Langmuir 1997, 13, 2541-2544
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Long Time Evolution of Adsorbed Protein Layers in a Thin Gap between the Two Mica Surfaces of an Automatic Surface Force Apparatus B. Gauthier-Manuel and J. P. Gallinet* Groupe Pluridisciplinaire d’E Ä tude des Interfaces, U.F.R. Sciences etTechniques, Laboratoire de Physique et Me´ trologie des Oscillateurs du CNRS associe´ a` l’Universite´ de Franche-Comte´ , 25030 Besancon Cedex, France Received November 18, 1996. In Final Form: February 25, 1997X A protein solution is confined between two solid ultrasmooth mica surfaces of an automated surface force apparatus (SFA). When the surfaces are slowly brought closer to each other (v e 0.2 nm‚s-1), the ordering and displacement of the molecular layers in the compressed protein solution can be observed by continuous recording of the refractive index of the medium between the mica surfaces of the SFA. At a pH near the protein pHi, an increase of the ionic strength in the medium induces an accumulation of the protein molecules against the imposed compression stress, and the mica surfaces recede. The refractive index of the medium, constant when the adsorbed layers are ordered, decreases step by step from a mean high value (around 1.55) to an intermediary value (1.45). We discuss this behavior in terms of growth of multiple nucleation centers.
Introduction Molecular layering against solid surfaces is a major phenomenon showing little reversibility.1,2 The regular orientation of the macromolecules in the first layer adsorbed onto a smooth solid surface often tends to line up the further protein interactions.2,3 Proteins can be studied as polyelectrolytes in solution.3 The accessible surface charges of a protein set the further protein-protein interactions which lead to clusters, amorphous aggregates, or crystal packing.4 Measurement of the intermolecular forces with a surface force apparatus (SFA) is a technique5 to study the alignment or the displacement of molecules between solid surfaces5,6,7 and completes other investigations such as electrochemistry.7 The automatic control of the displacement of the solid surfaces in the SFA permits the measurement of both the forces and the mean refractive index of the medium from multiple beam interferometry.8-10 From the experimental data, the fluctuations of the structural ordering of macromolecules can be described in the confined medium between the two solid surfaces submitted to a very slow compression stress.11 At a given pH and an ionic strength e10-3 M, Concanavalin A (Con A), a D-glucose/D-mannose-specific lectin from jackbean (Canavalia ensiformis) seeds,12 is easily adsorbed on solid mica surfaces.13-15 From the X-ray crystallographic structure, the Con A molecule consists of
two (dimer) or four (tetramer) identical protomers of mean molecular weight 25 000, folded into domelike structures (4.2 × 4.0 × 3.9 nm).12 Each subunit contains only two large antiparallel pleated β-sheets with three binding sites for respectively Mn2+ and Ca2+ 16 and the saccharide molecule.17 The dimers can be considered as anisotropic objects, approximately 8.4 × 4.0 × 3.9 nm in size, and the tetramers as isotropic objects.11 The first layer is packed into a compact organization and the accumulation of the following layers becomes more and more disordered. Thus, in the midway of the medium, the refractive index of the diluted protein solution is about that of the solvent (water).11 The number and the thickness of the first ordered protein layers vary with the pH and the ionic strength of the solution. The refractive index of the ordered Con A macromolecules increases to 1.55.11 The protein molecules can be accumulated against the imposed compressive force when the ionic strength increases in the interface of the solid surfaces of the SFA. We discuss the refractive index variations during this molecule accumulation from a change of the local pH of the medium to the pHi of the protein as predicted by the theory of Tanford and Kirkwood,18 and we suggest a crystal growth phenomenon from many nucleation centers.
Abstract published in Advance ACS Abstracts, April 1, 1997.
Several recent modifications from the original measuring device,19 allowed an easier use of the surface force apparatus (SFA). The experimental procedure has been extensively documented in recent reviews.5,6 The nature and the magnitude of the interact ions between two surfaces separated by a liquid medium are observed through the controlled displacement of two ultrasmooth cleaved muscovite mica sheets, silvered on the backside and glued to silica cylindrical lenses (mean radius ) 2 cm). The lenses are oriented in a crossed-cylindrical geometry, an orientation equivalent to a sphere-plane geometry at short
X
(1) Law, B. M. J. Colloid Interface Sci. 1990, 134, 1. (2) Haynes, C. A.; Norde, W. Colloid Surf., B 1994, 2, 517. (3) Fraaije, J. G. E. M., Lyklema, J. Croat. Chem. Acta 1990, 63, 517. (4) Littlechild, J. A. J. Phys. D, Appl. Phys. 1991, 24, 111. (5) Israelachvili, J., Ed. Intermolecular and Surface Forces; Academic Press: London, 1992. (6) Leckband, D.; Israelachvili, J. Enzyme Microbiol. Technol. 1993, 15, 450. (7) Lacour, F.; Torresi, R.; Gabrielli, C.; Caprani, A. Colloid Surf. B: Biointerfaces 1993, 1, 251. (8) Tolansky, S., Ed. Multiple Beam Interferometry; Clarendon Press: Oxford, 1948. (9) Israelachvili, J. J. Colloid Interface Sci. 1973, 44, 259. (10) Kekicheff, P.; Spalla, O. Langmuir 1994, 10, 1584. (11) Gauthier-Manuel, B.; Gallinet, J.-P. J. Colloid Interface Sci. 1995, 175, 476. (12) Reeke, G. N.; Becker, J. W.; Edelman, G. M. J. Biol. Chem. 1975, 250, 1525. (13) Afshar-Rad, T.; Bailey, A. I.; Luckham, P. F.; McNaughtan, W.; Chapman, D. Colloid Surf. A 1988, 31, 125. (14) Fitzpatrick, H.; Luckham, P. F.; Eriksen, S.; Hammond, K. J. Colloid Interface Sci. 1992, 149, 1.
S0743-7463(96)02018-5 CCC: $14.00
Materials and Methods
(15) Gallinet, J.-P.; Gauthier-Manuel, B. Eur. Biophys. J. 1993, 22, 195. (16) Hardman, K. D.; Agarwal, R. C.; Freiser, M. J. J. Mol. Biol. 1982, 157, 69. (17) Derewenda, Z.; Yariv, J.; Helliwell, J. R.; Kalb, A. J.; Dobson, E. J.; Papiz, M. Z.; Wan,T.; Campbell, J. M. EMBO J. 1982, 8, 2189. (18) Tanford, C.; Kirkwood, J. G. J. Am. Chem. Soc. 1957, 79, 5333. (19) Tabor, D.; Winterton, R. H. S. Proc. R. Soc. London, Ser. A 1969, 312, 435.
© 1997 American Chemical Society
2542 Langmuir, Vol. 13, No. 9, 1997
Gauthier-Manuel and Gallinet the interaction force F
F ) K(∆D - ∆D0)
Figure 1. Profiles of the fringes of equal chromatic order for computing of the refractive index. Y-coordinate: r ) radius in (µm) of the cross section of the white light pencil which passes through the two silvered mica surfaces. X-coordinate: wavelength of the visible light (λ in nm). m ) order of the fringes. When the solid mica surfaces are at molecular contact, the profiles of the fringes are deformed and then become parabolic when a liquid medium separates the mica surfaces. The surface separation is calculated by the equations D ) (1.024 modd + 1)∆λodd/2µmica and Dn2 ) (1.024meven + 1)∆λevenµmica/2. Here, the gap between the fringes at the molecular contact and with the compressed Con A solution (∆λ ) corresponds to D ) 9.1 nm and to a refractive index n ) 1.518. Fringe profiles were obtained from 3582 couples of experimental points. distance.5,20 The positions of the mica surfaces are controlled by a servo-looped piezoelectric ceramic in the range 0.1-5000 nm. The automatic data acquisition yields the interaction curves with very low thermal drifts because the experimenter does not stand near the device during the experiment. This setup cancels the hysteresis and the creep of the piezoelectric material. A CCD camera (array of 4096 pixels, 7 µm wide) simultaneously performs the measurement of two consecutive odd and even “fringes of equal chromatic order” (FECO). This FECO method offers a high sensitivity (e0.1 nm) in the direction perpendicular to the plane of contact area.9 The true distance measured by the FECO method is plotted as a function of the displacement of the piezoelectric ceramic. The reference position is determined by direct contact of the two mica surfaces in air and controlled with two different fringes, even and odd fringes successively (Figure 1). The fringe profiles are computed from the experimental data and show the data dispersion during the experiment from the molecular contact, which corresponds to the deformed fringes, to every moment of the mica surface moving. The parabolic profiles indicate that the two curved mica surfaces are brought under a controlled load without anomaly. A mean refractive index of the material between the mica surfaces can be computed from the FECO method.11 The wavelength λm° of the fringe number m, when the surfaces are at molecular contact, depends on the mica refractive index µmica and its thickness tmica. When the surfaces are separated to a distance D, the optical path increases and the fringes shift to the red. The simultaneous measurement of the shift ∆λ ) λm - λm° for an odd fringe (∆λodd) and an even one (∆λeven) allows the determination of the distance D and of the refractive index n of the medium between the two surfaces (Figure 1). With a gap small enough, approximations can be used for odd (order modd) and even (order meven) fringes
The force constant of the spring was K ) 84 N‚m-1. Under optimum conditions, the uncertainty on D is about 0.1 nm and the accuracy on n is 1%. In fact, this accuracy is seldom reached because the presence of an experimenter in the vicinity of a manually-operated SFA disturbs the mechanical stability of the two mica surfaces, and only discrete data are obtained.6,10,13 With the automatic SFA, continuous measurements are obtained, and the theoretical accuracy is reached.11,15,21 After the determination of the reference position, the mica surfaces are separated. A drop (30 µL) of an aqueous Con A solution (50 µg‚mL-1 of Con A type IV, Sigma ref. C-2010 diluted in degassed 10-3 M potassium phosphate buffer at pH 5.2) is fed between the two mica plates and allowed to adsorb during about 60 min. The measurement cell is filled up with the chosen buffer solution. Then the two mica surfaces are slowly brought closer to each other, and the force is recorded continuously at atmospheric pressure and 293 K. To minimize the hydrodynamic flow, the velocity of the displacement of the lower mica surface is as low as 0.1 nm‚s-1. Following this experiment, the surfaces are separated, the first buffer solution is removed, and the cell is refilled with the second degassed buffer solution (10-3 M potassium phosphate buffer at pH 7.2) to obtain the dimer-tetramer equilibrium of Con A.11 As in the same preceding conditions, the two mica surfaces are moved closer to each other very slowly (g8 h). The mica surfaces stop at 16.2 nm, and the mean refractive index of the confined medium under compression is between 1.50 and 1.55.11 To test the reversibility of the dimer-tetramer transformation, the mica surfaces are again separated at great distance (1 µm) and the measurement cell is filled up with a degassed 10-3 M potassium phosphate buffer at pH 5.2. After 3 h at rest, the mica surfaces are again slowly brought closer and the force is recorded. A third change of buffer is used to test the influence of an increase of the ionic strength at constant pH (pH 5.2, close to the Con A pHi 5.5): after the surfaces are stopped at the end of the preceding approach, the buffer is removed and replaced by a 10-1 M potassium phosphate buffer at pH 5.2. After 48 h at rest, the distance and the refractive index between the mica surfaces are recorded without any external displacement of the surfaces during 24 h.
Results
The accuracy of the refractive index depends upon the dispersion of the experimental points of the both even and odd fringe profiles. A test of the reliability of the mesurement is the value of 1.33 (water) obtained at large separation of the mica surfaces. The difference between the variation of the real distance ∆D when the lower surface comes close to the upper one and a known variation of distance ∆D0 with the piezoelectric ceramic yields
When the solid mica surfaces are moved toward each other in the presence of 10-3 M potassium phosphate buffer at pH 5.2, the interaction force increases with many jumps (Figure 2a). The force profile can be understood by plotting the concomitant variations of the refractive index (Figure 2b). Two different behaviors are observed during the mica surface displacement (distance D). When the distance D decreases from 200 to 97 nm, the force slowly increases and the refractive index shows serrated variations smaller than 0.037 with a maximal variation at the distance 150134 nm. Two anomalies of the force profile appear at 129-122.5 nm and at 108-103 nm: the interaction forces between the solid surfaces remain constant when the distance D decreases while the refractive index decreases. At D e 91 nm, we observe a relative ordered layer of Con A molecules which is diplaced at 79 nm. Between the compression of the multilayers (84-79 nm) the refractive index decreases. During the elimination of the following layer (79-71 nm), the index increases: this is the behavior observed with the most packed layers.11 The solid surfaces are stopped by multilayers of Con A molecules at 71 nm, and the interaction force, about 650 µN, is not sufficient to eject a new layer. The refractive index is near 1.37, a low value which indicates a layering yet slightly compact, compared to the value 1.55 observed with the most packed layers of the Con A dimers.15 After this experiment, the
(20) Hunter, R. J.; Ed. Foundations of Colloid Science; Clarendon Press: Oxford, 1987; Vol. 1, Chapter 4, pp 191-194.
(21) Gallinet, J.-P.; Gauthier-Manuel, B. Colloid Surf., A 1992, 68, 189.
D ) (1.024modd + 1)∆λodd/2µmica D n2 ) (1.024meven + 1)∆λevenµmica/2
Adsorbed Protein Layers
Figure 2. Interaction force and refractive index variations of a protein solution in a confined gap between two solid surfaces. Concanavalin A solution: 50 µg‚L-1 Con A in 10-3 M degassed potassium phosphate buffer at pH 5.2. Movement of the mica plates is controlled by two piezoelectric ceramics at a velocity ) 0.1085 nm‚s-1. Experimental points: 3582. (a) Computation of the force F in 10-6 N. (b) Mean refractive index n of Con A solution between the two mica plates. Computation of the refractive index from two optical fringes: odd fringe (order ) 33), even fringe (order ) 32).
buffer solution (potassium phosphate at 10-3 M and pH 5.2) is replaced with a new buffer solution at the same pH but at a final ionic strength 10-1 M. The mica surfaces stopped at D ) 71 nm are left 48 h at rest under a compressive stress of 650 µN. The rest time corresponds to the minimum time to achieve an ion-protein equilibrium in the confined medium between the mica surfaces. The diffusion length is about equal to the silica lens radius (1 cm). The length of diffusion L of the ions after 48 h is
L ) xDT ) 1.3 cm with D ) 10-5 cm2 s-1 This distance is near to the size of the lenses and to the mean distance run by the new buffer to reach the center of the light pencil. During 48 h, the distance between the mica surfaces decreased from 71 to 38 nm and the refractive index reached 1.55. The corresponding compressive force slightly decreased to 2.7 µN. Thus, the experiment can be regarded as constant pressure condition. During this time, an organization of the adsorbed molecules occurred and the density of the layers increased to a maximum packing of 10 layers of Con A dimers. The ulterior evolution of the odd FECO range number 33 on a long range time (14 h) is shown in Figure 3a. A spontaneous accumulation of Con A molecules pushed the mica surfaces by successive jumps. During the first jump, the refractive index remained nearly constant and at a high value (1.54). The accumulation of Con A molecules was probably ordered. Then, the jumps became irregular and faster. The refractive index decreased at each new matter accumulation, which then seemed little ordered (Figure 3b). Discussion Inside the gap between two mica plates filled with a Con A solution at pH 5.2 and a 10-3 M ionic strength, the variation of the interaction forces (Figure 2a) can be measured by the fluctuations of the mean refractive index (Figure 2b). The precision on the measurement of the refractive index is clearly seen on the scattering of the experimental points during a large experimental time (30 000 s). It is found to be equal to (1%. The force profile shows two sectors: first, a slow increase which becomes gradually greater when the distance D between the mica surfaces of the SFA decreases to 98 nm and presses the protein solution. Several irregularities of the force profile
Langmuir, Vol. 13, No. 9, 1997 2543
Figure 3. Con A molecules accumulation in a thin gap under a compression stress. A Con A solution (50 µg‚L-1, at pH ) 5.2 and at ionic strength µ ) 10-3 M) was confined between the two mica plates and then, after large separation of the two solid surfaces, a buffer (µ ) 10-3 M, pH ) 5.2) was introduced at the same pH but at a higher ionic strength (µ ) 10-1 M ) in the measurement cell. The device was left 48 h at rest before a new recording of the movement of the mica surfaces. Experimental points: 4095. (a) Displacement of the optical fringe of odd order 33 versus time: t ) 0 corresponds to the position of the mica plates after the last buffer change followed by a 48 h rest under a compressive stress of 650 µN (see Materials and Methods). (b) Concomitant variation of the mean refractive index of the confined medium between the two mica plates.
correspond to sudden serrated variations of the refractive index. The general slope of the force profile is similar to a DLVO profile obtained with charged macroions.5,20 At D g 98 nm, the irregular increase of the refractive index is synchronous with the increase of the interaction forces during a compression step. However, when the forces become constant during a new approach of the solid surfaces, the refractive index decreases, probably because of an unordered displacement of the Con A molecules in the gap. This behavior occurs at 129-123 nm and at 108103 nm and is completely different from the mechanism of molecule ejection of ordered Con A layers observed in a prior study.11 After an intermediary process with molecule layering (103 e D e 97 nm ) ending by a displacement of molecules when D varies from 97 to 91 nm, we find at D e 91 nm a mechanism of molecule ejection-molecule reorientation alternation that we previously described concerning the dimer-tetramer equilibrium submitted to a compression stress.11 When the distance D decreases, the compression of the protein layers corresponds to an increase of the force and to a concomitant decrease of the mean refractive index (84 nm e D e 79 nm). When the ejection of a Con A layer occurs, the index sharply increases while the force stays constant (79 e D e 77 nm). The ejection seems fast: the time between two experimental points is equal to 30 s. From Figure 2, a great difference between the two experiments appears here: when the mica surfaces stop at an interaction force of about 650 µN, the separation distance is very much higher (D ) 71 nm) than that observed with the dimer (D ) 16.2 nm) at the same pH 5.2 and the same ionic strength. Here, the layering is slight and the mean refractive index is low (1.37-1.41) compared to the value 1.50-1.55 obtained with compact ordered dimers.11 It is necessary to stop the mica surfaces during 48 h to observe an index of 1.55, a proof of a compact packing of molecules between the mica surfaces. But the 38 nm distance is twice that obtained with the dimers as mentioned in Materials and Methods: it is likely that the packing of the molecules is associated with an ordered matter accumulation. On Figure 3a, the first jump (13.2
2544 Langmuir, Vol. 13, No. 9, 1997
nm) immediately followed by a second jump (4.0 nm) goes on while the refractive index remains approximately constant at 1.55. This observation agrees well with an ordered accumulation of Con A molecules in the light beam of the interferometer. Then, during 8 h (about 30 000 s), a slow continuous deviation, which reaches 9.0 nm, could correspond to a reorientation from a “side-on” to a “sideoff” position of the Con A dimers in two layers. If Con A dimers are adsorbed “side-on”, one should observe two jumps of 4.0 nm each, but if the molecules are adsorbed “side-off”, or if a few tetramer molecules are still in equilibrium with the dimers, only one jump of about 8.4 nm is then possible. A thermal drift cannot be eliminated during this long period of time. At a pH g pHi of the protein, the net surface charge of Con A is negative and the layering is probably favored by the two cation sites occupied by Mn2+and Ca2+ on the surface of each Con A molecule: two sites per monomer and four sites per dimer.12,17 Most jumps in Figure 3 are a multiple of 4.0 nm (the thickness of the dimer) or 8.4 nm (dimer length or depth of the tetramer) because dimers and tetramers were in equilibrium in the preceding surface displacement (see Methods). When the proteins are charged (pH * pHi) the hydrodynamical size is a function of the surface charge and depends on the location of the protein in the gap. Then the size of the jumps can be unsteady. When the compression stress on the solid surfaces exceeds a given limit, the mechanical device, essentially the cantilever spring, becomes loose and the two solid surfaces recede from each other. Thus, the protein solution can enter in
Gauthier-Manuel and Gallinet
the light zone, and the mean refractive index decreases by jumps: this process occurs at 40 000 s. In this case, the phenomenon that most likely occurs is a molecule accumulation from nucleation centers in the periphery of the light zone observed by interferometry. The molecules are adsorbed in a disordered manner and the refractive index tends to that of water. As molecules attract strongly and particles, such as nucleation centers, attract weakly, the interaction potentials between molecules and particles in a medium can show secondary minima.11 This situation can exist here when the Con A molecules are at pH 5.2 near their pHi and in a buffer solution at a high ionic strength (10-1 M ). Nucleation is thus favored. Since the diffusion of phosphate ions is slow, nucleation occurs first in the periphery of the mica surface. The fluctuations due to a convection flow in the medium are unlikely to occur. Indeed, several observations with a microscopic cell showed that the convection pertubation was insignificant when the thickness of the confined solution is less than 10 µm.22 Acknowledgment. This work is supported by the CNRS (GDR 1092: “interactions faibles: solutions concentre´es et cristalloge´ne`se des macromole´cules biologiques”). LA962018M (22) Komatsu, H.; Miyashita, S.; Suzuki, Y. Jpn. J. Appl. Phys. 1993, 32, L1855.
