5554
J. Phys. Chem. 1996, 100, 5554-5561
Forces between Macroscopic Surfaces in Solutions of Calcium Binding Proteins S. J. Miklavcic,*,† E. Thulin,‡ and B. Jo1 nsson‡ Departments of Food Technology and Physical Chemistry 2, Chemical Center, Lund UniVersity, S-221 00 Lund, Sweden ReceiVed: June 2, 1995; In Final Form: October 12, 1995X
Forces have been measured between macroscopic sheets of mica exposed to aqueous solutions of the calcium binding protein, calbindin D9k. The study focuses on the nature of the long-range exponential double layer force and on the adsorption behavior of calbindin on mica. These two features have been monitored as a function of molecular protein composition. We have varied the primary structure of calbindin by genetic substitution of specific amino acids. In particular, we report on the effects of neutralizing individual negative amino acid groups. Advantage is also taken of the protein’s ability to bind calcium ions to reduce its net negative charge further. At a fixed protein concentration, in the absence of added salt, the variation in net charge affects the decay length of the exponential force at large separations, in the way expected from classical continuum theory. In fact, after spectroscopic analysis of the solutions we find that the measured decay length agrees with the Debye length. The force at short separations is greatly affected by the presence of calcium bound to the protein. Specifically, when the protein’s binding affinity is high, the presence of bound calcium provokes a very strong adhesive force between the protein-adsorbed mica sheets. This we attribute to an ion correlation effect, which in the biological literature would be termed calcium bridging. In this and other cases the conformation of adsorbed calbindin is greatly influenced by calcium.
Introduction The forces between charged surfaces are instrumental in determining the behavior of a great many colloidal and biological systems. As the surface charges originate from widely different sources, these forces can be modulated by the intervening solution in various ways, e.g., by adsorption, salt screening, pH change, dimerization, etc. This is used to advantage by nature and has also been utilized in technical processes. Understanding how solution conditions can modify surface interactions has long been the goal of surface and colloid chemists. Until recently their task has been hampered by the need to invert data obtained from experiments which are limited to observing only the macroscopic consequences of particle or surface interactions. Direct force measuring techniques, such as the surface force apparatus,1 the osmotic stress technique,2 and the atomic force microscope,3 however, now enable one to monitor the interaction itself. Since the introduction of such methods, a vast range of direct force studies have been reported in the literature dealing with increasingly complex systems.4-10 These have steadily tested and even exceeded the limits of valid application of the classical theory of electrical double layer interaction.11,12 This, the DLVO theory, provides qualitative and semiquantitative explanations for the force behavior in some simple systems, such as the interaction of two charged mica surfaces in a univalent electrolyte solution. However, under some conditions, e.g., with divalent ions, in particular divalent counterions, a qualitative discrepancy at short surface separations can exist between that theory and experiment. The reasons for this discrepancy are now well founded through more exact statistical mechanical calculations.13-15 Surpassing this, however, is the diversity of * To whom correspondence should be addressed at the Ian Wark Research Institute, University of South Australia, The Levels Campus, 5095 SA, Australia. † Food Technology. ‡ Physical Chemistry 2. X Abstract published in AdVance ACS Abstracts, February 1, 1996.
0022-3654/96/20100-5554$12.00/0
discrepancies in the long-range behavior of the force in systems containing highly asymmetric electrolytes. In particular, the rarely disputed exponential decay length of the force, the classical Debye length,12 has not always been confirmed in these solutions. In some cases where exponential forces are present, the measured decay lengths are significantly smaller while in others they are significantly larger. Theoretical work going beyond Debye-Hu¨ckel approximation has indeed shown that the Debye length is inadequate in describing the long-range decay in asymmetric electrolytes. The predicted correction reduces the decay length relative to that of Debye length regardless of whether or not the high-valency ion was a surface counterion.16 Subsequent, and more exact, analyses17-20 have largely confirmed this fact. Recently, Kekicheff and Ninham21 measured forces in cytochrome protein solutions (at normal solution pH mica is negatively charged and the solution is a +12:-1 electrolyte) and reported that their decay lengths do indeed differ from the Debye value by the amount prescribed by Mitchell and Ninham’s correction.16 On the other hand, force measurements in negatively charged surfactant solutions at concentrations above the cmc, wherein the micelles that form are highly charged surface co-ions, have produced decay lengths which are longer than expected.22-24 The above investigations provided the initial impetus for our present work on surface forces in protein solutions, the first results of which we present here. There are, however, more significant and pressing issues which we also aim to address in our continuing studies. Questions which relate to how a solution of proteins affects surface interactions either through their presence in solution or through surface adsorption, how the adsorption process is affected by other solution factors, and what final equilibrium structures adsorbed layers take. Most proteins will adsorb onto the mica surfaces used in the surface force apparatus. This does not necessarily complicate matters, but can be used to advantage since the technique can then provide some information on how a protein structure changes upon adsorption, and of its structural response to © 1996 American Chemical Society
Surface Forces in Solutions of Calbindin changing solution conditions. In this paper we present results of our study of the interaction of two mica surfaces in a solution containing the calcium binding protein, calbindin D9k. Calbindin D9k belongs to a class of structurally related Ca2+ binding proteins including both Ca2+ regulatory proteins such as troponin C and calmodulin and Ca2+ buffers like parvalbumin and calbindin D28k. A calbindin solution can be viewed as a highly asymmetric electrolyte solution with the protein being negatively charged, in contrast to cytochrome c.21 Thus, we have the potential (in principle) to investigate the long-range behavior of the force. To make possible a study of how small structural changes affect both the adsorption and the surface-surface interaction, we exercise our capability to genetically modify the protein by exchanging individual amino acid residues. In this paper we report on the effects of neutralizing negative amino acid groups. The fact that calbindin binds two Ca2+ ions with high affinity provides a second mechanism for modification of its adsorption and screening properties. The double layer interaction between calbindin-covered mica surfaces can therefore be modified experimentally either using site-directed mutagenesis of ionic residues or changing the composition of the solvent. Suitable combinations of these means may provide a unique set of experimental data that can be used for successfully investigating proteins at surfaces in general. An often-neglected problem with protein samples, produced in-house or purchased, is that they are seldom pure, with many types of impurities. For example, we have found, via agarose gel electrophoresis, that cytochrome c samples purchased from Fluka contain two protein forms with different net charge. Normally a freeze-dried protein also contains considerable amounts of various salts. However, excessive refinement procedures, aimed at reducing this salt content, may be counterproductive by causing, for example, deamidation and changes in the protein net charge.25 Thus, it is virtually impossible to make a protein solution “salt-free” and there will always remain on dissolution a residual concentration of simple electrolyte, typically of the order of 10 times the protein concentration, and often more. These facts have to be considered, and the salt and protein concentrations have to be known quantitatively from atomic absorption spectroscopy and acid hydrolysis, before any quantitative discussion of the interaction of mica surfaces in a protein solution can be made. Materials and Methods Materials and Chemicals. In these experiments we used optically clear muscovite ruby mica, obtained from Associated Commodity Corp. Ltd (NY) and epoxy resin Epikote 1004 from Shell Pty. Ltd., to glue down the mica sheets onto silica disks. Ethanol of spectroscopic grade from Kemetyl (Sweden) was used in the cleaning procedure. Deionized and distilled water used in all experiments was passed through Millipore water system consisting of an ion-exchange cartridge, Organex-Q, activated charcoal filter, and nucleopore filters. On each occasion, the first few liters were flushed to avoid the risk of contamination incurred with stored water. Water that then passed was collected and deaerated to prevent bubble formation in the SFA chamber. The measured conductivity of the water was about 0.5 µS/m with a pH of between 5.5 and 5.7 due to absorbed carbon dioxide. Sodium chloride of analytical grade was obtained from Merck. Calbindin D9k. Calbindin D9k is an intracellular globular protein of 75 amino acid residues with a molecular weight of around 8500. The tertiary structure of calbindin in solution is known,26 with NMR and X-ray measurements suggesting it remains close to the crystal state structure.27,28 Calbindin takes
J. Phys. Chem., Vol. 100, No. 13, 1996 5555
Figure 1. Schematic plot of the tertiary structure of calbindin protein illustrating the two helix-loop-helix “EF hands”. The binding sites are situated in the “loop” areas of the EF hands, which are here shown to be occupied by two spherical calcium ions.
the form of an approximate sphere with diameter of 3 nm (Figure 1). It possesses 30 apolar groups and 45 charged or polar amino acids. Most of the hydrophobic groups are found inside the four R-helices which make up calbindin’s two EF hands, while 12 negative charges are located on the two terminal loops in the vicinity of the calcium binding sites. The Ca2+ binding sites are composed of a characteristic helix-loop-helix motif; the EF hand29 and a pair of EF hands are capable of binding two Ca2+ ions with high affinity and positive cooperativity. The sites are surrounded by several negatively charged glutamate and aspartate residues and the net charge of the apo form of the recombinant wild-type protein is -7, or -8 if the methionated N-terminal is formylated, as we will assume below. An examination of the structure reveals that all the charged amino acids are close to the surface of the protein. This protein we refer to as wild-type or M0. In the calbindin mutant, M11, used in this study three negatively charged amino acids, glutamate (E) or aspartate (D), in the vicinity of the binding sites have been replaced by their neutral counterparts, glutamine (Q) or asparagine (N), respectively (E17Q + D19N + E26Q, the number refers to the position in the primary sequence where the groups reside). Since the exchanged amino acids are sterically very similar and located on the surface of the protein, this choice ensures the smallest structural perturbation, as confirmed by NMR chemical shift assignments.30 Mutant proteins were produced in E. coli by using synthetic genes as described in full elsewhere,31 and purified according to ref 32. Protein purity was checked against SDS and agarose gel electrophoresis, isoelectric focusing, and 1H NMR. In the pH range 6-8, the wild-type calbindin has a net charge of -8; the mutant M11, with three negative residues neutralized, therefore has a net charge of -5 in this same pH range. Apart from the (positive) counterions, the protein solutions are accompanied by additional salt, whose concentration must be determined by quantitative analysis at the end of each experiment. In these systems calcium ions are particularly difficult to avoid since all glassware release calcium ions under sufficient incentive. Both the wild-type, denoted M0, and the M11 forms are very strong calcium binders, binding two calcium ions per molecule.
5556 J. Phys. Chem., Vol. 100, No. 13, 1996 In pure water the binding constants are 2 × 108 and 3.7 × 108 for the wild-type protein and 6.3 × 106 and 5.0 × 106 M-1 for M11.30,31 We have also studied the force behavior in aqueous solutions made of these calcium-loaded forms. The binding is so effective that these two proteins have net charges of -4 and -1, respectively. The difference in preparation between calcium-loaded and apo forms of the two proteins occurs in the last ion exchange step of purification procedure,32 in which a calcium buffer is used. The calcium-loaded protein solution is passed through Sephadex G25 in doubly distilled water in order to remove excess salt. The apo protein form is obtained by treating this solution with a 10-fold excess of EDTA at pH 7.5. Finally, EDTA is removed by passing the solution through a Sephadex colon, to which a saturated NaCl solution (treated with Chelex 100 to remove traces of Ca2+) has been applied. The volume ratio of protein:NaCl:Sephadex G25 used in the last step was 1:5:60. We point out a problem connected with strong calcium binding proteins. In solution they are able to induce leaching of calcium ions from otherwise inert surfaces. Pyrex or silica glass is particularly problematic in this respect and so whenever possible we have avoided its use. Any glass that could not be avoided in the experimental procedure was first treated with an EDTA solution, in order to extract any calcium ions on or within the interface, and then thoroughly rinsed with Millipore water. Such measures are, however, not foolproof. In dealing with protein adsorption, especially whenever mutations are involved, a key question is that of stability. Any decrease in a protein’s structural stability may lead to enhanced adsorption and reconformation upon adsorption. It is well documented33,34 that not only is the basic calbindin protein structure maintained with the mutation, the stability against denaturation is actually increased with the apo form of the mutant M11. Even more important in the context of this paper, the calcium-loaded forms are extremely stable. Stability has been judged by circular dicroism spectra taken in high concentrations of urea (4-6 M): it is not possible to denaturate the calcium-loaded forms at all, even in 8 M urea solutions.33,34 In summary, we employ solutions of extremely stable proteins as asymmetric electrolytes of valency -8:1, -5:1, -4:1 and -1:1. In these experiments we have a known protein concentration and study the behavior of the forces with changes in valency. Surface Force Measurements. The forces between mica surfaces were measured using Israelachvili’s technique, described in full elsewhere.1,36 Back-silvered mica surfaces were mounted on cylindrical silica disks in the cross configuration. The separation of the mica surfaces was determined interferometrically,37 while the force between the surfaces, F, was derived from the deflection from its equilibrium position, of a spring holding one of the silica disks. The magnitude of any adhesion was estimated by the amount of “pull-off” force required to separate the surfaces to infinite distance. Due to the large radii of curvature of the cylinders (2 cm) compared to the separations studied the force can be related, to sufficient accuracy, to the interaction free energy per unit area between plane surfaces, E, using the Derjaguin approximation38
F ) 2πRE where R is the mean radius of curvature. For this reason, in all the experimental figures we have plotted the force normalized by the measured mean radius of curvature. The surface separation was controlled by means of a piezoelectric crystal. In particular, cited values of proteinprotein contact are those obtained with the piezo, since
Miklavcic et al. application of the coarser electric motor present in the device at or near contact can induce damage to adsorbed protein layers leading to artefactual results. Flat adhesive mica-mica contact in air is used as a test of surface cleanliness. A similar condition is required to hold in water. Provided that such conditions are met, the apparatus is filled with the desired electrolyte solution. For these studies, bulk protein solutions were prepared external to the SFA and the entire chamber (35 mL) filled. With such bulk exchanges one normally need only wait 2-3 h before recording the first measurements. No time dependence of the forces was observed after this period. Individual studies were repeated several times on different positions within an experiment and with different pairs of mica sheets in new solutions (representing new experiments). Whenever the surfaces did not come into adhesive contact, the force profile on separation was recorded. Measurements were taken very slowly to ensure that the system had sufficient time to establish equilibrium after a change of separation. Usually, at very large distances separation changes were made at rates of 0.1 nm/s, by changing the voltage applied to the piezo at rates of about 0.3-0.7 V/s. However, no variations in the results were found with rates marginally outside this range. All the measurements were carried out in a thermally insulated room, kept at a constant temperature of 22 °C. An individual force run was started only when the thermal drift of the surfaces was negligible. This is a crucial factor in our study, as otherwise misleading determination of decay lengths can result. The zero of separation is taken to be that found in water. We have compared the measured forces with theoretical fits using the DLVO theory to determine E. The calculations are based on the numerical algorithm of Chan et al.39 for the electrical double layer interaction between two charged planar surfaces at a fixed surface potential, Ψ0, or fixed surface charge density, σ, immersed in a univalent electrolyte of bulk molar concentration, c0. The van der Waals force is calculated using nonretarded Hamaker theory with the constant A ) 2.2 × 10-20 J. In cases where there is evidence of protein adsorption the fitting parameters (values of surface potential, Ψ0∞, and charge, σ∞, for surfaces in isolation) were taken to originate at a plane further in to the solution corresponding to a distance of the adsorbed layer thickness. No adjustment of the origin of the van der Waals force was performed for reasons which we make clear later. The Debye screening parameter is defined as
κ2 ) ∑qi2e2ci/(r0kT)
(1)
where e is the elementary charge, r and 0 are the relative permittivity and the permittivity of vacuum, respectively, k is Boltzmann’s constant, and T is the temperature. The summation is taken over all charged species i of concentration ci (actually a number density, m-3) and valency qi. The analysis provides a value of the decay length of the double layer force, κ-1, with the surface charge and surface potential considered only as effective parameters. Results and Discussion When an aqueous solution is introduced between two mica surfaces, an electrical double layer repulsion and a dispersion of van der Waals attraction compete. This is the case for deionized and distilled water at a pH of about 5.5 (Figure 2): an exponentially decaying repulsive force dominant at large separations, which is overcome at small separations by the van der Waals attraction bringing the surfaces into contact. Some features of this force profile to be noted for comparison with subsequent results are (i) the decay length of the repulsion which
Surface Forces in Solutions of Calbindin
Figure 2. Semilogarithmic plot of the force between two crossed mica sheets in the presence of purified distilled water (filled diamonds) and a solution of 0.5 mM NaCl (open triangles). The pH of these solutions were 5.5-5.7. The solid lines denote theoretical DLVO fits assuming constant surface charge (upper curve of each set) and constant surface potential (lower curve of each set). The mean-field model assumes a univalent electrolyte solution and the following parameter sets. For the water comparison Ψ0∞ ) 80 mV, σ∞/e ) 88.7 nm2, and c0 ) 7 × 10-5 M with κ-1 ) 36 nm. For the NaCl comparison Ψ0∞ ) 110 mV, σ∞/e ) 14.6 nm2, and c0 ) 5 × 10-4 M with κ-1 ) 13.5 nm.
is traditionally expected to be the Debye screening length, κ-1, (ii) a region in which the force cannot be measured due to an inherent mechanical instability, and (iii) the contact separation, i.e., the distance of closest approach. Included in Figure 2 is the measured force curve in a 0.5 mM NaCl electrolyte solution. It is difficult to obtain the expected decay length in the case of water: a concentration of about 5 µM monovalent electrolyte, an appropriate concentration for water at pH 5.5, corresponds to a Debye length of about 170 nm. In order to measure exponential forces one generally is required to measure distances out to the order of several decay lengths. For water this is, however, impractical under usual circumstances and since it lies outside our main interest we have not endeavored to achieve an accurate value. In 0.5 mM electrolyte solution, on the other hand, the decay is accurately determined. The classical DLVO model predicts a decay length of 13.5 nm based on the Debye formula, and this is precisely that found in our experiments. Common to both the water and salt data is the jump into contact from finite separations to within an angstro¨m or two of the contact achieved in air. Once in contact the surfaces cannot be separated by means of the piezoelectric crystal, characteristic of a very strong van der Waals adhesion. Also in Figure 2 are DLVO fits to both sets of experimental data. In the case of 0.5 mM NaCl, very good quantitative agreement can be obtained assuming standard parameter sets consistent with literature values.5 In the case of water, the difficulty associated with measuring weak forces at very large distances affects not only the determination of the decay length but also the magnitude. For this reason we have not obtained the value of Ψ0∞ representing the magnitude of the force, claimed by Pashley5 (cf. our value of Ψ0∞ ) 80 mV and Pashley’s 150 mV). Wild-Type Calbindin. Replacing the salt solution with a solution containing both 0.5 mM NaCl and 7 µM of wildtype calbindin results in the disappearance of the jump instability with the surfaces never achieving mica-mica contact (Figure 3). With the piezo, the surfaces cannot be made to approach closer than a distance of about 6.0 ( 0.2 nm. Comparing this with the X-ray crystallographic size of calbindin D9k27,28 suggests the adsorption of a single layer of protein to each surface. In this system we found no hysteretic behavior in the force profile: inward and outward runs were identical, with no observed adhesion between the protein layers.
J. Phys. Chem., Vol. 100, No. 13, 1996 5557
Figure 3. Semilogarithmic plot of the force in the presence of a solution of 0.5 mM NaCl (open triangles) and a solution of 0.5 mM NaCl and approximately 0.1 mg/mL calbindin protein (filled diamonds). The solid lines denote DLVO theoretical fits assuming constant surface charge (upper curve) and constant surface potential (lower curve) with the parameters Ψ0∞ ) 100 mV, σ∞/e ) 15.77 nm2, c0 ) 6.25 × 10-4 M, and κ-1 ) 12.1 nm. In this fit the plane of charge and/or potential was shifted out 3.0 nm per surface to account for an adsorbed protein layer. The inset shows forces measured on approach (filled symbols) and separation (open symbols) 24 h after the salt/protein solution had been replaced with pure water. The change in separation seen at high compressional loads is likely due to enforced protein layer reconfiguration. We did not pursue this feature. The DLVO fits assumed the values Ψ0∞ ) 70 mV, σ∞/e ) 100 nm2, and c0 ) 5.5 × 10-5 M with κ-1 ) 40.6 nm. The same shift in surface charge plane was assumed.
To confirm the presence of adsorbed layers of calbindin, the sodium chloride/calbindin solution was drained from the apparatus and replaced with pure water and the system monitored over a period of 24 h. Three force measurements conducted during this time gave characteristic double layer repulsions of very large decay lengths, the last of which is shown in the inset to Figure 3. Compared with the situation prior to exposure of protein, the origin of the force is shifted out by approximately twice the thickness of an adsorbed layer. Furthermore, the force is again nonhysteretic. That is, we could measure both inward and outward double layer repulsions, both of which are shown here. A discontinuity in slope at about 15 nm out from mica contact is now very much evident compared with the salt/protein case. In fact, with these results one can appreciate the demarcation between the short-range force, likely of steric origin, and the long-ranged, salt-screened double layer interaction. Although it is somewhat surprising to find a negatively charged protein adhering to a negative surface, other instances have been reported in the literature.40-43 The adsorption process is either driven by nonelectrostatic forces alone or aided by dipolar attractive interactions with the surface; together these are obviously strong enough to overcome the net electrostatic protein-mica and protein-protein repulsion. We should expect the adsorption process to be salt dependent; that is, increased salt content should reduce the protein-mica and protein-protein repulsion and favor adsorption. We have performed experiments with wildtype calbindin with and without added salt, and at this and at one higher pH value in the absence of salt, in order to demonstrate this mechanism, and found that only in salt-free, high pH solution is there evidence of removal or partial removal of the protein layer(s) (Figure 4). Consequently, we attribute the depletion of protein to a combination of diminshed electrolyte screening and increased surface-protein charge interaction. The 0.5 mM NaCl and 0.1 mg/mL force curve can been fitted with DLVO theory assuming a univalent electrolyte with a
5558 J. Phys. Chem., Vol. 100, No. 13, 1996
Miklavcic et al. TABLE 1: Salt and Protein Concentrations (in µM) in Calbindin Solutionsa M0 protein Na+ K+ ClCa2+
Figure 4. Linear plot of the short-range force between two crossed mica sheets in 0.1 mg/mL solutions of calbindin protein. In the case represented by open diamonds 0.5 mM NaCl has been added at a pH of 5.5-5.7. Two cases without added salt are also shown pH adjusted to 8.5-9.0 (filled diamonds) and 7.0 (open circles).
nominal concentration of 0.625 mM (κ-1 ) 12.1 nm) and a surface potential of 100 mV, the latter assuming the plane of charge to be 3.0 nm out from the origin of the van der Waals plane, consistent with the presence of one adsorbed protein layer on each surface. Comparison between constant surface charge and constant surface potential models of the double layer force would suggest that the protein layer/electrolyte interface is neither, but that the charge layer undergoes regulation. We remark that if, instead, the charge plane were set to be the same as for the NaCl case, i.e., the same position as the van der Waals plane, then the data at large separations can be fitted equally well, but now assuming a surface potential twice as large, at 210 mV. However, at smaller separations (less than 15 nm) the experimental data lies considerably above the theoretical curves, which could be interpreted as a result of a steric contribution to the total force associated with the interaction between adsorbed protein molecules. Although we have shifted the surface charge plane (and potential plane) to coincide with the extremity of the protein monolayers, we have deliberately kept the van der Waals’ plane at the mica surfaces. We have found that moving the origin of the van der Waals force to the protein interface results in a much poorer fit to the curves at short separations: the theoretical curves turning attractive while the experimental forces stay repulsive. Further, we found that with the origin of the van der Waals plane at the mica surface the dispersion contribution to the total force is negligible for separations greater than 6 nm, the distance of closest approach. So, although one would expect the Hamaker constant to be affected by the protein layers present between the mica surfaces, its exact value is not important as the overall effect is not measurable. The slight difference in the decay length of the forces in NaCl (13.5 nm) and NaCl with calbindin (12.1 nm) does not permit any conclusive study of the protein contribution to the screening. For this reason, in the remainder of the presentation, we focus attention on the forces in solutions of protein alone. From this point on we have to know the protein concentration and also the concentration of accompanying salt quite accurately: a protein concentration calculated on the basis of the weight of the freeze-dried protein is not sufficient. For this reason we have performed an acid hydrolysis of the apo and calciumloaded forms of wild-type calbindin together with atomic absorption measurements and photometric titrations in order to determine the protein and salt concentrations. The data are collected in Table 1. We note that the calbindin concentration determined via acid hydrolysis as opposed to simply weighing the freeze-dried sample differs by as much as 50%. For
M11
Ca free
Ca loaded
Ca free
7.0 ( 0.9 117 ( 2 5 ( 0.2 85 ( 15 10 ( 1
9.0 ( 0.5 43 ( 2 6 ( 0.2 28 ( 15 28 ( 1
5.5 ( 0.2 100 ( 2 4(2 28 ( 15 3(1
Ca loaded
a The protein concentration is determined from acid hydrolysis, and the salt concentrations are obtained from atomic absorption measurements and photometric titration. No data has been obtained for the case of calcium-loaded M11, nor is any attempt worthwhile, as this system is on the limit of resolution of the SFA in accurately determining the decay length.
Figure 5. Semilogarithmic plot of the force in the presence of 7 µM wild-type calbindin. Diamond symbols represent forces measured on approach (filled) and on separation (open). Open circles represent forces measured on approach only of mica surfaces in a 9 µM solution of calcium-loaded wild-type calbindin. The pH of these solutions were 7.0 ( 0.5.
TABLE 2: Theoretical and Experimental Decay Lengths for the Different Proteins Studied.a protein
measured decay (nm)
M0 M0 + Ca2+ M11 M11 + Ca2+
20.0 28.5 26.5 30.0
Debye length (nm) protein only
protein + salt
19.2 32.0 33.5
21.1 ( 1.5 26.6 ( 0.5 25.3 ( 1.5
a The pH of the solution was adjusted to be 7.0 ( 0.5 Temperature was taken to be 22 °C. Decay lengths are given in nanometers. The theoretical values are based on the quantitative analysis of protein and salt concentration performed on the experimental solutions after the measurements were made.
cytochrome c purchased from Fluka or Sigma the values differ by an order of magnitude! Quantitative analyses of the protein samples are imperative. In Figure 5 we show the force obtained with a solution containing wild-type calbindin at a pH of 7 ( 0.5. A nominal concentration of 0.1 mg/mL assuming a molecular weight of 8500 leads to a protein concentration of 12 µM, while acid hydrolysis of the same sample gave a value of 7 ( 0.9 µM. Using the latter value and a valency of -8, we find a screening of 19.2 nm in good agreement with the experimental value of 20.0 nm (see Table 2). However, it is to be remarked that this agreement is purely accidental as we have yet to include the component of screening due to the accompanying salt. Now we have the unusual situation that including salt, specifically calcium ions, leads to a longer screening length. The experimental result is mildly reminiscent of the case of CTAB micellar solutions,22-24 in which it was found that only dissociated
Surface Forces in Solutions of Calbindin counterions and free cationic surfactants contributed to the decay length; the highly charged micelles themselves were excluded from the region between the surfaces and therefore did not contribute to the screening. We first considered this is a possibility here with the protein taking the place of the micelles. Fortunately, we could avoid this radical interpretation, as a quantitative analysis of the solution provided the following more likely explanation. Each calbindin molecule has the ability to bind two calcium ions, thus reducing its valency from -8 to -4. Using the best possible estimates of salt and protein concentration we find κ-1 to be 21 nm, still in good agreement with experiment. The protein, of which 5 µM is calcium-loaded with 2 µM in apo form, and the remaining background salt contribute approximately equally to the screening length. Included in Figure 5 is our measured force curve for a solution of the calcium-loaded form of M0. The net protein valency is now -4; we would therefore expect that the repulsion decays with a larger characteristic length, as is indeed found. We note from the figure that, just as was the case for the forces between protein-adsorbed surfaces in water (Figure 3, inset), a distinct change in slope occurs near protein contact, again suggesting different force contributions at smaller separations. The magnitude of the force as measured by Ψ0∞ is decreased with the calcium loaded system as one would expect with the adsorption of a lower net valent species: Ψ0∞ drops from 115 mV with the adsorption of apo form calbindin, to 83 mV with the adsorption of calcium-loaded calbindin. Fitting the force curve for the calcium-loaded protein system to the DLVO model (or alternatively to an exponential, but at sufficiently large distances) we obtain a value for the decay length of 28.5 nm. Comparing this to the calculated value of 32.0 nm for a 4:1 electrolyte shows that we lack some contribution to the screening. With the additional salt the expected decay length is reduced to 26.6 nm in better agreement with experiment. Apart from the difference in decay lengths of the forces at large separations, these two systems also exhibit quite different force behavior when the surfaces are at small separations. In the apo protein system, once in layer-layer contact the mica surfaces maintain a stable separation (≈6.0 nm). And even after 20-30 min in protein-protein contact, taken to be the separation where an upturn in the force is evident, the surfaces can be separated with the piezoelectric crystal and the outward or decompression force measured, being identical to the force found on approach. Unequivocally the system is in equilibrium. In contrast, with calcium-loaded M0, although the surfaces are brought together with the piezo against a repulsive, albeit weaker, double layer force, experiencing a similar barrier at around 6.0 nm, they are not stable at this separation. Over a period of less than 1 min, initially being left at 6.0 nm at a constant applied load, the surfaces approach spontaneously to a final separation of about 3.0 nm. This spontaneous movement of the two surfaces toward one another is portrayed in the inset to Figure 6 by the arrow positioned between the two parallel broken lines. There is clearly a rearrangement of both the interlayer and intralayer configuration of protein molecules characterized by a slow relaxation time. Because the calciumloaded protein is considerably more stable than the apo form, it would appear unlikely that the observed change in separation is due to tertiary structural changes. At this final equilibrium distance the surfaces exhibit a strong adhesive force which cannot immediately be overcome by application of the piezo. That is, gradually reversing the applied voltage over the full possible range is ineffectual in separating the surfaces directly; in fact, there is no measurable change in
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Figure 6. Linear plot showing the adhesive force found in 7 µM solution of calcium-loaded wild-type calbindin. Filled triangles represent the forces measured on approach, while open triangles represent forces measured on separation. The inset shows an enlargement of the region near contact; the arrow between the two parallel vertical lines relates schematically to our observations that after the surfaces are brought together, they spontaneously move toward each other under a constant load, after which the strong adhesion is found. The decompression data represent results of two separate experiments, while for the forces measured on approach only one force run is shown. This is in order to highlight the surface movement under constant load. Other inward runs which are not shown for reasons of clarity end at different force magnitudes, corresponding to the magnitudes of the pulloff forces which are shown.
separation, as displayed in the main body of Figure 6. Again after a period of some minutes left in this extreme condition, the surfaces do in fact detach and reach an equilibrium separation of between 450 and 500 nm. From these values we determined an adhesive force of 3.0-3.5 mN/m to be compared with the value of 2 × 10-2 mN/m, based on the van der Waals attraction between mica sheets44 at a separation of 3 nm. Clearly, the adhesion has its origin in an independent mechanism. At this stage we have negatively charged surfaces and a surplus of divalent calcium ions in the intervening solution. From this we may speculate that the strong adhesion measured is due to an ionic correlation mechanism sometimes seen in systems with divalent counterions,7,13-15 which is often referred to as calcium bridging (in the case of calcium) in the biological literature. Further work examining the salt dependence of this adhesion is in progress to establish this fact conclusively. Calbindin Mutant M11. In Figure 7 we show data for the forces measured in solutions of M11, both the apo as well as the calcium-loaded form. In these cases the proteins are expected to have a net negative charge of -5 and -1, respectively. Again, as is to be expected, the decay lengths of the long-range repulsions are larger than found with the wildtype protein, with calcium-loaded M11 giving the largest decay length of all, and the magnitudes of the forces are smaller. Exponential fits gave decay lengths of 26.5 and 30.0 nm, respectively. A theoretical comparison is possible only in the case of apo form M11, due to the lack of quantitative data for the protein and salt concentration in the calcium-loaded system. Similar to the force behavior in the solution of calcium-loaded M0 protein, the larger decay lengths strongly highlight the transition between the double layer repulsion and the shorter ranged steric forces between the protein layers. The transition appears sharper when the protein charge is lower. A qualitative difference between this and the wild-type system is the shortrange behavior of the force in both cases of apo M11 and calcium-loaded M11. Note that compared with the solution of calcium-loaded M0, an equilibrium force barrier is present at 6-7 nm in M11 solutions despite the fact that the charges on these two respective proteins differ by only one unit. On the
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Figure 7. Semilogarithmic plot of the force in a solution containing approximately 0.1 mg/mL of the calbindin mutant M11. The pH of these solutions were 7.0 ( 0.5. Nablae symbols represent force measurements on approach (filled) and on separation (open). Circular symbols similarly represent forces measured in a solution of the calcium loaded form of M11, and again filled symbols represent compression runs and open symbols denote decompression runs.
other hand, with the solution of calcium-loaded M11, the surfaces approach continuously to an equilibrium separation of approximately 3 nm, the same as that found with calcium-loaded M0. This is further evidence that the bound calcium ions are responsible for the observed behavior. But, in contrast to the case of calcium-loaded M0, the force in calcium-loded M11 is nonhysteretic. Both compression and decompression curves for the latter system are shown in Figure 7. Why this should be has not been unequivocally established. However, what is certain is that this finding all but eliminates the possibility of any involvement by van der Waals forces since it is unlikely that the minor perturbations introduced to the mutant’s amino acid composition would have such an enormous effect on the electromagnetic absorption properties of the two proteins, giving rise to such different strengths of the dispersion force. The answer must lie in the electrostatics. The suggestion made earlier on the role of Ca2+ ion correlations is not wholly inconsistent with this result: with the M11 system we are less likely to expect very highly charged protein surface layers, the charge density (per protein) having been reduced by a factor of 4. It is known that a net attraction between charged surfaces due ion correlations requires high surface charge densities.13-15 The fact that M11 is also a much weaker calcium binder than M0 is also likely to be of significance. Final Remarks and Conclusions Protein solutions always contain a variety of charged species. As mentioned earlier, these can contribute to a background electrolyte solution, possibly on the order of 10 times the protein concentration. As can be verified by a simple calculation based on the Debye formula (eq 1), provided the net protein charge is sufficiently high so that qp2 . 2Csalt/Cp (the subscript p refers to protein), the relative importance of free salt is minor. The problem of knowing the amount of accompanying ions is then also minor. However, for proteins at the isoelectric point or under solution conditions not too far removed from it, the accompanying salt can significantly affect the system’s behavior. Such is our case, especially since the trace amounts of free calcium present in solution are sufficient to reduce the net charge of calbindin. Nevertheless, as a general rule, in order to perform a quantitative comparison between measured and theoretical double layer forces one must determine the salt and protein concentrations by quantitative analysis. Once this was done in our study we found that the Debye formula correctly predicted
Miklavcic et al. the long-range repulsive decay between the mica surfaces in calbindin D9k solutions. Although we were unable to produce any contributing experimental evidence of the MitchellNinham16 decay correction here, due to the interference of calcium ions, we shall continue to pursue this line of study using other well-characterized proteins at our disposal. The force behavior at short range is considerably more complex, as a result of the irreversible adsorption of negatively charged calbindin onto negatively charged mica surfaces. The presence of both free and bound calcium ions is very much evident; they seem to play a crucial role in determining the adhesive strength, possibly via an ionic correlation mechanism. An undoubtedly positive feature of this work is the usefulness of employing genetically modified mutant proteins to ascertain molecular origins of observed behavior. In the results presented here, for example, it is the comparison of the triply-mutated M11 with calcium-loaded wild-typestheir charge differing by only one unitswhich pointed out the importance of Ca2+ ions in the surface adhesion. Even the long-range component is sensitive to the effect of amino acid neutralization. It is quite obvious that there remains considerable scope with which to study protein-surface interactions using site-directed mutagenesis. Acknowledgment. We gratefully acknowledge financial support provided by the National Board for Industrial and Technical Development (NUTEK) and the Swedish Engineering Science Research Council (TFR). We thank Ka˚re Larsson, Tommy Nylander, and Plamen Petrov for very useful discussions and their general interest in this work. We are especially grateful for the technical assistance provided by Plamen Petrov. References and Notes (1) Israelachvili, J. N.; Adams, G. E. J. Chem. Soc., Faraday Trans. 1 1978, 74, 975. (2) Parsegian, V. A.; Rand, R. P.; Fuller, N. L.; Rau, D. C. Methods Enzymol. 1986, 127, 400. (3) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Langmuir 1992, 8, 1831. (4) Israelachvili, J. N. Faraday Disc. 1978, 65, 20. (5) Pashley, R. M. J. Colloid Interface Sci. 1981, 83, 531. (6) Pashley, R. M.; Israelachvili, J. N. J. Colloid Interface Sci. 1984, 101, 511. (7) Marra, J. Biophys. J. 1986, 50, 815. (8) Parker, J. P.; Claesson, P. M. Langmuir 1994, 10, 635. (9) Afshar-Rad, T.; Bailey, A. I.; Luckham, P. F.; MacNaughton, W.; Chapman, D. Colloids Surf. 1988, 31, 125. (10) Rau, D. C.; Lee, B.; Parsegian, V. A. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2621; Rau, D. C.; Parsegian, V. A. Biophys. J. 1992, 61, 246. (11) Derjaguin, B. V.; Landau, L. Acta Phys. Chim. URSS 1941, 14, 633. (12) Verwey, E. J. W.; Overbeek, J. Th. G. Theory of the Stability of Lyophobic Colloids; Elsevier: Amsterdam, 1948. (13) Guldbrand, L.; Jo¨nsson, Bo; Wennerstro¨m, H.; Linse, P. J. Chem. Phys. 1984, 80, 2221. (14) Kjellander, R.; Marcelja, S.; Akesson, T.; Jo¨nsson, Bo. J. Chem. Phys. 1991, 97, 1424. (15) Kekicheff, P.; Marcelja, S.; Senden, T. J.; Shubin, V. J. Chem. Phys. 1993, 99, 6098. (16) Mitchell, D. J.; Ninham, B. W. Chem. Phys. Lett. 1978, 53, 397. (17) Kjellander, R.; Mitchell, D. J. Chem. Phys. Lett. 1992, 200, 76; J. Chem. Phys. 1994, 101, 603. (18) Attard, P. Phys. ReV. E 1993, 48, 3604. (19) Ennis, J.; Kjellander, R.; Mitchell, D. J. J. Chem. Phys. 1995, 102, 975. (20) Attard, P. AdV. Chem. Phys., in press. (21) Ke´kicheff, P.; Niham, B. W. Europhys. Lett. 1990, 12, 471. (22) Pashley, R. M.; McGuiggan, P. M.; Horn, R. G.; Ninham, B. W. J. Colloid Interface Sci. 1988, 126, 569. (23) Kekicheff, P.; Richetti, P. Prog. Colloid Polym. Sci. 1992, 88, 8. (24) Richetti, P.; Kekicheff, P. Phys. ReV. Lett. 1992, 68, 1951. (25) Chazin, W. J.; Ko¨rdel, J.; Thulin, E.; Hofmann, T.; Drakenberg, T.; Forse´n, S. Biochemistry 1989, 28, 8646. (26) Ko¨rdel, J.; Forse´n, S.; Chazin, W. J. Biochemistry 1989, 28, 7065.
Surface Forces in Solutions of Calbindin (27) Szebenyi, D. M. E.; Obendorf, S. K.; Moffat, K. Nature 1981, 294, 327. (28) Szebenyi, D. M. E. & Moffat, K. J. Biol. Chem. 1986, 261, 8761. (29) Kretsinger, R. H.; Nockolds, C. E. J. Biol. Chem. 1973, 248, 3313. (30) Linse, S.; Johansson, C.; Broden, P.; Grundstro¨m, T.; Drakenberg, T.; Forse´n, S. Biochemistry 1991, 30, 154. (31) Brodin, P.; Grundstro¨m, T.; Hofman, T.; Drakenberg, T.; Thulin, E.; Forse´n, S. Biochemistry 1986, 25, 5371. (32) Johansson, C.; Brodin, P.; Grundstro¨m, T.; Thulin, E.; Forse´n, S.; Drakenberg, T. Eur. J. Biochem. 1990, 187, 455. (33) Akke, M.; Forse´n, S. Proteins 1990, 8, 23. (34) Wendt, B.; Hofmann, T.; Martin, S.; Bayley, P.; Brodin, P.; Grundstro¨m, T.; Thulin, E.; Linse, S.; Forse´n, S. Eur. J. Biochem. 1988, 175, 439. (35) Linse, S.; Brodin, P.; Johansson, C.; Thulin, E.; Grundstro¨m, T.; Forse´n, S. Nature 1988, 335, 651. (36) Parker, J. P.; Christenson, H. K.; Ninham, B. W. ReV. Sci. Instrum. 1989, 60, 3135.
J. Phys. Chem., Vol. 100, No. 13, 1996 5561 (37) Israelachvili, J. N. J. Colloid Interface Sci. 1973, 44, 259. (38) Derjaguin, B. V. Kolloid Z. 1934, 69, 155. (39) Chan, D. Y. C.; Pashley, R. M.; White, L. R. J. Colloid Interface Sci. 1980, 77, 283. (40) Matuszewska, M.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1981, 84, 403. (41) Cosgrove, T.; Obey, T. M.; Vincent, B. J. Colloid Interface Sci. 1986, 111, 409. (42) Bonekamp, B. C.; Hidalgo Alvarez, R.; de las Nieves, F. J.; Bijsterbosch, B. H. J. Colloid Interface Sci. 1987, 118, 366. (43) Nylander, T.; Kekicheff, P.; Ninham, B. W. J. Colloid Interface Sci. 1994, 164, 136. (44) Israelachivili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1991.
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