Forces between model polypeptides and proteins adsorbed on mica surfaces

Colloids and Surfaces, 31 (1988) 125-146 Elsevier Science Publishers B.V., Amsterdam

125 -

Printed

in The Netherlands

Forces between Model Polypeptides and Proteins Adsorbed on Mica Surfaces T. AFSHAR-RAD*,

A.I. BAILEY and P.F. LUCKHAM+

Department of Chemical Engineering and Chemical Technology, Imperial College, London SW7 (United Kingdom) W. MACNAUGHTAN**

and D. CHAPMAN

Department of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF (United Kingdom) (Received

15 May 1987; accepted in final form 4 July 1987)

ABSTRACT The forces of interaction between proteins and model polypeptides adsorbed onto mica have been measured as a function of the distance of separation between the mica surfaces in aqueous solution. Results were obtained for the basic polypeptide, poly-1-lysine, of molecular weight 4,000 and 75,000, cytochrome c, concanavalin A and myelin basic protein. For cytochrome c no interaction between the adsorbed protein layers was noted until the surfaces were separated by 12.5 nm whereupon an attraction was measured, indicating that the negatively charged mica has been neutralised by adsorption of the positively charged cytochrome c. For all the other proteins, and the poly-1-lysine the force at long range was repulsive and could be explained simply in terms of electrical double layer theory. At short surface separations attractions were noted in all cases (although for concanavalin A calcium and manganese ions needed to be present). These attractions can be accounted for by van der Waals forces for all the systems, with the exception of myelin basic protein for which an attraction ten times larger than that predicted by van der Waals forces was measured. We propose that this additional attraction may be due to hydrophobic interactions between the adsorbed myelin basic protein layers.

INTRODUCTION

Interactions between proteins adsorbed onto, or incorporated into cell membranes are of great importance in many biological and medical fields [ 1,2]. In areas such as cell recognition [ 31 it will be the proteins and glycoproteins on *Present adress: Rutherford Appleton Laboratory, Chilton, Didcot, Oxon. OX11 OQX, United Kingdom. **Present address: R.H.M. Research Ltd, The Lord Rank Research Centre, Lincoln Road, High Wycombe, Buckingham, United Kingdom. +To whom all correspondence should be sent.

0166-6622/88/$03.50

0 1988 Elsevier Science Publishers

B.V.

126

the outside of the cell which will detect the presence of an approaching cell. Also in antibody-antigen binding, the specificity of an antibody for a particular antigen is dependent upon protein-protein interactions [ 41. In this paper we report direct measurements for the interactions between adsorbed protein layers. To achieve this, we have used a technique developed by Tabor and Winterton [ 51 to measure van der Waals forces between mica surfaces and subsequently used by Israelachvili and co-workers [ 6-81 to study the interactions between mica surfaces immersed in liquids. Recently this technique has been used to measure the interactions between mica surfaces bearing adsorbed layers of synthetic polymers [g-12]. We have chosen to study several different proteins. A model polypeptide, poly-1-lysine, of two molecular weights was investigated in order to highlight differences between random polypeptides and specific proteins. We have investigated the properties of cytochrome c. This is a well-studied protein which is found in the internal membrane of mitochondria in all eukaryotic cells [ 131. The amino acid sequence of cytochrome c from several different organisms are known. The plant lectin concanavalin A was also chosen. The lectins are proteins which bind specifically to certain carbohydrates, in the case of concanavalin A the specificity is for terminal mannose or glucose residues [ 141. Interactions between adsorbed layers of concanavalin A is a prerequisite to the investigation lectin sugar interactions. Finally, the interactions between adsorbed layers of myelin basic protein have been measured. This protein is found in the cytoplasmic space of the myelin sheath which surrounds the nerve axons. It has been suggested that myelin basic protein is responsible for wrapping the myelin membrane tightly around the nerve axons [ 151, if such a proposal is correct then one would except a strong attractive force between adsorbed layers of myelin basic protein to be present. EXPERIMENTAL

Procedure The apparatus used to perform these experiments was similar to that used by Israelachvili and Adams [6] and will not be described in detail here. All parts of the apparatus which came into contact with experimental solutions were thoroughly cleaned prior to each experiment using the following procedure: metal and teflon parts were sequentially soaked in a Decon solution, rinsed with several litres of water and immersed in absolute alcohol; glassware was cleaned in sulphachromic acid and rinsed thoroughly with water. Cleaved mica sheets ( 1: 3 pm thick), were mounted on glass discs of cylindrical profile using an epoxy glue (Epokite 1003). The apparatus was then assembled in a laminar flow cabinet, within a class 1000 clean room and sealed. (The mica cleaving was also carried out in a class 1000 clean room. ) The ap-

127

paratus was removed from the clean room and placed on an anti-vibration table. Water was introduced into the apparatus and after allowing approximately an hour for thermal equilibration to occur, the forces between the bare mica surfaces were measured. It was observed that at close separations ( ~5 nm) the surfaces experienced a. strong attraction and jumped together. This was taken to be the point at which the two mica sheets were in molecular contact, i.e. when the surface separation D=O. It was at this stage that the presence of any contamination, e.g. dust particles, mica flakes, bacteria, etc., could be detected; only experiments which were free of such contamination were taken to the next stage. The surfaces were then separated to ca 2 mm, the water withdrawn and replaced by a protein or poly-1-lysine solution of the required concentration. The surfaces were allowed to incubate in the solution for between 0.5 and 24 h to allow adsorption to occur, following which the force profiles were redetermined. In some cases the protein solution was drained, replaced by the solvent (water or electrolyte solution) and the force redetermined. No desorption was ever observed, except in the case of poly-1-lysine, the details for which are given in the text. All the results reported here were obtained from at least two, generally more, completely independant experiments, i.e. with fresh mica surfaces.

Materials The mica used was best quality FS/GS, grade 2, Muscovite ruby mica, obtained from Mica and Micanite Ltd, London. The water was double-distilled and passed through a Mill;-& filtering unit. Cleaning solvents were of analytical grade and obtained from James Burroughs Ltd. The electrolyte solutions were prepared using water and AnalaR grade materials (ex. B.D.H.). The myelin basic protein (MBP) rabbit brain, was obtained from CalbiochemBehring Ltd. The other proteins and polypeptides were obtained from Sigma Ltd. The cytochrome c, type IV, product number C 7752, was obtained from horse hearts and extracted without the use of trichloroacetic acid; concanavalin A, type V, product number C 7275, was highly purified material containing less than 0.1% manganese and calcium chloride; the poly-1-lysine hydrobromide samples had molecular weights of 4,000 and 75,0000 g mol-l. The molecular characteristics of the proteins used are given in Table 1. All materials were used without further purification. RESULTS

All the force profiles are presented as F/R against D. The quantity F/R is the force normalised to the radius of curvature of the mica surfaces, and enables comparison between experiments using different mica curvatures. Ac-

128 TABLE 1 Characteristics of proteins studied MBP M.W. Dimensions (nm) Conformation Isoelectric pH D, (nm) Refractive index at D, Amount adsorbed (mg m-‘) Area on surface (nm’) Jump distance (nm) Adhesion energy (mN m-‘) Surface potential (mV )

18,500 1.5x 15.0

Cytochrome c

Concanavalin A

Poly-1-lysine

12,000

50,000 (dimer)

4,000 and 75 000 -0.5X6and 0.5x115 straight chain 10.5 < 1.0 -

3.0x3.4x3.4

8.0x4.0x4.0

ablate spheroid dimer elipsoidal = 10.5 10.5 5.5 3.0 6.5 7.7 1.44 f 0.02 1.42 f 0.02 1.42 + 0.02 1.2kO.4 2.2 z!z0.6 2.9kO.6 23+7 10+3 31f6 15.0 6.0 no jump upto 2.0 N l.Oin 4 mM Ca2+/Mn2+ 50f5 0 50+_5

< 0.5 5.0 20.0 80+5

cording to the Derjaguin approximation [ 161, the value of F/R is equal to 2xE, where E is the corresponding energy per unit area between two flat surfaces. The results obtained when mica surfaces were immersed in water were similar to those already published by ourselves [ 17,181 and Israelachvili and Pashley [ 7,8] and have not therefore been reproduced graphically here. The force was long range, commencing at surface separations of N 125 nm and increased approximately exponentially with decreasing surface separation until the surfaces were 1: 5 nm from contact where an attraction was noted and the surfaces ‘jumped’ spontaneously into molecular contact. Poly-l-lysine The force-distance profiles between mica surfaces following incubation in a 25 mg dmm3 poly-1-lysine solution, molecular weight 4,000 g mol-‘, in water for 2 h are shown in Fig. 1. Similar profiles were recorded when the surfaces were equilibrated for up to 24 h. Measurements commenced at large surface separations (D N 300 nm ) ; within experimental error (typically N 20 ,uN m- ’ ) no forces were detected down to D = 125 nm, where an exponentially increasing force was observed. At D = 5 nm the surfaces experienced a strong attractive force causing them to jump to within 1 nm of molecular contact of the mica sheets. On separation of the surfaces a large adhesive force was measured. The inset in Fig. 1. illustrates the data on a linear scale, and shows the strength of the attraction. The force-distance profiles between mica surfaces following incubation in a 12 mg dmV3 poly-1-lysine solution, molecular weight 75,000 g mol-‘, in water

129

0

25

50

Dhm)

75

Fig. 1. The interaction force profile between two curved mica sheets bearing poly-1-lysine, molecular weight 4,000 g mol-‘, adsorbed from water, at a solution concentration of 25 mg dmm3. The results are fitted (for details see text) assuming a surface potential of 80 mV and an electrolyte concentration of 7.9 x 10m5 mol dmm3, for a constant surface charge (full line) and a constant surface potential (dotted line). The surface charge density is 0.0152 electrons nm-‘. The inset shows the force on a linear scale and shows the strength of attraction.

for 2 h are shown in Fig. 2. (Similar profiles were recorded when the surfaces were equilibrated for up to 24 h.) These results are essentially identical to those of the lower molecular weight poly-1-lysine. Replacement of the poly-1-lysine solution by water results in a reduction of the force due to some desorption of the polyelectrolyte. This effect has been discussed by us in a previous publication [ 171. Myelin basic protein Figure 3 shows a logarithmic plot of the force against distance for MBP adsorbed on mica in water. The force commenced at surface separations of approximately 75 nm and increased exponentially with decreasing distance until the surfaces were N 17 2 1.0 nm from the mica contact, where an attractive force between the surfaces was observed, and the surfaces ‘jumped’ to a

,l-----0

25

50

15

0 (nm)

Fig. 2. The interaction force profile between two curved mica sheets bearing poly-l-lysine adsorbed from water, molecular weight 75,000 g mol-‘, at a solution concentration of 12 mg dme3. The results have been fitted (for details see text) assuming a surface potential of 80 mV and an electrolyte concentration of 1.0 X 10e4 mol dmm3, for a constant surface charge (full line) and a constant surface potential (dotted line) interaction.

position 3.2 + 0.2 nm from contact. Further compression of the surfaces resulted in a steeply rising repulsive force, with the surface separation decreasing by only a further 0.2 nm. At this separation the refractive index was measured from the fringes of equal chromatic order, which are also used to measure the surface separation [ 6,9] and found to be 1.44 5 0.02. On separation of the surfaces a strong adhesive force was measured. When the surfaces had separated by 0.2 nm the surfaces ‘jumped’ apart by several microns. In order to represent this very strong attractive force, the inset in Fig. 3 shows the data plotted on a linear force scale. The attraction was found to vary between 6 and 15 mN m-‘, but had a typical value of 8 mN m-l. The smaller attractions were possibly due to incomplete coverage of the surface by protein, although this variation could not be detected from the refractive index of the material between the mica sheets.

131

10

I

I

15 D(nm) Fig. 3. The interaction force profile between two curved mica sheets bearing myelin basic protein adsorbed from water, at a solution concentration of 5 mg dmm3. The results are fitted (for details see text) assuming a surface potential of 50 mV and an electrolyte concentration of 3.0 x lo-* mol dmv3, for a constant surface charge (full line) and a constant surface potential (dotted line) interaction. The inset shows the force plotted on a linear scale and shows the strength of the attraction. The surface charge density is 0.015 electrons nm-‘. The different symbols represent the results of two independent experiments. 25

50

Concanavalin A Figure 4 shows a logarithmic plot of force against distance for concanavalin A adsorbed on mica. In water, the force commenced at surface separations of 85 nm, and increased exponentially with decreasing distance until the surfaces were -8 nm from the mica contact whereupon further compression of the surfaces resulted in a steeply rising repulsive force, with the surfaces only being compressed by a further 0.5 nm. At a surface separation of 7.7 nm the refractive index was 1.42 + 0.02, as measured from the fringes of equal chromatic order [ 6,9]. On separation of the surfaces no attraction was observed, as was the

132

lO(

10

50

25

15

Fig. 4. The force profile between two curved mica sheets bearing concanavalin A adsorbed from water, at a solution concentration of 5 mg dme3 (filled symbols). The results are fitted (for details see text) assuming a surface potential of 50 mV and an electrolyte concentration of 4.0 x 10m4,for a constant surface charge (Ml line) and a constant surface potential (dotted line) interaction. The surface charge density is 0.017 electrons nm-‘. The open symbols are the results obtained in 4 mA4 Ca2+/Mn2+. The inset shows the force plotted on a linear scale and shows the strength of the attraction. The different symbols represent the results of two independent experiments. case with poly-1-lysine

and myelin

basic protein,

and similar

force profiles

were

In order for the lectin concanavalin A to bind with the sugars glucose and mannose, calcium and manganese ions need be present. Therefore we repeated the above experiment in the presence of a 4 x 10m3 molar solution of these ions, however under these conditions there was no adsorption of the protein. In order to investigate the forces between ‘activated’ adsorbed layers of concanavalin A, the protein was adsorbed from water and the ions (4 mM) added subsequently. These results are also presented in Fig. 4 (open symbols). A repulsion was observed at surface separations of 20 nm which increased exponentially with decreasing distance until the surfaces were 8.0 nm apart where again a much steeper repulsion was noted. measured

on decompression.

133

2000

-

F'El

@Nm) 1000

-

O-

5

1d :

>

.

15

2 0

.

l

D(nmj

: :

-1000

-

-2000

-

1' I / I : ,'

Fig. 5. The interaction force profile between two curved mica sheets bearing cytochrome c adsorbed from water, at a solution concentration of 5 mg dmm3. The results for different experiments are shown by different symbols.

A weak attraction was observed on approach, and a small adhesion ( < 1 mN m-’ ) was observed as the surfaces were separated (see inset in Fig. 4 which shows the data on a linear force scale ) .

Cyctochrome c

When cytochrome c was adsorbed on mica no measurable long-range repulsion was observed. A linear plot of F/R against distance is shown in Fig. 5. Within experimental error no force was detected until the surfaces were 12.0 + 1.0 nm from the mica contact, where an attraction was observed and the surfaces ‘jumped’ by approximately 6 nm to a separation of 6.5 + 0.3 nm. On further compression of the surfaces a steeply rising force was seen, with the surface separation decreasing by only 0.5 nm. At this separation, the refractive index was 1.42 5 0.02, as measured by the fringes of equal chromatic order [ 6,9]. On separation of the surfaces an attractive force was measured. The surfaces separate by 1.0 nm before they jump apart by 1: 200 nm. The attraction had a value of 1.8 5 0.3 mN m-l. For all the proteins studied, no changes in the force distance profiles were detected on replacement of the original protein solution by water.

134 DISCUSSION

Long-range interactions Probably the most striking feature of the results presented here is the similarity of the force profiles at large surface separations, in particular the gradient of the log force (8’) versus distance (II) profiles, for all systems studied, with the exception of cytochrome c. Such a similarity is expected between charged surfaces immersed in polar liquids. (Simple DLVO theory us that the slope of the force profile is to a first approximation independent of the surface proporties and only depends upon the bulk solution properties, which in the work reported here was water.) Therefore we have used DLVO theory [ 19,201 to estimate the force of interaction between the two protein-covered surfaces. In the model adopted here the total force was calculated by summing the van der Waals attractive force and the electrical double layer repulsive force. The van der Waals force was estimated using the equation, F=AR/GD’

(1)

which is a reasonable approximation to the more developed Lifshitz theory. For the Hamaker constant, A, a value of 2 x 10m2’ J was taken; this value was taken as being a reasonable maximum value for the Hamaker constant of protein-covered mica surfaces [ 211. The electrical double layer component of the force between protein-covered mica surfaces was estimated by using an algorithm which estimates, from a non-linear Poisson-Boltzmann equation, the force between two flat plates as a function of their surface separation for conditions of both constant surface potential and constant surface charge [ 221. The non-linear Poisson-Boltzmann equation may be written as d2 Y -=sinh dx2

Y

(2)

where Y is given by the expression (zeryl]zBT) and x is KD In the above expressions z is the valency of the ion considered, e the electronic charge, v the surface potential, hn Boltzmann’s constant, T the absolute temperature, K the Debye-Hiickel parameter and D the surface separation. Using the above approach, theoretical force distance profiles were calculated for all the systems studied, with the exception of cytochrome c for which no long-range repulsion was observed. The experimental points were fitted choosing a surface potential which corresponded closest to the experimental data (to an accuracy of 5 mV) . Both constant charge (full curves) and constant potential (broken curves) boundary conditions were considered. The exact conditions of the calculation are given in the figure legends, to summarise though, it was found that a surface potential of 50 mV was the best fit for the

135

myelin basic protein and concanavalin A, whilst a potential of 80 mV fitted the experimental data of the poly-1-lysine best. For poly-l-lysine and myelin basic protein it may be seen that the data is best fitted assuming a constant surface charge model, whilst the concanavalin A results obey a constant surface potential force law. Whether this is a charge effect is questionable. It is possible that there is some steric component to the force at short range which increases the repulsive force. Adsorbed conformations of proteins A second general feature of the results is that a short range ( < 10 nm) the results cannot be fitted by DLVO theory. A much stronger repulsive force is observed. This corresponds to the adsorbed protein layers themselves overlapping and giving rise to a steric repulsion. Thus the surface separation where the steep repulsion is first noted corresponds to twice the adsorbed layer thickness of the protein. The results, summarised in Table 1, show there is no obvious relationship between the dimension perpendicular to the mica surface and the molecular weight of the proteins. However, there is a strong correlation between the dimensions of the protein in solution and the distance of closest approach of the surfaces D,. Poly-l-lysine The thickness of an adsorbed layer of poly-1-lysine was independent of molecular weight over the range studied (4,000-75,000). It was difficult to detect and was certainly less than 1 nm. There appears to be a lack of any steric component in the force profile, the results being explainable in terms of electrical double layer and van der Waals forces only. The results are consistent with the polypeptide laying flat on the surface in a structureless form with the dimension perpendicular to the surface being that of the hydrocarbon backbone of the polypeptide chain, a value of about 0.5 nm. In solution at the pH of the experiments (pH 5.5 t 0.5) poly-1-lysine is a highly charged molecule. The charges are mutually repulsive and each charge will experience the charge of a neighbouring segment if the two charges are separated by a distance less than l/x. If the charges are separated at distances less than l/x,then over a distance of l/lc the molecule will be rigid. In the current experiments, where we have an electrolyte concentration of N 1 x 10e4 mol dme3 l/lc is s: 35 nm. Thus the polyelectrolyte will be rigid over some 35 nm. In the experiments reported here the fully extended chain lengths of the poly-1-lysine is N 6.0 nm for molecular weight 4,000 and 115 nm for molecular weight 75,000. In both cases then the molecule will be in a very extended configuration (a rod shaped molecule) at the electrolyte concentrations of the experiments and adsorbs as a rod, flat on the surface. It is interesting to compare these results with our earlier data [ 121 where poly-1-lysine (molecular

136

weight 90,000), was adsorbed onto mica from a 10-l mol dram3 potassium nitrate solution. Under these conditions a long-range steric repulsion was noted on a first approach of the surfaces. In these experiments l/lc is 2: 1.0 nm, so that the molecule is rigid over only 1.0 nm, thus the molecule will exist in solution as a random coil, and one is not surprised that the adsorbed layer thickness is larger. This is in accord with recent theoretical predictions by Papenhuizen et al. [ 231. Myelin basic protein

The size and shape of the MBP molecule have been measured in solution using a variety of techniques. Hydrodynamic [ 241 and X-ray studies [ 251, together with model building show the molecule to be a prolate ellipsoid or ‘hairpin’ structure of dimensions 1.5 x 15 nm. Perhaps of more relevance to the present experiments are the dimensions of MBP when adsorbed on surfaces, mainly lipid surfaces. Previous X-ray work [ 26,271 has shown that the overall structural dimensions, if not the exact conformation remain the same when MBP is associated with a surface. The present work confirms these observations, the distance of closest approach being between 3 and 3.4 nm. Concanavalin A

An X-ray analysis of concanavalin A shows that below pH 5.6, the concanavalin A tetramer dissociates to two dimeric structures of dimensions 8.0 x 4.0 x 4.0 nm [ 281. The pH of our experiments was between 5.0 and 5.5, and indeed the distance of closest approach which we observed (8 nm) is consistent with a monolayer of concanavalin A dimers adsorbed to each of the mica surfaces, with a short dimension oriented in a perpendicular fashion (tetramers have a tetrahedral structure and so would be expected to give a larger dimension). In the presence of doubly charged ions (calcium and manganese) which are known to activate the protein, we observed an attraction. This may be indicative of a structural change on addition of these ions, although we did not detect any change in the surface separation. Cytochrome c

Cytochrome c is a well-studied protein, a complete crystallographic structural analysis being available for several specific cytochrome c molecules [ 291. The molecule resembles a flattened sphere with dimensions of 3.0 x 3.4~ 3.4 nm. From our distance of closest approach D, = 6.5 + 0.3 nm, the molecule would still appear to be in the native form when adsorbed to the mica surface. In all cases the conformation of the adsorbed protein, as measured from the surface separation where a strong steric repulsion is observed, is entirely consistent with the protein being adsorbed in a conformation similar to that in solution. This can be noted by comparing the adsorbed protein layer thickness

137

0,/2 to one or other of the solution dimensions of the protein. Of course all the proteins may be grossly denatured and this correlation be fortuitous, although we doubt this. Similarly there is no evidence of multilayers of adorbed proteins. Adsorbed amount

The amount of protein adsorbed can be readily calculated from the refractive index data. For example in the case of myelin basic protein the refractive index of the material between the two mica surfaces when they were separated by 3.2 + 0.2 nm was 1.44 ? 0.02. The refractive index of pure protein is 1.52 [ 121 thus we calculated that this material is composed of 60% protein and 40% water and indicates that 1.2 mg me2 of protein has adsorbed on mica. Furthermore, since myelin basic protein has a density 1387 kg m-‘, and a molecular weight of 18,500, we calculate an area per molecule of 23 nm2. The degree of hydration of proteins in aqueous solution for proteins varies between 15% and 50% [ 301; therefore there appears to be fairly complete coverage of the mica surface. (Remember that in solution the myelin basic protein is a rod shaped molecule with dimensions 15.0 x 1.5 X 1.5 nm.) Similarly adsorption values for cytochrome c of 22 mg m-‘, and concanavalin A of 2.9 mg mm2 were calculated. There was no significant change in the refractive index with distance for poly-1-lysine indicating a very low adsorption certainly less than 0.5 mg rnm2. Again this data is consistent with the proteins being adsorbed in a similar conformation to their solution configuration. The results for the area per molecule and the amount adsorbed are summarised in Table 1. Surface &urge effects

When mica is placed in an aqueous solution, potassium ions leave sites on the surface resulting in a net negative charge. From the crystal structure of mica one would expect there to be two charges per nm2, however Israelachvili and Adams [ 61 have measured that only upto a l/2 charge per nm2 is actually present. At the pH of the experiments, concanavalin A, myelin basic protein and of course poly-1-lysine are all positively charged. From the calculated surface area per molecule data we can derive some insight into the net charge of the protein surface. The data for cytochrome c is particularly suitable for this type of analysis. The force-distance profiles indicate that there is, within the error of the experiment, no net chage on the surface. The surface area of one molecule of cytochrome c adsorbed on mica is N 30 nm2, at pH 5.5 (the pH of our experiments) cytochrome c has a net positive charge of 12, i.e. one charge every 2-3 nm2 of surface. From the data of Israelachvili and Adams [ 61, we would except a maximum surface charge density of one electron every 2 nm2, thus these data are consistent. Concanavalin A only has one positive charge at the pH of the experiments, and therefore the adsorbed layer of the protein is

138

net negatively charged. Myelin basic protein, however, has net positive charge of 31. The molecule occupies some 23 nm2 on the surface, which would correspond to approximately 1.5 charges per nm2. Thus it seems likely that the net charge on an adsorbed layer of myelin basic protein on mica is positive. Using the equation o= (2n~k~T/n)~/~sinh( Y0/2)

(3)

the magnitude of the effective surface charge density, C, corresponding to a situation where the surfaces are at a large separation, can be calculated. These values are given in the figure legends of the force profiles. Attractive forces between adsorbed protein layers The attractive forces measured between the adsorbed layers of the proteins, in particular those for myelin basic protein, are probably the most significant feature of these results. In order to semi-quantitatively analyse our results we have fitted our data to a theoretical estimation of the attractive force assuming the attractive force to be due simply to van der Waals forces [ Eqn (1) 1. From the literature, a range of Hamaker constants for proteins and hydrocarbons interacting across water has been obtained. These values range from 1.0 to 2.2 x 10m2’ J [ 211. A reliable indicator of the attractive force between surfaces is the distance at which the surfaces begin to jump into contact. At this distance dF,,/dD = dFDLvo /dD + k

(4)

where k is the constant of the spring on which one of the pieces of mica is attached. Fh gives the force operating in addition to the force predicted using the DLVO theory. We can readily obtain d.FddD at the jump distance from the results. To calculate the slope of the van der Waals force as a function of distance, dF,,/dD = -AR/3D3 was plotted against D (Fig. 6). For poly-1-lysine, and cytochrome c the attractive force is well explained by van der Waals forces. Only in the case of MBP there is a force in excess of that predicted by the van der Waals attraction. At a protein-protein surface separation of 14 nm, it was found to be an order of magnitude greater which is well above the error of the measurements. This may be due to a hydrophobic effect, similar to those observed previously [ 301, since other explanations, for example charge effects and protein bridging, are quite simply incapable of accounting for the powerful attraction. We also note that there has been a suggestion that the dimerisation of myelin basic protein involves hydrophobic side chains [ 151. Work is in progress to investigate the force law for the attraction. For example, a hydrophobic interaction is to be expected to yield an exponential dependance of the force with distance [ 311, whilst van der Waals forces give an inverse power dependance. The results for the adhesion force between the adsorbed protein layers, i.e.

139

0

5

Dlnm)

10

15

Fig. 6. A plot of log,, [ d(F/R)/dD] against surface separation. The calculated non-retarded van der Waalsforce [ Eqn (l)] for hydrocarbon,A=0.8X 10b2’ J, andmica,A=2.2 X 10e20 J, surfaces separated by water are shown by the two continuous curves. Also shown are the values for the attractions between the protein-covered surfaces. (+, MBP; l ,cytochrome c; n , poly-l-lysine 4,000; 0, poly-1-lysine 75,000.) This clearly demonstrates that the attractive forces observed between cytochrome c and poly-I-lysine are due to van der Waals forces, whilst the attraction between MBP-covered surfaces is an order of magnitude greater.

the force needed to separate the protein layers, are presented on the insert to the force profiles, where the data is plotted on linear scales and are summarised in Table 1. Again the protein with the strongest adhesion is the myelin basic protein. The myelin basic protein results are of great significance when one remembers the proposed role of myelin basic protein in nature, namely that the protein is adsorbed on the inner side of the cytoplasmic membrane of the Schwann cells of the myelin sheath and that the protein is responsible for maintaining the structure of the lipid sheath. These results, together with our earlies studies on the interaction between lipid bilayers bearing myelin basic protein [ 321 confirm that adsorbed layers of myelin basic protein are attractive at separations of less than 15 nm, and that the proposed role of the protein is feasable.

140 CONCLUSION

In conclusion, it would appear that the proteins studied here bind to a mica surface without major disruption of structure. At surface separations greater than the adsorbed protein layer thickness, all the principal features of the force profiles were well described by electrical double layer and van der Waals forces. Only in the case of myelin basic protein did the attractive force appear to be in excess of van der Waals attraction. We suggest that this attraction is hydrophobic in nature and is the first direct measurement of the strength of the hydrophobic interaction between biological molecules. The results are the first direct measurement of attractive forces between biologically specific molecules. The strong attraction measured for myelin basic protein is significant when one considers the role it has in stabilising the myelin sheath. ACKNOWLEDGEMENTS

We wish to thank the Nuffield Foundation and the Wellcome Trust for their financial support of this work.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

S. Hakamori, Ann. Rev. Biochem., 50 (1981) 733. M. Hair (Ed.), The Chemistry of Biosurfaces, Marcel Dekker, New York, 1971, Vol I and II. M. Monsigny, C. Kieda and AS!. Roche, Biol. Cell, 47 (1983) 95. J.M. Bird and S.J. Kimber, Dev. Biol., 104 (1984) 449. D. Tabor and R.H.S. Winterton, Proc. Sot. London Ser. A, 312 (1969) 435. J.N. Israelachviii and G.E. Adams, J. Chem. Sot. Faraday Trans. 1,74 (1987) 975. R.M. Pashley and J.N. Israelachvili, CoIIoids Surfaces, 2 (1981) 169. R.M. Pashiey, Adv. Coiioid Interface Sci., 16 (1982) 57. J. Klein and P.F. Luckham, Macromolecules, 17 (1984) 1041. P.F. Luckham and J. Klein, Macromolecules, 18 (1985) 721. J. Klein and P.F. Luckham, Colloids Surfaces, 10 (1984) 65. P.F. Luckham and J. Klein, J. Chem. Sot. Faraday Trans. 1,80 (1984) 865. P. Mitchel, Science, 206 (1979) 1148. C.W.M. Grant and M.W. Peters, Biochim. Biophys. Acta, 779 (1984) 403. E. Roboz-Einstein, D. Robertson, J. Dicaprio and W. Moore, J. Neurochem, 9 (1962) 353. B.V. Derjaguin, KoIIoid Z., 69 (1934) 155. T. Afshar-Rad, A.I. Bailey, P.F. Luckham, W. MacNaughtan and D. Chapman, Colloids Surfaces, 25 (1987) 263. T. Afshar-Rad, A.I. Bailey, P.F. Luckham, D. Chapman and W. MacNaughtan, Biochim. Biophys. Acta, (1987) in press. E.J.W. Verwey and J.T.G. Overbeek, Theory of the Stability of Lyophobic Cohoids, Eisevier, Amsterdam, 1948. B.V. Derjaguin and L. Landau, Acta Phys. Chim USRR, 14 (1941) 633. J. Visser, Adv. Colloid Interface Sci., 3 (1972) 331. D.C. Chan, R.M. Pashley and L.R. White, J. Colloid Interface Sci., 77 (1980) 284.

141 23 24 25 26 27 28 29 30 31 32

J. Papenhuizen, H.A. van der Schee and G.J. Fleer, J. Colloid Interface Sci., 104 (1985) 540. R.M. Epand, M.A. Moscarello, B. Zierenberg and W.J. Vail, Biochemistry, 13 (1974) 1264. G.W. Brady, N.S. Murthy and D.B. Fein, Biophys. J, 34 (1981) 345. W. MacNaughtan, K.A. Snook, E. Caspi and N.P. Franks, B&him. Biophys Acta, 818 (1985) 132. J. Sedzik, A.E. Blaurock and M. Hochli, J. Mol. Biol., 174 (1984) 697. G.M. Edehnan, B.A. Cunningham, G.N. Reeke, J.W. Becker, M.J. Waxdal and J.L.Wang, Proc. Nat. Acad. Sci. U.S.A., 69 (1972) 2580. R.E. Dickerson, T. Takano, D. Eisenberg, O.B. Kahai, L. Samson, A. Cooper and E. Margalaish, J. Biol. Chem., 246 (1971) 1511. P.G. Squire and M.E. Himmel, Arch. Biocbem. Biophys., 196 (1979) 165. R.M. Pashley, P.M. McGuigan, B.W. Ninham and D.F. Evans, Science, 229 (1985) 1088. T. Afshar-Rad, A.I. Bailey, P.F. Luckham, W. MacNaughtan and D. Chapman, Spec. Discuss. Faraday Sot., (1987) 239.

142

DISCUSSION Th.F. TADROS (ICI Plant Protection Division, Bracknell, United Kingdom) In your calculation of the force using the DLVO theory you had to assume a value for vu0and this could be an overestimate of the real value. Therefore, the fit with the experimental data may not be indicative of lack of steric effects. Moreover, the attraction at short distances could be due to depletion effect of “squeezing out” of the solvent in between the protein layers. P.F. LUCKHAM (Imperial College, London, United Kingdom) The first part of your comment is correct, although I would find it strange for the steric component to be independant of molecular weight and to have the same distance dependance of the force as a double layer interaction. I do not believe the attraction to be due to depletion of polyelectrolyte or protein because the protein concentration in solution is very low (l-10 mg 1-l j , also I believe that depletion effects are unlikley to be measured on the surface forces apparatus since the surface area of the two surfaces are very high N 100 pm2, this implies that the protein has to diffuse several microns in order to deplete the gap between two approaching surfaces. This will take longer than the time scale observed for the attraction which is of the order of a second. Bridging is a possibility in the polyelectrolyte case and probably does occur and is in addition to the van der Waals force. A. SILBERBERG (W eizmann Institute of Sciences, Rehovot, Israel) (i) At the ionic dilution which you use you clearly have a Debye layer thickness which is broader than the polymer layer. In fact your repulsions are larger without than with polymer. Is it not your conclusion that, if the polymer can “hide” in the shadow of the double layer, the only effect observed is a change (a reduction) in surface charge. One has to come quite close to the surface before one first “sees” the polymer. Since these are polyelectrolytes they are easily very weakly adsorbed at low ionic strength. Hence, the flexible polylysine is adsorbed flat and “bridges” will only be induced when the two mica surfaces are very close to each other. (ii) Very low ionic strength is dangerous to use with globular protein and the effect of this should be investigated before the conformation of the adsorbed species is considered. Even with polylysine there is an effect due to shielding. When spaced out sideways the charges on the polylysine are probably some 30 A or more apart. Hence they are effectively shielded from each other at 0.1 M monovalent salt solutions, but begin to “see” each other as the ionic strength is reduced. Hence there is a qualitative change in the polymer conformation as this criteria is crossed.

143

P.F. LUCKHAM (Imperial College, London, United Kingdom) (i) In my reply to Dr Tadros’s question I have said that bridging of poly-llysine between the surfaces is possible. Personally I believe this does occur in our system, but as the force is short range it is difficult to be certain as the van der Waals attractive forces between the surfaces will be present in addition to the bridging. (ii) I agree that low ionic strengths are not ideal. We have performed experiments with myelin basic protein in 10m2 mol dmp3 KNO, and, with the exception of a reduced double layer, observe similar behaviour to water. Also the results for Con A in 4 n&f Ca2+ and Mn2+ show the protein is in a similar, although not identical conformation. In addition, the distance of closest approach of the surfaces for all the proteins is consistent with the protein being adsorbed in its native conformation. J.A. WATERS (ICI Paints, Slough, United Kingdom) What checks were made that the proteins are uniformly adsorbed over the surface of the mica. Could it reside on the surface as globules which make a contact angle with the surface, even possibly greater than 90’ C? There might be a driving force for the latter which arises because the sum of the three appropriate interfacial energies might be a minimum. Such a mechanism for adsorption could lead to increased apparent thickness for the layer. P.F. LUCKHAM (Imperial College, London, United Kingdom) No checks on uniformity ofthe protein adsorbed were made. I would point out however that the surface area of the experiment is some 100 pm2 and therefore I am sure that over this scale there is uniform adsorption. Our results are consistent with the proteins being adsorbed in a monolayer on the surface without any gross conformational, change of the protein from solution. Thus I do not believe that we have large aggregates of protein on the surface. R.J. DAVIES (Cavendish Laboratory, Cambridge, United Kingdom) (1) You explain the dependence of adsorbed layer thickness by a screened electrostatic persistence length argument for M.W. 75,000 and M.W. 90,000 poly-1-lysine in 10v4 and 10-l M, electrolyte, respectively. However, the data you have published (J. Chem. Sot. Faraday Trans. 1,80 (1984) 865) for the M.W. 90,000 polymer in 10m3A4electrolyte does not tie in with this theory, or does it? (2) Specific energies measured by the “force machine”, apart from giving errors of + 30% in the magnitude of coverage, are uncalibrated by an independent technique. Perhaps, with more such devices coming into use, it is time that the method was assessed.

144

P.F. LUCKHAM (Imperial College, London, United Kingdom ) (1)I agree to some extent although of course the screening length at 10m3 mol dmm3is less than 10B4 mol dmm3. I point out though that the results presented by L. Dix here show the same results as I have obtained with poly-llysine. (2) I agree with this point. The only attempts to do this so far has been by Terashima using a microbalance technique (H. Terashima, J. Klein and P.F. Luckham, in C.H. Rochester and R.H. Ottewill (Eds) , Adsorption from Solution, 1982, p. 299). Experiments on ground mica may be useful although edge effects and surface area errors may affect the results. 0. NEHME (Cavendish Laboratory, Cambridge, United Kingdom) Polylysine at low ionic stength is an extended coil due to the charge repulsion, but it is not a rigid rod. The radius of gyration measured for poly-1-lysine is much smaller than its length. It is erroneous to assume that the “rigid-rod” conformation in the bulk solution gives a flat conformation when adsorbed on the surface. My data presented in the adsorption poster shows that the layer thickness was about 200 A and independent of molecular weight. P.F. LUCKHAM (Imperial College, London, United Kingdom) Your data covers a wide molecular weight range for poly-1-lysine adsorbed to polystyrene latex. I find the absence of a molecular weight dependance strange, particularly as the adsorbed layer thickness is 20 nm. In order to have an extended chain of 20 nm we need to go to molecular weight of 15,000. Thus I would not expect the 4000 molecular weight poly-1-lysine to have this adsorbed layer thickness! I would also like to make the point that polystyrene latex is a very different surface to mica and as we saw in Prof. Killmann and Dr Maier’s paper the adsorbing surface can strongly influence any measured adsorbed layer. I would stress that our poly-I-lysine experiments where the poly-1-lysine is adsorbed from water are explainable simply in terms of electrostatic and van der Waals interaction; we see no steric component and therefore conclude that the polyelectrolyte adsorbs flat. I accept your point concerning the polyelectrolyte in solution though and thank you for bringing this to my attention. S. GRANICK (University of Illinois, Urbana, IL, U.S.A.) (1) The poly-1-lysine findings of you as well as of the Cambridge group suggest that the amount adsorbed diminishes with decreasing surface separation. One must beware of estimates of amount adsorbed which are based on what is observed at small separation. (2)A question about your physical picture of the state of the adsorbed proteins. The salt concentration and temperature are far from physiological, and moreover the structure may be altered by the state of being adsorbed. Have

145

you done experiments to probe these possible influences? Furthermore, much is known about the specific structure, in solution, of the proteins you study. Do you believe all proteins adsorb with the same side pointing into solution; are any specific interactions suggested? P.F. LUCKHAM (Imperial College, London, United Kingdom) (1) Desorption (of poly-1-lysine) may occur, as the surfaces approach, we have no evidence one way or the other for this though. (2) We have done some experiments under more physiological conditions, e.g., Con A in 4 mM Mn2+ and Ca2+ ions and MBP at 10m2mol dmm3 KN03. There are some small differences under these conditions, e.g., the double layer thickness is reduced in both cases and an attraction is observed at close separation, for Con A. The distance of closest approach (D,) in all cases is the same, however. Also these D, values correspond closely to solution properties of the proteins obtained, in some cases under close to physiological conditions. The data can be explained by the protein being very close to its solution configuration. Thus it is unnecessary to invoke any conformation change. It must be remembered that these molecules are ‘designed’ to be active at an interface. It is known that Con A is active when adsorbed onto gold and silica particles, it would be unlikely to be inactivated on mica. We cannot at present say how the proteins are adsorbed with absolute confidence. We can say that MBP is laying on the surface with its 1.5 nm dimension perpendicular and Con A with its 4.0 nm dimension perpendicular. MBP is a basic protein, these basic amino acids may orient towards the mica leaving the hydrophobic amino acids exposed to the solution (the contact angle of the protein surface is - 70” C ) . G.J. FLEER (Agricultural University, Wageningen, The Netherlands) The results reported by both Dr Luckham and Dr Dix seem to be in qualitative agreement with theoretical models and also with adsorption measurements of polylysine onto negatively charged surfaces: without added salt the layers are thin, the adsorption is small and there is no molecular weight dependence. At higher salt concentrations more similarity with uncharged polymers is expected, implying a clear molecular weight dependence. Have you observed such a trend? P.F. LUCKHAM (Imperial College, London, United Kingdom) For force measurements at higher salt concentration (10-l mol dmm3KN03), the repulsive force is due to polymer only since the Debye length is so short that little repulsive interaction is observed. There is a clear dependence of the range of the force on molecular weight. Between ca 4000 and 564,000 molecular weight poly-1-lysine there is a linear relationship between the onset of the force and log (molecular weight).

146

W. NORDE (Agricultural University, Wageningen, The Netherlands ) In discussing the surface charge effects you suggest that the charge on the protein-covered surface is more or less the sum of the charges of the surface and the protein molecule before adsorption. From electrophoretic measurements we have evidence that surface charge effects in adsorption of globular proteins are much more complicated. For instance, a negative protein that adsorbs on a negative surface usually results in a complex that is far less negative than the bare surface and also less negative than the protein molecule before adsorption. This phenomenon is due to small ions (in this example cations) that are incorporated in the relatively compact adsorbed protein layer in order to avoid accumulation of net charge in the low dielectric contact region between the protein molecule and the sorbent surface. P.F. LUCKHAM (Imperial College, London, United Kingdom) I agree entirely with these comments, our discussion is only intended as a guide to suggest the nature of the surface charge. Results for Con A in 4 mA4 Ca2+ and Mn2+ indicate that the surface is net negative, that cytochrome c is neutral and that poly-1-lysine is positively charged. We believe that MBP is also positively charged but have no evidence for this. H.N. STEIN (Eindhoven University of Technology, Eindhoven, The Netherlands) You find a larger attraction than can reasonably be accounted for by van der Waals forces. We found the same from rheological experiments. I did not hear you mention the possible existence of entanglement of protruding chains. What reasons do you have for excluding this possibility? P.F. LUCKHAM (Imperial College, London, United Kingdom) To be honest we have not seriously considered this possibility. I would not rule it out completely though. I would expect for the case of MBP (myelin basic protein) that the molecule retains its solution configuration, the distance of closest approach of the surfaces suggests this, making entanglements likely. In addition, if entanglements were involved on separation of the surfaces I would expect the surfaces to move slowly apart. We do not observe this. What we see is a reduced force for the same surface separation.