19 The Permeability of Adsorbed- and Spread-Membrane Protein (Spectrin-Actin)
Bioelectrochemistry: Ions, Surfaces, Membranes Downloaded from pubs.acs.org by YORK UNIV on 12/13/18. For personal use only.
Films to Ions M A R T I N B L A N K , L . SOO, and R. E . A B B O T T Department of Physiology, Columbia University, 630 W. 168 St., New York, NY 10032
The surface properties of spectrin and actin films suggest that these erythrocyte-membrane proteins are present in vivo as an interacted network that also may constitute a significant ion-permeability barrier. Using polarography, we have shown that the ionic permeability of adsorbed and spread spectrin and actin monolayers at the mercury/water interface at different pH's is consistent with an isoelectric point of 5.5. At pH 7.4, the spectrin and actin layers are relatively impermeable to anions (with an estimated permeability of about 10 cm/sec for an adsorbed film) in the range where the erythrocyte membrane is very permeable. Our observations suggest that the specialized anion carrier molecules in the membrane penetrate an anion-depleted spectrin and actin layer. -4
the last few years there have been many advances i n analyzing the I n components and the structure of the red cell membrane (1,2). Many lipid and protein components have been localized i n the structure (3) and several individual proteins have been isolated, e.g. spectrin (4) and actin ( 5 ) . It is now well established that the spectrin component of the erythrocyte membrane is derived from the cytoplasmic side, contains actin, and accounts for about a third of the membrane protein. It is relatively easy to extract and appears " . . to form an anastomosing network beneath the erythrocyte membrane" ( 5 ) . The recently obtained surface-packing information and the surface rheological properties of spectrin-actin (S + A ) monolayers support the 0-8412-0473-X/80/33-188-299$05.00/l © 1980 American Chemical Society
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view that it is an important functional component of the membrane that is present as an interacted cross-linked network on the inner face ( 6 ) . Such a layer i n the cell membrane may constitute a significant ionpermeability barrier. T o examine this possibility we have obtained information about ion transport rates through this layer. W e have been studying the transport of molecules and ions across monomolecular films of lipids and proteins for some time, and w e now have a reasonable understanding of the physical factors that influence the rate ( 7 ) . (It should be mentioned that our early studies of monolayer permeability provided us with sufficient insight into monolayers to predict the correct magnitudes of other important properties, such as the selfdiffusion coefficient of lipids (8) i n membranes and the flipping frequency (9) of lipid molecules between layers.) More recent studies of monolayer permeability to ions have provided information about effects of surface charge density, electrolyte concentration, layer thickness, etc., the fundamental factors governing electrical resistance i n very thin membranes and i n interfacial regions (10,11). These results can be summarized and explained in terms of electrical double-layer theory (12). Experimental Preparation of S + A . The large number of proteins that are present i n the erythrocyte membrane can be separated into a number of well characterized components. The components associated w i t h SDS polyacrylamide gel Bands 1 and 2 and called spectrin can be obtained without the aid of surface-active agents (see Figure 1). It is possible to extract this material from erythrocyte ghosts by using low salt and also by incubating with E D T A . The extraction treatment generally produces an additional low-molecular-weight component (Band 5 on S D S polyacrylamide gels) that has been identified as actin. This preparation constitutes about one third of the membrane protein and the major components of the endofacial layer of the erythrocyte membrane. In our experiments, we used standard techniques i n preparing spectrin and actin. Human erythrocytes were obtained from freshly outdated blood bank blood. Following centrifugation and removal of the plasma and buffy coat, the erythrocytes were washed w i t h cold isotonic buffer ( 3 0 m M sodium phosphate ( p H 7.4), 117mM N a C l , and 2.8mM K C 1 ) . The washed, packed cells were hemolyzed i n 12 vol cold 8 m M sodium phosphate, p H 7.4, for 30 m i n at 4 ° C , and ghost membranes isolated by the method of Dodge et al. (13). The washed ghosts were stored frozen at — 20°C overnight and then extracted with 10 vol O . l m M sodium E D T A , p H 8.0, for 20 min at 37°C. Centrifugation at 78 X 10 g (30 min, 4 ° C ) yielded a clear supernatant (typical protein concentration about 0.2 m g / m L ) which was S + A (Bands 1, 2, and 5) as judged by the Coomassie Brilliant Blue bands seen after SDS-polyacrylamide gel electrophoresis ( P A G E ) (see Figure 1). This solution was used to form spread films at an air/water interface or was diluted to nanomolar concentrations for experiments on adsorbed films. 3
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Figure I . Two tracings of the densities of the bands seen with proteins from the erythrocyte membrane after SDS treatment and PAGE. The numbers correspond to the major peaks on the upper tracing—a sample of proteins from the whole membrane. The lower tracing is of an S + A preparation used in these experiments.
Some experiments were done with bovine serum albumin ( B S A ) i n order to compare our measurements on S + A films with those of a known substance. The B S A was obtained from Sigma and used without further treatment. T r o u g h Measurements. The experiments were performed with a Langmuir-type trough and barriers made of Teflon. A Teflon barrier was attached to an apparatus which varied the area at a rate of 10 cm /sec. The original area for spreading the film was 300 c m and the experiments were performed at a temperature of 25°C and i n the p H range of 3-8. The surface tension was measured with a Sanborn ( M o d e l 311A) transducer and an attached sand-blasted platinum plate. The Teflon apparatus and a glass rod were cleaned w i t h detergent, rinsed several minutes i n running cold water and finally i n doubly distilled water. The surface tension was recorded after filling the trough with O.liV N a C l solution, sweeping the surface, and setting up the dipping plate. Precise amounts of S + A spreading solution were de2
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livered from L a n g - L e v y pipets and spread down the glass rod to the surface. The glass rod then was rinsed several times with 0.1N N a C l solution. After waiting 5 min, the movable Teflon barrier was started with a speed of 1.58 mm/sec or 1.58 cm /sec and the surface-tension readings were recorded automatically. W h e n S + A films were spread i n the polarographic cell, film thicknesses were estimated assuming that the partial molar volume of a protein on a surface is equal to that i n solution, 1.3 c m / g . Combining this figure with our value for the surface concentration of a close-packed S + A film, 1.4 X 10" g / c m , we obtain a thickness of 18 A per monolayer (6). Ion Transport Through Monolayers. The movements of ions through oriented monolayers was studied by determining the effect of an adsorbed or spread monolayer on the polarographic reduction of an ion at a mercury/water interface (10). The permeating ions are shown i n Table 1 along with the p H ranges i n which they were used. (The complex ions of C u assume different charges depending on the anions present.) To overcome the experimental limitations in connection with the need for soluble substances as films, we extended the technique devised by Pagano and Miller (14), which utilized the polarographic technique in conjunction with the conventional surface film balance. This makes it possible to form films at an air/water interface under conditions where the surface concentration and charge are well characterized, and then to study the ionic permeability of these films at a mercury/water interface. W e therefore have studied the permeability of both adsorbed protein films and spread films formed directly in the polarography cell. In both types of experiments the results were interpreted in terms of a transport rate constant, k , using a method described earlier (10). A t polarizations where the rate of electroreduction is high, the diffusion current across an electrode covered by a monolayer is given by the diffusion of the depolarizer to the surface and by the rate of crossing the monolayer. This problem has been treated formally like the kinetic current, where the electrode processes are replaced by the crossing of the monolayer, and the solution of the differential equation for the dropping mercury electrode is the same as given by Koutecky (15), who tabulated X = (k t /D ) as a function of the reduced current J/J , where J and 7„ are the diffusion-controlled instantaneous currents in the presence and absence of a film, respectively. Knowing the time, t, and D, the diffusion coefficient, we used his tabulated data for calculating k from the reduced current. 2
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Figure 2. The surface pressure, IT (in dynes/centimeter), vs. the surface area of spread S + A monolayers (meters /milligram) at 25°C. The curves refer to: (O), compressed monolayers on 0.1 M NaCl; (A), monolayers formed by additions of S + A at constant area on 0.1 M NaCl; (A), monolayers formed by additions of S -f- A at constant area on 0.1 M phosphate buffers. 2
Results W h e n we deposit the S -f- A from a O . l m M E D T A solution on to a O . l m M saline surface at a p H of 5.8 (approximately the isoelectric point of S + A ) and compress the film on a standard surface balance, we obtain the surface pressure data shown i n Figure 2. If we extrapolate to zero surface pressure, the intercept is about .7 m / m g . O n the same abscissa, we also have plotted the surface pressure-surface concentration curves obtained by incremental spreading of S -f- A at a constant area. The curves are really quite different from the compressed films and the extrapolated area at zero surface pressure is considerably larger. These results indicate that the structure of an S + A film is very dependent upon the conditions under which it is formed, an observation that w e shall underscore i n the ion transport measurements. W h e n the S + A films are formed directly i n the polarography cell at the air/water interface and their permeabilities to ions are determined at different pH's, the values of k are shown i n Figure 3 as functions of the surface concentration. As observed previously i n the case of adsorbed films (16), at p H 3 the S -f- A film is relatively impermeable to cations and at p H 6.4, to anions. The values of k are also about the same order 2
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Figure 3. The permeability, k (in centimeters/second), of spread S + A films to C u ions as a function of the surface concentration of S + A (in milligrams/meter ). At pH 3.0 the data apply to the permeation of cations while at pH 6.4 they apply to anions. c
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of magnitude for both types of films (see Figure 4 ) . However, there is an enormous difference between the thickness of the impeding layers. I n the spread films (see Figure 3 ) , there are over ten monolayer thicknesses on the k plateau regions, while for the adsorbed films (see Figure 4) there is not even one complete monolayer present. One can estimate (16) that it would take about 17 sec at the highest S + A concentration to achieve a complete monolayer, a drop time that we cannot achieve. Although the spread S + A monolayer is apparently much looser than the adsorbed layer, it is still much more compact than a spread serum albumin film. Ion transport measurements at p H 3 equivalent to those i n Figure 3 are shown i n Figure 5 for B S A films. Apparently, one must go to about 100 times the surface concentration (or thickness) of S -J- A layers i n order to observe a comparable value of k for B S A . The p H dependence of adsorbed S -f- A films (when compared at the same S -f- A solution concentration and mercury drop times) show the expected selectivity around p H 5.5. W h i l e the earlier results (16) up to a p H of about 6 suggested a much steeper rise i n selectivity on the alkaline 0
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side, the more expanded set of observations show greater symmetry. The decrease i n selectivity at p H 9 may be associated with the swelling of the monolayer or the slow degradation of the protein (see Figure 6 ) . Discussion
The Structure of S + A Interfacial Films. I n this chapter we have presented data that relate to four different types of film. In Figure 2, the standard n - A curve with the intercept at .7 m / m g is obtained by spreading a known amount of S + A at almost zero surface pressure, and slowly compressing. The other curve, usually called a n - C curve, is done at constant area w i t h increments of S + A deposited into the already existing film. The n - A curve reflects a film where the molecules are spread out more effectively, whereas the n - C film contains many molecules that have been incorporated under a significant surface pressure 2
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Figure 4. The permeability, k (in centimeters/second), of adsorbing S -f A monolayers at pH 3 to C u ions as a function of time (in seconds). The concentrations listed refer to the S + A in solution (asuming a molecular weight of 200,000). c
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and so have not been able to spread out to the same extent. These two curves are similar to published data for B S A films (17), and lead us to conclude that S -f- A films are subject to the same packing restrictions on a surface. The third type of film is the one studied i n Figure 3. It is spread like i n the n - A experiment, but at such high concentration that the effect is like many layers of a n - C film. The S + A molecules do not have the opportunity to spread out at the interface, and because of the many layers i n these films, the S -f- A molecules do not experience the same asymmetry between hydrophilic and hydrophobic regions as is present i n a single layer. These spread films undoubtedly contain very poorly packed S -f- A molecules, and the very high permeabilities bear this out. Still another type of film is used i n Figure 4. This fourth type is adsorbed at the mercury/water interface and probably is closest to the n - A film at higher areas. The adsorbing S + A molecules always arrive at an expanding interface and the film never reaches the full monolayer concentration. Therefore, there is ample room for the molecules to spread and readjust their orientations at the surface. This film is therefore very different in structure from the one used i n Figure 3, and the permeabilities indicate this as well. The S + A film that is of greatest interest to us, of course, is the complex structure on the inner face of the red cell membrane. Using our
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Figure S. The permeability, k (in centimeters/second), of spread BSA films to C u ions at pH 3 as a function of the surface concentration of S + A (in grams/meter ) c
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Figure 6. The ratio of the reduced current for the permeation of adsorbed S + A monolayers by cations to the reduced current for anions as a function of the pH. The ratio (+/—) is a measure of the selectivity of the S -f A monolayer for cations relative to anions. surface measurements, we calculate that this layer is probably about two monolayers thick (6). In addition to the thickness, the natural S + A film is very different from the four films we have discussed already. It is in contact with the membrane lipids on one side and a concentrated hemoglobin solution on the other. Furthermore, it appears likely that some of the other membrane proteins, i n particular the Band 3 protein, may extend through this layer. It is difficult to decide which type of surface film the natural S + A film resembles most closely. The number of S + A molecules present i n the membrane is closer to the adsorbed film and the packing is believed to be more orderly (2). Also, the ease with which the S + A molecules can be removed from the membrane by low ionic strength media, suggests a loose and more accessible structure. F o r these reasons we feel that the n - A type of film below 10 d y n / c m pressure resembles most closely the natural S -f- A film, and that the data of Figure 4 are more relevant to the membrane state than those of Figure 3. It appears somewhat unreasonable that this less concentrated ( i n two dimensions) but more ordered surface structure is less permeable than
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Figure 7. The diffusion coefficient, D (in centimeters /second), of C u ions through anS + A film at pH 3 as a function of the estimated thickness of the film (in angstroms)
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the many-layered one formed by the rapid spreading method at constant area. Apparently the major factor controlling the flow of ions through the layer is the surface charge (12), and this is probably optimized during a more orderly adsorption process, as opposed to rapid spreading. Protein dissolution in the spread films at higher pressure probably contributed to the greater permeability, but recent experiments indicate that only about 30% of the S + A layer dissolves under the conditions of the experiments. The Permeability of S -f- A Films. As already mentioned, the k values in Figures 3 and 4 refer to different types of films of S - f A and also to different thicknesses. The magnitude of k for spread S -f- A films (see Figure 3) reaches a plateau value of about 10" cm/sec at relatively high surface coverages. For the adsorbed films (see Figure 4 ) , the k decreases with time and S - f A concentration, but shows no plateau. This is to be expected, since the form of the current-time curve indicates that the S + A film is still below the saturated monolayer stage (10). Higher concentrations of S -f- A and longer drop times cannot be reached experimentally, but we can estimate the time at which the S + A monolayer would be complete and also the approximate magnitude of k at that point. Extrapolating from the behavior of B S A under the same conditions, we have determined the time of formation of a complete monolayer as a function of the B S A concentration, and have extrapolated those results to the time required for the formation of a S + A monolayer at the highest S + A concentration studied. Using this information, the shortest time (as seen i n Figure 3) for a full monolayer of S + A is 17 sec, and k would be about 10" cm/sec on extrapolation (16). c
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The permeabilities of the two types of S + A films differ by an order of magnitude, so extrapolating to the same layer i n the red cell membrane, involves a choice between a resistance of the membrane layer to ion flow that is either negligible or significant. For reasons given in the previous section, the adsorbed type of film is probably more closely related, i n which case k ~ 10~ cm/sec and significant. This, of course, would mean that the special anion carrier molecules (Band 3 in the SDS gel) i n the membrane would have to extend through the S - f A layer i n order to be effective. There is some evidence that the S + A layer i n vivo does constitute a significant ion-permeability barrier. Lepke and Passow (18) have shown that internal trypsin can cause a slight increase in anion equilibrium exchange in erythrocytes. In any case, the S -f- A layer i n vivo represents a significant anion-depleted (cation-rich) zone in the membrane, which could cause unusual effects in ion transport kinetics (19). c
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The Variation of Permeability with Film Thickness. In early studies of monolayer permeability to water and gases a very strong dependence of the transport rate on the thickness of the film was found
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(7). These observations suggested that as one increases the film thickness there is a transition of monolayer properties to bulk properties. In a more recent study of the viscoelasticity of spread S + A films of different thicknesses (6), we found that when the surface properties were converted to equivalent bulk values (by introducing the film thickness) there was also a variation w i t h thickness. The viscosity coefficient and elastic modulus became independent of thickness at about 80 A , or four monolayers, while the residual yield decreased with thickness over the entire range of the measurements. These results were interpreted to mean that the molecular interactions that account for the properties must become less effective within a layer as the number of layers increases and there are more interactions between layers. W e can examine our permeability data in the same way and calculate a diffusion coefficient upon multiplying the transport rate coefficient (in centimeters per second) by the estimated film thickness ( i n centimeters) to yield a diffusion coefficient (in cm /sec). The results of this calculation, shown in Figure 7, indicate that the diffusion coefficient increases with the thickness of the S -J- A film over the entire range of measurement. This result is directly i n line with the variation of the residual yield in our rheological studies (6), where the original layer had the strongest orientation and interplanar interactions, and all subsequent layers showed less orientation and weaker interactions. The subsequent layers apparently become more permeable as a result of this change i n structure. 2
Acknowledgment This work was supported by Research Grant P C M 76-11676 from the National Science Foundation. Literature Cited 1. Steck, T . L . J. Cell Biol. 1974, 62, 1. 2. Kirkpatrick, F. H. Life Sci. 1976, 19, 1. 3. Zwaal, R. F. A . ; Roelofsen, B.; Colley, C. M . Biochim. Biophys. Acta 1973, 300, 159. 4. Marchesi, V. T . ; Steers, E . Science 1968,
159,
203.
5. Tilney, L . G.; Detmers, P. J. Cell Biol 1975, 66, 508.
6. Blank, M . ; King, R. G . ; Soo, L.; Abbott, R. E . ; Chien, S. J. Colloid Inter-
face Sci. 1978, 69, 67. 7. Blank, M . Prog. Surf. Membr. Sci. 1979, 20, 789. 8. Blank, M . ; Britten, J. S. J. Colloid Sci. 1965,
9. 10. 11. 12.
20,
789.
Blank, M.; Britten, J. S. Chem. Phys. Lipids 1973, 10, 286. Miller, I. R.; Blank, M. J. Colloid Interface Sci. 1968, 26, 34. Sweeney, G. D.; Blank, M . J. Colloid Interface Sci. 1973, 42, 410. Britten, J. S.; Blank, M. Bioelectrochem. Bioenerg. 1977, 4, 209.
13. Dodge, J. T . ; Mitchell, C., Hanahan, D . J. Arch. Biochem. 1963,
14. Pagano, R.; Miller, I. R. J. Colloid Interface Sci. 1973, 45, 126.
110,
119.
19.
15. 16. 17. 18. 19.
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Koutecky, J. Collect. Czech. Chem. Commun. 1953, 18, 597. Blank, M.; Soo, L.; Abbott, R. E. J. Electrochem. Soc. 1979, 126, 1487. Mussellwhite, P. R.; Palmer, J. J. J. Colloid Interface Sci. 1968, 28, 168. Lepke, S.; Passow, H. Biochim. Biophys. Acta 1976, 455, 353. Blank, M.; Britten, J. S. Bioelectrochem. Bioenerg. 1978, 5, 528.
Explanatory Note The authors note that their article in the Journal of Membrane Biology, 1979, 47, 185-193, provides more detailed information about the packing of S + A at an interface. RECEIVED October 17, 1978.
