential and electrical resistance measurements with NaCl solutions: Membrane potential measured for a pepper cuticle in Na+-form or PC(Na+) membrane with different NaCl constant solutions is shown in Fig. 4. Fixed charge concentration, ion transport numbers and membrane permselectivity were also determined by means of Eqs. (5), (1) and (6), respectively, and their values are also written in Table 1. Results show a clear decrease in the fixed charge concentration, but lower values of the cation transport number and membrane permselectivity were also obtained. Membrane electrical resistance, Rm, as a function of the external salt concentration for native and PC(Na+) fruit pepper cuticles is shown in Fig. 5, where a decrease in the membrane resistance values when the concentration increases can be observed, which is mainly attributed to the effect of the electrolyte taken up by the membrane [20,21]. Rm values for the PC(Na+) cuticle are around 60% lower that those determined for the native one. Mem-
brane potential and electrical resistance results show a loss of the barrier character corresponding to the pepper cuticle as a result of chemical treatment. The effect of temperature on native cuticles was also determined from membrane potential and electrical resistance measurements with NaCl solutions: Fig. 6 shows a comparison of Dfm versus ln(C i/0.005) obtained for a cuticle before and after annealing at 60°C: as a result of annealing the membrane, an almost linear relationship between Dfm and ln(C i/C o) was obtained, instead of the parabolic one found for the nonannealed native membrane; according to Eq. (1) these results would indicate that for the
Fig. 3. Membrane potentials at C =0.005 M NaCl. A comparison between experimental () and calculated () values (subtracting the Donnan potential contribution).
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Fig. 4. Membrane potential versus ln(C i/C o) for the PC(Na+) cuticle with NaCl solutions at different concentrations: () 0.001 M, () 0.005M, ( ) 0.01 M and (X) 0.05 M.
Fig. 6. Membrane potential, Dfm. versus ln(C i/C o) at C= 0.05 M NaCl. A comparison between: () native cuticle, () native cuticle after annealing at 60°C.
annealed sample the membrane potential corresponds to a diffusion potential, and the Donnan potential contribution can be neglected. The electrical resistance values for the annealed membrane were also lower than those for the native sample and a ratio Rm(annealed)/ Rm(native) : 0.7 was obtained. These results also indicate a more open structure of the pepper cuticle due to thermal treatment. It should be pointed out that both chemical and thermal treatments produce a decrease in the barrier behaviour of cuticular membranes, which could
be related to conformational changes in the macromolecular arrangement of the cutin matrix, and they can affect the transport of electrolyte through cuticles.
Fig. 5. Membrane electrical resistance as a function of the NaCl concentration: () native cuticle; () cuticle in Na+ form.
5. Conclusions Membrane potential measurements for isolated pepper cuticles with NaCl and CaCl2 solutions allow the determination of some electrokinetic parameter: A small negative fixed charge (around 2× 10 − 3 M) in the membrane. Due to its small value, the Donnan exclusion of the co-ions only appears at low concentrations (CB 10 − 2 M), while for concentrations higher that 10 − 2 M the membrane potential is practically due to the different mobilities of the ions in the cuticular membrane. Concentration dependence of transport numbers in the cuticle for both electrolytes. Results show that pepper cuticles present higher selectivity for Na+ than for Ca2 + ions. The effects of both chemical and thermal treatments on membrane potential and electrical resistance values were also studied. A decrease in both kinds of measurements occurred as a consequence of treatments. In both cases, results show a loss of
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the barrier function of the cuticle, which must be attributed to some changes in the macromolecular arrangement of the cuticular membrane.
Acknowledgements The authors thank the DGCYT (project PB941492) for financial support.
References [1] P.J. Holloway, The Plant Cuticle, Academic Press, New York, 1982. [2] J. Schonherr, K. Eckl, H. Gruler, Planta 147 (1979) 21. [3] A. Chamel, B. Gambonet, C. Genova, A. Jourdain, J. Environ. Qual. 13 (1984) 384. [4] J. Schonherr, Planta 128 (1976) 113. [5] J. Schonherr, M.J. Bukovac, Planta 109 (1973) 73. [6] J. Schonherr, R. Huber, Plant Physiol. 59 (1977) 145. [7] M.T. Tyree, T.D. Scherbatskoy, C.A. Tabor, Plant Phys-
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iol. 92 (1990) 103. [8] A. Heredia, J. Benavente, Biochim.Biophys.Acta 1062 (1991) 239. [9] W.E. Shafer, M.J. Bukovac, Plant Physiol. 83 (1987) 652. [10] J. Benavente, A. Munoz, A. Heredia, J. Membrane Sci. 139 (1998) 147. [11] T. Teorell, Discuss. Faraday Soc. 21 (1956) 9. [12] K.H. Meyer, J.F. Sievers, Helv. Chim. Acta 19 (1936) 649. [13] N. Laksminarayanaiah, Chem. Rev. 65 (1965) 491. [14] S.G. Schultz, Basic Principles of Membrane Transport, Cambridge, University Press, 1980. [15] H.-U. Demisch, W. Pusch, J. Colloid Interface Sci. 69 (1979) 247. [16] Y. Kimura, H.J. Lim, T. Ijima, Angew. Makromol. Chem. 138 (1986) 151. [17] J. Benavente, Nonequilib.Thermodyn. 15 (1990) 213. [18] M.D. Reboiras, J. Membrane Sci. 92 (1994) 75. [19] A. Rees, E.J. Welsh, Angew.Chem. Int. Ed. Engl. 16 (1977) 214. [20] J.R. Ramos-Barrado, J. Benavente, A. Heredia, Arch. Biochem. Biophys. 306 (1993) 337. [21] J. Benavente, J.R. Ramos-Barrado, A. Heredia, Colloids Surfaces 140 (1998) 333.
Fixed charge and transport numbers in isolated pepper fruit cuticles from membrane potential measurements: Donnan and diffusion potential contributions J. Benavente a,*, A. Mun˜oz b, A. Heredia b, A. Can˜as a b
a Departamento de Fı´sica Aplicada, Facultad de Ciencias, Uni6ersidad de Ma´laga, E-29071 Ma´laga, Spain Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias, Uni6ersidad de Ma´laga, E-29071 Ma´laga, Spain
Abstract Membrane potential of native isolated pepper cuticles were measured with NaCl and CaCl2 solutions for a wide range of concentrations (10 − 3 BC(M)B10 − 1). From membrane potential results the fixed charge concentration, X, and the transport number of the ions in the cuticular membrane were obtained. Taking into account the X value, an estimation of the Donnan potential contribution to the membrane potential was carried out; it was found that the Donnan exclusion of co-ions only occurs at low concentrations, while at concentrations higher than 10 − 2 M the membrane potential is mainly due to the different mobility of the ions in the cuticular membrane (diffusion potential). Concentration dependence for both fixed charge concentration and transport numbers was also considered. The influence of chemical and thermal treatments on membrane potential and electrical resistance was also studied. A decrease in the experimental values of both parameters as a consequence of chemical treatment was found; results obtained with an annealed pepper membrane also show a reduction in the electrical resistance and in the contribution of the Donnan potential to the measured membrane potential. These facts indicate a more open structure for the pepper cuticles due to both different treatments. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Chemical and thermal treatments; Electrokinetic parameters; Fruit cuticles
1. Introduction Plant cuticles are biological polymer membranes of heterogeneous composition [1] which cover the aerial organs of terrestrial plants. They * Corresponding author. Tel. + 34-5-2131929; fax: +34-52132000. E-mail address: j – [email protected] (J. Benavente)
constitute the interface between the environment and the plant cells and, although their main function is to minimise water loss from plants when stomata are closed, the cuticles are not impermeable to water (or electrolyte solutions) and under some conditions, they cannot prevent wilting. In fact, their transport properties are of great biological and practical interest as can be seen from different studies dealing with cuticular transpira-
0927-7757/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 9 9 ) 0 0 2 7 5 - 7
424
J. Bena6ente et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 159 (1999) 423–430
tion, uptake of nutrients, or herbicides, fungicides and xenobiotics applied to the foliage [2,3]. The cuticular membrane model generally adopted is a porous membrane with the pore size depending mainly upon swelling in the presence of water [4]. Scho¨nherr and Bukovac [5] showed that cuticular pores have polar regions composed by the following components: (i) cutin, which is a weakly polar compound due to hydroxyl and unesterified carboxyl groups; (ii) polyuronic acids associated with cellulose and pectin in the secondary cuticle; and (iii) some cuticular waxes with polar substitutents. It was reported that at a pH\ 3 the cuticles have a net negative charge, which may affect the sorption and transport of electrolytes [5,6]. These results also indicate an asymmetrical behavior of the two morphological surfaces of the cuticular membrane [7,8]: the inner surface, contacting the cell wall, appears with less hydrophobic character that the highly hydrophobic outer surface, covered by waxes. In this paper, the membrane potential for isolated fruit cuticular membranes (pepper cuticles) with NaCl and CaCl2 solutions at different concentrations was studied, which allows one to get information about characteristic membrane parameters such as membrane effective fixed charge concentrations and ion transport numbers. Contributions of both, Donnan and diffusion potentials to the measured membrane potential, are also estimated. Since fruit cuticles in their native stage contain different cations sorbed in their matrix (exchangeable cations) [5], which could interact with the electrolyte solutions during transport mechanism masking the results, membrane potential and electrical resistance for a pepper fruit isolated cuticle in sodium-form (Na+ ions should only be present in the cuticular matrix) were also measured. The comparison of the results obtained with native and sodium-form cuticles shows how changes in the cuticular matrix can affect the electrokinetic parameters. On the other hand, temperature effects on the structure of native cuticles were also studied from comparison of membrane potential and electrical resistance results for native and annealed isolates cuticles.
2. Experimental
2.1. Material Different samples of astomatous cuticles from pepper (Capsicum anuum, L.) fruits were used. The cuticles were enzymatically isolated following the procedure described by Shafer and Bukovac [9]. The cuticles will hereafter be called PC(Na+). Exchangeable cations were removed from a cuticle sample by shaking in a 1 M HCl solution for 15 min (three changes) followed by washing with deionized water to remove sorbed HCl. The cuticle in the sodium form, PC(Na+), was obtained after equilibration with 10 − 2 M NaCl in Tris buffer solution at pH= 7.0 in a bath shaker for 24 h at room temperature. Thereafter the cuticles were washed five times in 10 ml of deionized water (10 min each) to remove sorbed Na+ ions. In order to determine the effect of temperature on electrokinetic parameters, a native isolated cuticle was annealed at 60°C for 2 h. Experiments were carried out using NaCl and CaCl2 (Merck™, 99.5% purity) aqueous solutions at different concentrations at constant temperature t= 25.09 0.3°C and pH 6.7 9 0.3. Before use, the cuticles were kept for at least 48 h in distilled water, and later they were immersed for 12 h in a solution of the appropriate concentration.
2.2. Membrane potential and electrical resistance measurements The measuring cell is similar to that indicated in Ref. [10], and it basically consists of two halfcells, of 10 cm3 each, with a membrane area of 0.7 cm2. To minimize the concentration-polarization at the membrane/ solution interfaces, a magnetic stirrer with a stirrer rate of 500 rpm was placed at the bottom of each half-cell. Membrane potential measurements were carried out keeping constant the concentration of the solution at one side of the membrane, C2, and increasing gradually the concentration of the solution at the other side, C1 . Six different values for the constant concentration were considered (C2 = 10 − 3, 2 × 10 − 3, 5 × 10 − 3, 10 − 2, 5 × 10 − 2 and
J. Bena6ente et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 159 (1999) 423–430
10 − 1 M), while C1 ranged between 5 × 10 − 4 and 5× 10 − 1 M. In order to see if the membrane asymmetry can be detected from membrane potential measurements, the constant concentration was placed in contact with both the inner, C1, and the outer, C o, membrane surfaces; in all measurements, the electrode placed into the solution in contact with the outer membrane surface was grounded, this means: Df = f(C i) −f(C o). The electrical resistance measurements were carried out using an alternating current bridge (Wayne Kerr, Automatic Precision Bridge, model B905) at a frequency of 1 KHz, which was connected to the cell via Ag/AgCl electrodes. Resistance values were determined for different NaCl and CaCl2 solutions, for concentration ranging between 10 − 3 and 5× 10 − 2 M, having the solution at both sides of the membranes the same concentration. Resistance measurements were made with the cuticular membranes placed in the membrane holder (Rmd) and without them (Rd), the difference between both values was taken as the membrane resistance. In both experiments three series of measurements were carried out with each native membrane sample for each constant concentration, and the results presented in this paper correspond to the average of those values.
where u + and u − are the cationic and anionic mobilities, z + and z − their valencies, respectively; R and F are the gas and Faraday constant, and T the temperature of the system. t + and t − are the cation and anion transport numbers in the membrane, and they are related to the mobilities by ti = zi ui /i zi ui ; since water transport is not considered, t + and t − represents the apparent transport numbers in the membrane.- a Donnan potential difference at each membrane/solution interface, which is due to the different concentration of co-ions (ions with the same charge as the membrane) in the electrolyte solution and in the membrane (CB and C m B , respectively). Assuming diluted solutions (ai : Ci), and taking into account the conditions of electroneutrality in the solution and in the membrane, the following relation between the distribution of co-ions in the membrane and the solution can be derived as a function of the concentration of fixed charge in the membrane, X, [13,14] ( zB /zA) m rB = (C m B /CB)= [ ZB CB/( zB C B + zX X)]
(2) where zA, zB and zX are the counter-ions, co-ions and membrane charge valencies, respectively. The expression for the Donnan potential is DfDon = f m − f=(RT/zBF)ln(rB)
(3)
In general, the potential difference measured at both sides of a membrane or ‘membrane potential’, Dfm, can be represented by
3. Theory
Dfm = DfDoni + Dfdif + DfDono
According to the Teorell – Meyer – Sievers theory [11,12], the electrical potential difference measured at both sides of a charged membrane when it is separating an electrolyte solution of different concentrations (C1 and C2) basically consists of two terms:- a diffusion potential across the membrane, which is due to the different mobility of the ions in the membrane and, for diluted solutions (concentrations are used instead of activities) it can be expressed as [13] Dfdiff =(RT/F)([u + / z + ]− [u − / z − ])ln(C1/C2) = (RT/F)([t + / z + ]− [t − / z − ])ln(C1/C2)
425
(1)
a diffusion potential across the membrane, Dfdif, and two Donnan potentials, DfDoni and DfDono, one at each membrane/solution interface. Taking into account Eqs. (1) and (3), the following expression for the membrane potential can be written [13]: Dfm = (RT/wzF){ln(C1[(1+4y 21)1/2 + 1]/C2 [(1+4y 22)1/2 + 1]+wU ln[(1+4y 21)1/2 − wU] /(1+4y 22)1/2 − wU)}
(4)
w is +1 or −1, for anionic or cationic membranes, respectively, and yj = zjksCj /wX, ks being
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the salt partition coefficient (ks :1); the parameter U is related to the transport number of the ions in the membrane (U =[t + / z + ]− [t − / z − ]). The fitting of the membrane potential values measured at different salt concentrations allow us to determine the effective fixed charge and the parameter U. Because of the ion transport number represents the amount of current transported for one ion with respect to the total current crossing the membrane, tj =Ii/IT, this means t + + t − = 1, cation and anion transport numbers (apparent transport numbers) can also be obtained from membrane potential measurements as a function of the external salt concentration.
Average values of the fixed charge concentration and cation transport numbers in native pepNa + Ca2 + and t + , are shown per cuticles, X, t + in Table 1 for the whole range of concentrations. The negative fixed charge must be due to the dissociation of COOH- groups from the fatty acids in the polyesters and from -OH phenolic groups [6], depending on the electrolyte, no significant differences were found. However, important differences in the values of cation transport numbers for both electrolytes were obtained, that Na + is the value for t + was much higher than that
4. Results and discussion Fig. 1(a and b) shows the experimental values for the membrane potential, Dfm versus ln(C i/C o) at different values of the constant concentration, with NaCl and CaCl2 solutions, respectively. As can be seen from these pictures, the experimental values correspond to different parabolas, with the maximum/minimum shifting to the left, when the constant concentration increases. A comparison of the Dfm values obtained at a given concentration (0.05 NaCl) for both reverse external situations is shown in Fig. 2. From this picture, some differences in Dfm values depending on the membrane surface in contact with the constant concentration can be observed, being this effect more evident at low concentrations. From Eq. (4), and taking into account the external condition for the curve, the fixed charge concentration in the membrane can be obtained [15]: X=2UCext/(1 − U 2)1/2
(5)
where Cext is the concentration at the maximum/ minimum of the curve [16 – 18]. The parameter U can be obtained by the slope of the experimental points measured at high external concentrations (Cext X), when the Donnan potential contribution can be neglected; from this value, the transport numbers of the ions in the cuticular membranes were also obtained.
Fig. 1. Membrane potential, Dfm, versus ln(C i/C o) for native pepper cuticles at different NaCl concentrations for both external reverse situations. ( +) 0.001 M, () 0.005M, () 0.01 M, (X) 0.02M and ( ) 0.05M. (a) C i =cte; (b) Co = cte.
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the formation of highly hydrated environments due to the calcium solvation potential. Membrane permselectivity, Ps( + ), which is a measure of the selectivity of the counter ions over the co-ions, can be obtained from transport numbers [13]: o o )/(1 − t + ) Ps( + ) = (t + − t +
(6)
o +
where t represents the transport number of the cations in solution. Ps( + ) values for both electrolytes are also indicated in Table 1, where the higher selectivity of pepper cuticles for Na+ ions is clearly shown. Values in Table 1 are the average of at least 18 measurements (three with each membrane sample) and the interval of errors account for changes in both the variability of the native pepper cuticles and the measurements themselves, for this reason small differences in these parameters due to the membrane asymmetry or concentration dependence can be masked. In order to verify these two points, concentration dependence for the average values of the cation transport numbers obtained with a given membrane sample for both external reverse situations (C o = constant or C i =constant) are shown in Table 2. An important decrease of t + when the concentration increases was obtained (around 20 and 40% for NaCl and CaCl2 solutions, respectively); however, no clear influence of membrane asymmetry on t + values was obtained, since the differences found for both external reverse situations are always included in the error interval. Influence of Donnal potential on the experimental membrane potential results can be determined by means of Eq. (2), once the value of the fixed charge in the membrane was obtained. Fig. 3 shows a comparison of the experimental values
Fig. 2. Membrane potential, Dfm, versus In(C i/C o) for native pepper cuticles at different CaCl2 concentrations for both external reverse situations. ( +) 0.001 M, () 0.005 M, () 0.01 M, (X) 0.02M and ( ) 0.05 M. (a) C i = cte; (b) C o =cte. Ca2 + , which does not differ very much for t + from its value in solution. Differences must be due to the high affinity of Ca2 + for some polysaccharides (mainly polyuronic acids) present as components of the cuticular matrix [19] as well as
Table 1 Na+ Ca2+ and t+ , and cation permselectivity, Averages values of the fixed charge concentration, X, cation transport numbers, t+ + Ps(+) for native and Na -form pepper cuticles Ion type
NaCl CaCl2
Na+-form cuticle
Native cuticle X×103 M
t+
Ps(+)
X×103 M
t+
Ps(+)
−2.09 0.6 −1.69 0.5
0.719 0.06 0.4590.06
52% 9%
−(0.690.2)
(0.63 90.04)
40%
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Table 2 Concentration dependence of cation transport numbers for a pepper fruitcuticle CaCl2
NaCl C
C o = cte t+
C i =ctet+
C o =cte t+
C i =cte t+
0.0055 0.0060 0.0075 0.015 0.030 0.045 0.055
(0.83 90.04) (0.76 90.03) (0.75 90.04) (0.74 90.03) (0.71 90.02) (0.69 90.02) (0.67 90.02)
(0.87 9 0.04) (0.78 9 0.04) (0.77 9 0.03) (0.76 90.04) (0.72 9 0.02) (0.71 9 0.02) (0.70 90.02)
(0.56 90.03) (0.53 9 0.04) (0.52 9 0.04) (0.46 9 0.03) (0.43 9 0.02) (0.41 9 0.02) (0.39 9 0.02)
(0.59 90.04) (0.57 90.04) (0.55 90.04) (0.40 90.03) (0.39 90.02) (0.36 90.02) (0.36 90.02)
and those obtained by subtraction of the Donnan potential at both membrane – solution interfaces. Almost linear relationships between Dfm and ln(C1/C2) were obtained for the calculated values, which agrees with the assumption that, in this case, the membrane potential is due to the diffusion potential. The possible influence of chemical treatment on membrane structure and electrokinetic parameters was also studied from membrane potential and electrical resistance measurements with NaCl solutions: Membrane potential measured for a pepper cuticle in Na+-form or PC(Na+) membrane with different NaCl constant solutions is shown in Fig. 4. Fixed charge concentration, ion transport numbers and membrane permselectivity were also determined by means of Eqs. (5), (1) and (6), respectively, and their values are also written in Table 1. Results show a clear decrease in the fixed charge concentration, but lower values of the cation transport number and membrane permselectivity were also obtained. Membrane electrical resistance, Rm, as a function of the external salt concentration for native and PC(Na+) fruit pepper cuticles is shown in Fig. 5, where a decrease in the membrane resistance values when the concentration increases can be observed, which is mainly attributed to the effect of the electrolyte taken up by the membrane [20,21]. Rm values for the PC(Na+) cuticle are around 60% lower that those determined for the native one. Mem-
brane potential and electrical resistance results show a loss of the barrier character corresponding to the pepper cuticle as a result of chemical treatment. The effect of temperature on native cuticles was also determined from membrane potential and electrical resistance measurements with NaCl solutions: Fig. 6 shows a comparison of Dfm versus ln(C i/0.005) obtained for a cuticle before and after annealing at 60°C: as a result of annealing the membrane, an almost linear relationship between Dfm and ln(C i/C o) was obtained, instead of the parabolic one found for the nonannealed native membrane; according to Eq. (1) these results would indicate that for the
Fig. 3. Membrane potentials at C =0.005 M NaCl. A comparison between experimental () and calculated () values (subtracting the Donnan potential contribution).
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Fig. 4. Membrane potential versus ln(C i/C o) for the PC(Na+) cuticle with NaCl solutions at different concentrations: () 0.001 M, () 0.005M, ( ) 0.01 M and (X) 0.05 M.
Fig. 6. Membrane potential, Dfm. versus ln(C i/C o) at C= 0.05 M NaCl. A comparison between: () native cuticle, () native cuticle after annealing at 60°C.
annealed sample the membrane potential corresponds to a diffusion potential, and the Donnan potential contribution can be neglected. The electrical resistance values for the annealed membrane were also lower than those for the native sample and a ratio Rm(annealed)/ Rm(native) : 0.7 was obtained. These results also indicate a more open structure of the pepper cuticle due to thermal treatment. It should be pointed out that both chemical and thermal treatments produce a decrease in the barrier behaviour of cuticular membranes, which could
be related to conformational changes in the macromolecular arrangement of the cutin matrix, and they can affect the transport of electrolyte through cuticles.
Fig. 5. Membrane electrical resistance as a function of the NaCl concentration: () native cuticle; () cuticle in Na+ form.
5. Conclusions Membrane potential measurements for isolated pepper cuticles with NaCl and CaCl2 solutions allow the determination of some electrokinetic parameter: A small negative fixed charge (around 2× 10 − 3 M) in the membrane. Due to its small value, the Donnan exclusion of the co-ions only appears at low concentrations (CB 10 − 2 M), while for concentrations higher that 10 − 2 M the membrane potential is practically due to the different mobilities of the ions in the cuticular membrane. Concentration dependence of transport numbers in the cuticle for both electrolytes. Results show that pepper cuticles present higher selectivity for Na+ than for Ca2 + ions. The effects of both chemical and thermal treatments on membrane potential and electrical resistance values were also studied. A decrease in both kinds of measurements occurred as a consequence of treatments. In both cases, results show a loss of
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the barrier function of the cuticle, which must be attributed to some changes in the macromolecular arrangement of the cuticular membrane.
Acknowledgements The authors thank the DGCYT (project PB941492) for financial support.
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