solution interface: effect of d.c. potential and a.c. field

Colloids and Surfaces 0927-7765/93/$06.00

B B~ornterfaces.

;CI 1993 ~

1 (1993) 277-282 Elsevter Science Pubhshers

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Admittance measurements on protein layers adsorbed at the Pt/solution interface: effect of d.c. potential and a.c. field H. Matsumural,

J.M. Kleijn”

Department of Physical and Colloid Chemistry, 6703 HB Wageningen, The Netherlands

(Received

10 December

1992; accepted

Wageningen

Agricultural

University,

Dreijenplein

6,

12 May 1993)

Abstract The effect of changes m the electric field on the structure of protem layers adsorbed at the Pt electrode/solution interface has been investtgated by means of admtttance measurements. The measurements have been performed over a wide range of d.c. potenttals, at dtfferent frequenctes and amphtudes of the ax. electrtc field, and at vartous pH values. The protems used, cytochrome C and serum albumm, doffer constderably with respect to then molecular masses, points of zero charge and structure stabihttes. In contrast to serum albumin, cytochrome C has a relatively strong electric dtpole moment. Nevertheless, the results for the two protems are very stmilar. Both proteins stay adsorbed at the Interface over the d.c. potenttal range studied and at every pH. irrespecttve of any electrostattc repulsion. Apparently, for both protems factors other than electrostatic interactions are dominant in their final binding to the Pt/solutton Interface. In hne with this, no mdtcattons were obtamed that the orientation of adsorbed cytochrome C molecules ts modulated by low-frequency (2OOGlOOO Hz) reversal of the electrtc field of the interface. A strong ac. field leads to irreversible structural changes m the adsorption layer for both proteins. Key words.

Admittance

measurements:

Cytochrome

C; Electric field effect; Protern

actions between

Introduction The mechanism of protein adsorption onto solid surfaces is very complex and is affected by various physical and chemical interactions between protein, surface and solvent [1,2]. Norde and coworkers [336] have systematically studied the relation between protein structure stability and adsorption behaviour. They found that the adsorption of proteins that have a very stable native structure (so-called “hard” proteins) is governed by dehydration of hydrophobic areas of the sorbent surface and the protein exterior and electrostatic inter*Correspondmg author. ‘Permanent address: Electrotechnical Shi, Japan.

adsorptton.

Laboratory,

Tsukuba-

Serum albumm

protein

and surface. These proteins

do not adsorb onto hydrophilic surfaces unless there is electrostatic attraction between protein and surface. For proteins that have a much less stable native structure (“soft” proteins) conformational rearrangements during adsorption are an additional factor promoting adsorption (entropy gain). As a result the latter type of protein tends to adsorb also on hydrophilic, electrostatically repelling surfaces. Performing protein adsorption experiments at the surface of an electrode offers the possibility to vary systematically the potential of the interface and might give further insight into the role of electrostatic interactions in the overall adsorption process. Presently, in our laboratory the influence

chrome

C in solution

and in the adsorbed

state

of the electric potential of the interface on the initial adsorption rate and adsorbed amounts (detected by reflectometry) of various proteins is

(on ultra fine silica particles) same [13], the corresponding

being studied. In this paper we describe

albumin differ substantially [13,14], revealing a reduction in x-helix content of 20&40% upon

field at the interface

the effect of the electric

on adsorbed

protein

layers,

i.e. the electric field effect after protein adsorption has taken place. Admittance measurements on protein layers adsorbed at the Pt/solution interface have been performed over a wide range of d.c. potentials and at different frequencies and amplitudes of the ac. electric field. Under appropriate experimental conditions (see Materials and Methods section) the capacitance of the electrode interface can be derived from the cell admittance. Because the inner part of a (native) protein molecule is apolar, an adsorbed protein layer may manifest itself as a low permittivity layer between the metal surface and the solution and it is expected that the capacitance of the interface drops when protein adsorption takes place [7]. It should be noted, however, that even a complete monolayer of protein molecules still contains an appreciable amount of solvent and electrolyte. Therefore, the overall interfacial capacitance in the presence of a protein adsorption layer might still be largely determined by the capacitance of the metal/ solution interface [8,9]. In this study we used two different types of protein:

cytochrome

C and

serum

albumin.

The

are practically the spectra for serum

adsorption. Materials and methods All chemicals used were analytical reagent grade. Horse-heart cytochrome C was purchased from Boehringer-Mannheim and bovine serum albumin (BSA) from Sigma. Both proteins were used without further purification. The concentration of protein used in each experiment is 1.0 g ll’, which is more than sufficient to reach plateau adsorption [2]. The supporting electrolyte was KClO, (Merck) at a concentration of 0.1 M. and buffer reagents were biphthalate (J.T. Baker) for pH 4 and boric acid (Merck) for pH 9. Cell and electrodes A three-electrode cell was used together with a potentiostat, a wave generator and a phasesensitive analyser (Solartron 1255 HF). As a working electrode a Pt wire (length 5 mm unless indicated otherwise, counter-electrode reference

electrode

diameter 1 mm) was used. The was a Pt plate (3 cm’) and the was Ag/AgC1/3.5

M KCl.

All

cytochrome C molecule is relatively small and almost spherical (A4 = 12 500 Da, diameter about 3 nm). Its point of zero charge is at pH 10. Due to

potentials are referred to this electrode. The temperature of the solution (about 50 ml) was kept at (20 f 0.2)‘C. Before and during every experiment

an inhomogeneous distribution of charged groups, it has a relatively strong electric dipole moment, which amounts to 325 Debye (1.08 x 10m2’ C m) at pH 7 [lo]. There are strong indications that the protein does not undergo major structural rearrangements upon adsorption [IO-131. Serum albumin (M = 69 000 Da) is much larger than cytochrome C and has an ellipsoidal shape (11.6 nm x 2.7 nm x 2.7 nm); it has no electric dipole moment, its point of zero charge is at pH 5.5, and it changes its structure upon adsorption [2]. While circular dichroism (CD) spectra of cyto-

remove oxygen. The solution was continuously stirred using a magnetic stirrer. The Pt working electrode was cleaned by heating in a gas flame and immersing in a 10 vol.% HNO,/lO v01.%~H202 aqueous solution for one night. Protein adsorption took place at the open circuit potential of the Pt electrode. Admittance measurements as a function of d.c. potential or a.c. amplitude were conducted about I h after protein was added to the cell. (It was found that the admittance stabilises within 20-30 min after protein addition.)

the

solution

was

purged

with

nitrogen

gas

to

H. Matsumura

and J.M. KleiJn/Colloids Surfaces B, Biointerfaces 1

1993) 277-282

Calculation

of interfacial

If measurements are performed in the potential range where the electrode behaves as an ideally

adsorbed at the Pt electrode at every V,, value and at every pH, irrespective of any change in electrostatic interactions. This is in accordance with the results of Morrissey et al. [15], who measured

polarisable

(ellipsometrically)

interface

capacitance

(double

interfacial capacitance C and tance R, can be derived from

layer

C

region),

the solution

the resis-

OC

= = oCsin

(1 + &C2R31/2

1 13= F cos 0 s

(1)

Under linear conditions Y is the admittance of the electrode/solution interface; Rd is the detector resistance. v,(t) and x(t) are the output and input signals respectively, w is the angular frequency of the input signal and 8 = tan- ’ (Im[ Y]/Re[ Y]). The output signal was analysed in terms of 1Yj and 8. Results and discussion The effect of the bias potential V,, (at an a.c. amplitude A,, of 0.01 V) on the admittance of the bare and protein-covered electrodes is demonstrated in Fig. 1, for neutral pH (no buffer added), pH 4 and pH 9. In the presence of a protein adsorption layer, (YI is lower over the whole bias range examined. Apparently, both proteins stay

279

adsorbed

amounts

of

serum

albumin and other proteins on a Pt electrode as a function of applied potential. The adsorbed amounts (about 2 mg m 2 for serum albumin) did not change until a certain “onset potential” was reached (+0.6 V vs. SCE for serum albumin). Above this potential enhanced adsorption was found. Desorption as a result of changes in the potential was not observed. In Fig. 1 the variation of IYI with V,, reflects properties of the Pt electrode/solution interface: neither the type of protein nor its ionisation state has a significant influence on the shape of the curves. Obviously, the interfacial capacitance is still largely determined by elements that govern the capacitance of the bare electrode (see Introduction section). Admittance plane plots (0~ 1000 Hz) were obtained in the bias range from -0.5 to + 1.5 V. Vdc= + 1.25 V, direct currents were Above observed, in accordance with the d.c. voltammogram of the Pt electrode in 0.1 M KC104. From the admittance plots in the double layer region C and R, were calculated using Eq. (1). Below 200 Hz, the values obtained for C and R, increase strongly

103 IYI /Ix

Fig. 1. Dependence

of 1YI

OII

the bias potential (200 Hz, A,, = 0.01 V). (a) No buffer added; (b) pH 4; (c) pH 9; 0, 0, 1.0 g 1-l cytochrome C; a, 1.0 g 1-l BSA.

bare Pt electrode;

with decreasing frequency for both the bare and the protein-covered electrodes. Obviously, the simple double layer model is not correct in this frequency range. This might be due to the contribu-

chrome C oxidase and cytochrome C reductase). From total internal reflection fluorescence (TIRF)

tion of some kind of exchange reaction, resulting from the presence of surface inhomogeneities.

solution interface the orientation of cytochrome C molecules can be affected by the interfacial potential during the process of adsorption. However,

impurities and/or an oxide film at the surface [S]. At higher frequencies this contribution is suppressed and R, has a fairly constant value of about 120 R, which is the same for the bare and the protein-covered electrodes (see Fig. 3). In the presence of protein the capacitance C is lower than for the bare electrode at any I$, value. In Fig. 2 this is illustrated for cytochrome C at I$, = -0.25 V. BSA gives essentially the same results. but the drop in C is slightly smaller. It has been suggested [lo] that the electric dipole moment of cytochrome C is of physiological importance: it would cause the molecule to orient itself in the electric fields of its redox partners (cyto500

,

I

I

I

I

I (a)

Rp2

250 0

0

1 0

I

I

200

400

I

600

I

I

800 frequency

1000

C&F

2.5

1 0

I

200

I

400

Fig. 2. Frequency dependence 0.25 V); 0. bare Pt electrode;

I

600

I

800 frequency

of R, and C (A,,=O.OI ::I. 1.0 g 1-l cytochrome

once

adsorbed,

the

orientation

is not

changed

anymore by variation of the potential. Apparently, the final binding of the molecules to the interface is too tight to allow reorientation. The findings from the experiments described here are in line with these earlier observations. If the adsorbed molecules were able to adjust their orientation, they would follow the ac. field at low frequencies and the capacitance of the adsorption layer would be at its corresponding upper limit. Above a certain “dispersion frequency” (related to the rotational diffusion coefficient of the molecules at the interface), the protein molecules would not follow the electric field any longer. Then the capacitance of the adsorption layer would be lower. We did not find anything like a “dispersion frequency” in C in the frequency range ZOO- 1000 Hz at any Vdcvalue. However, there are two important points to consider: firstly, only if the capacitances for the two situations are sufficiently different. would it be possible to monitor this; secondly, it is still possible that a “dispersion frequency” below which cytochrome C reorients, does exist m the low-frequency range where Eq. ( 1) is no longer applicable. However, the fact that cytochrome C does not desorb at any I$, value strongly suggests a tight

/Hz

5

0

orientation measurements conducted in our laboratory [13] it was found that also at an electrode/

I 1000 /Hz V, I&= C

binding of the molecules to the interface. A number of experiments was conducted at a higher a.c. amplitude of 0.4 V. It must be noted that under this non-linear condition the term “admittance” for Y is not appropriate and C and R, calculated using Eq. (1) are less well defined. Figure 3 shows / YJ at 0.4 V a.c. amplitude over a wide bias range before and after adsorption of cytochrome C at the Ptlsolution interface. Above +0.75 V and below -0.5 V bias direct currents are observed. Now. the effect of the adsorbed protein layer is dependent on the bias potential

H Matsumura

and J.M

Klerjn/Colloids

Surfaces B

3

3-0.5

0

05

Fig. 3. IYI as a function

1

15 V&/V

of the bias potential at lugh ax. amplitude (&=0.4 0, bare Pt electrode: 0, 1.0 g 1 1 cytochrome

and the pH of the solution. At pH 9 )Y( is lower in the case of protein adsorption, as expected. At pH 4 there is hardly any effect and at neutral pH (no buffer added) above a bias potential of about 0 V 1Y[ is higher with protein adsorbed on the electrode than for the bare electrode. Practically the same results are found for serum albumin (shown only for pH 4, Fig. 3(b)). In the double layer region the frequency dependences of C and R, are comparable to those at the lower A,, value of 0.01 V (Fig. 2). In Fig. 4 the effect of the a.c. amplitude on ) YI is shown. In the presence of an adsorbed layer of cytochrome C or BSA, 1Yj increases with increasing A,, to a plateau value. However, when A,, is decreased again, the admittance does not return to its initial value for low A,,. For the bare Pt electrode hysteresis is negligible. Simple desorption of protein molecules at high a.c. amplitudes cannot be the cause of the results presented in Fig. 4: such a process would lead to an increase in interfacial capacitance in the direction of that for the bare electrode, never to capacitance values higher than that. Moreover, the process would be reversible: going back to low a.c. amplitude would result in readsorption of protein. A possible explanation for our findings at high a.c. amplitude is as follows. As long as the protein molecules are adsorbed more or less in their native

IYI x

V, 200 Hz). (a) No buffer added; C; C, 1.0 g I- ’ BSA.

(b) pH 4, (c) pH 9;

103(R-Q I

I

I

I

I

(a) 8

6

4 0

0.1

0.2

0.3

0.4

0.5

0.6 %cM

20

18

16

14 0

0.1

0.2

0.3 ‘%CM

Fig. 4. Effect of the ax. amphtude on 1Yj (1 kHz, Vdc=0.5 V. no buffer added). (a) 5 mm Pt electrode; 0, bare Pt electrode; 0, l.Og 1-l cytochrome C. (b) 15 mm Pt electrode. C, 1.0 g I-’ BSA.

H Matsumura and J.M

282

conformation, they form a low permittivity adsorption layer, resulting in an interfacial capacitance lower than for the bare electrode. Co-adsorbed counterions are localised predominantly in the “gap” between protein and Pt surface, keeping the net charge density in this region low [9]. A high amplitude a.c. field might induce drastic conformational changes in the adsorbed protein layer. (A relatively fast alternating electric field is far more disruptive than a d.c. electric field: the protein molecules do not have enough time to adapt the charge of their ionisable groups; also the effect of local heating is not ruled out.) The compact structure is lost and part of the surface-bound segments desorb. Furthermore, co-adsorbed counterions are no longer localised in the region between protein and electrode surface and counterion polarisation [ 16,171 becomes feasible. Instead of a low permittivity adsorption layer, a (strongly) polarisable layer is formed, leading to an increase of the interfacial capacitance, in some cases even beyond the capacitance of the bare Pt electrode. The hysteresis observed in Fig. 4 might be due to such a large deformation of the protein structure that refolding to a more compact structure and readsorption of segments do not occur or take a long time. Conclusions It is not possible

KleljniColloids

desorption

of cyto-

chrome C or serum albumin by changing the potential of the interface. Apparently, factors other than electrostatic interactions are dominant in the binding of these proteins to the Pt/solution interface. In line with this we did not find any indication that the orientation of cytochrome C, which has a relatively large electric dipole moment, is modified by the electric field of the interface. This result can be compared with our earlier TIRF experiments

B Biornterjhcrs

I

f IYY3) .?77-.?82

[12], from which it was found that cytochrome

C,

once adsorbed at a SnO, surface, could not be forced into another orientation by variation of the electric potential of the interface. A strong a.c. field seems to induce irreversible structural changes in the adsorption serum albumin.

layers

of both

cytochrome

C and

Acknowledgements The authors wish to thank Dr. H.P. van Leeuwen and Dr. W. Norde for carefully reading the manuscript and for valuable suggestions. References I 2 3 4 5 6 7 8 9

10

to induce

Sutfaws

11 12 13 14 15 16 17

BA. Ivarsson and K.I. Lundstr6m. CRC Cnt. Rev. Blocompat.. 2 (1985) 1. W. Norde, Adv Colloid Interface Scl., 25 (1986) 267. T. Aral and W Norde. Collcnds Surfaces, 51 ( 1990) I; 17. H. Slurahama, J. Lyklema and W. Norde, J. Colloid Interface Sci.. 139 (1990) 177. W. Norde, T. Aral and H. Shlrahama. Blofouhng, 4 (1991) 37. W Norde and J. Lyklema. J. Biomater. Sci Polym. Edn.. 2 (1991) 183. P. Bergveld. Blosensors Bloelectromcs, 6 (1991) 55. B A. Ivarsson, P.-O. Hegg. K.1 Lundstrom and U. JGnsson, Colloids Surfaces, 13 (1985) 169. J.G.E.M. Fraaqe. Interfacial thermodynamics and electrochemistry of protem partltloning m two-phase systems. Ph.D. Thesis, Wagenmgen Agrrcultural University, 1987. W.H. Koppenol and E. Margohash, J. Biol Chem., 257 [ 1982) 4426. J.A. Reynaud, I. Tavermer. LT. Yu and J M. Cachet. Bloelectrochem. Bloenerg.. 15 (1986) 103. J.G.E.M. Fraaije, J.M. Kleqn, M. van der Graaf and J.C. Dijt, Blophys. J., 57 (1990) 965. A Kondo, S. Oku and K. Hlgashltam. J. Collotd Interface SC]. 143 (1991) 214. W. Norde and J.P. Favler, Colloids Surfaces, 64 (1992) 87. B.W. Morrlssey. L.E. Smith. R.R Stromberg and CA. Fenstermaker. J. CoIlold Interface SC].. 56 (1976) 557 A. Minakata, N. Imai and F. Oosawa. Blopolymers. 1I (1972) 349. A. Mmakata. T Nishlo and H. Nakamura. Ferroelectncs, 86 (1988) 7.