Effects of Cosolvents and pH on Protein Adsorption on Polystyrene Latex: A Dynamic Light Scattering Study

Journal of Colloid and Interface Science 221, 25–37 (2000) Article ID jcis.1999.6560, available online at http://www.idealibrary.com on

Effects of Cosolvents and pH on Protein Adsorption on Polystyrene Latex: A Dynamic Light Scattering Study Da Song and Daniel Forciniti1 Department of Chemical Engineering, University of Missouri-Rolla, Rolla, Missouri 65401 E-mail: [email protected] Received March 1, 1999; accepted September 30, 1999

Our aim is to elucidate the effect of hydrophobic and electrostatic interactions on protein adsorption by changing solvents and pH. To achieve this goal, two well-characterized proteins, human immunoglobulin G (IgG) and human serum albumin (HSA), were adsorbed on uniform polystyrene beads (PS) from different cosolvents at different pH. The size of the proteins and the thickness of the adsorbed layers were measured by using dynamic light scattering (DLS). Since these two proteins have different isoelectric points (pI) and surface polarities, our studies demonstrate the delicate balance that exists among protein/ surface and protein/protein forces which determines not only the amount adsorbed but also the orientation of the proteins at the surface. Polystyrene latex is an excellent substrate because of its homogeneous size, large surface area, negligible solubility in water, and invariant surface characteristics (3–6). IgG and HSA were chosen as our model proteins because they have been extensively studied by different methods, such as electrophoretic mobility, ellipsometry, electron spin resonance spectroscopy, infrared bound fraction measurement, quasi-elastic light scattering, reflectometry, sedimentation, small-angle X-ray scattering, reflection Fourier transform infrared radiation (FT-IR), atomic force microscopy, small-angle neutron scattering, and Monte Carlo simulations, and because they have different surface properties and shapes. Protein interactions with solid surfaces involve a complex interplay of Van der Waals forces, hydrogen bonding, and electrostatic and hydrophobic forces (7). Previous studies of protein adsorption on solid surfaces show that both hydrophobic and electrostatic interactions determine the amount of protein adsorbed (8) and that the effect of electrostatic interactions becomes more important when the adsorption is performed at low ionic strength where the screening effect of added salts is weakened (9). Studying protein adsorption behavior in aqueous mixtures of organic cosolvents (such as methanol and glycerol) is a useful method to learn about the effect that hydrophobic interactions have on protein adsorption. By changing the solvent properties rather than the characteristics of the surface, we can isolate the effect of the solvent in protein/surface interactions

Dynamic light scattering was used to study the adsorption of two proteins with different surface properties (IgG and HSA) on negatively charged polystyrene latex. The proteins were adsorbed from water and from water/methanol and water/glycerol mixtures at various pH. Some striking differences between the adsorption behaviors of the proteins were observed. Whereas the thickness of the adsorbed layer of HSA was extremely sensitive to pH and solvent composition, that of IgG was not. IgG mainly showed an end-on orientation on polystyrene whereas several different surface orientations are suggested for HSA under different conditions. The addition of methanol inhibited the adsorption of HSA on the latex, but it did not affect the adsorption of IgG. In contrast, the addition of glycerol increased the thickness of the adsorbed layers of both proteins. So, the orientation of IgG on the latex is insensitive to pH but is a function of the kind of solvent whereas both pH and solvent strongly affect the adsorption of HSA. This is a puzzling result since both cosolvents should equally affect the adsorption of both proteins if the dominant forces for adsorption are the same. Therefore, we concluded that, whereas hydrophobic interactions are the dominant force in the adsorption behavior of HSA, van der Waals forces are the main forces involved in the attachment of IgG to the lattices. °C 2000 Academic Press Key Words: dynamic light scattering; protein adsorption; polystyrene; human serum album; immunoglobulin G; cosolvent; hydrophobic interaction; electrostatic interaction.

INTRODUCTION

Protein adsorption at the solid–liquid interface has been studied extensively (1, 2). Because the adsorption mechanisms of the protein mixtures observed in biological systems are very complex and hard to elucidate, protein adsorption is generally studied by using simple systems consisting of one or more wellcharacterized proteins, a well-characterized adsorbent, and a well-defined aqueous solution. Even so, small changes in the experimental conditions, such as pH, ion strength, and temperature, generate totally different results.

1

To whom correspondence should be addressed. 25

0021-9797/00 $35.00

C 2000 by Academic Press Copyright ° All rights of reproduction in any form reserved.

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SONG AND FORCINITI

while keeping the surface properties invariant (6). These two solvents decrease the dielectric constant of water, but they have an opposite effect on hydrophobic interactions, which are the result of the presence of the nonpolar groups of the protein in water which forms a clathrate cage type structure around these groups and causes them to approach one another (10). The addition of glycerol to an aqueous suspension of latex and proteins strengthens hydrophobic interactions between protein molecules and the solid surface. In contrast, the addition of methanol weakens them. The addition of these cosolvents affects not only the amount adsorbed (protein/surface interactions are relevant here) but also lateral interactions (protein/protein interactions are relevant here). For example, Tilton et al. (6) studied the adsorption of ribonuclease A (Rnase A) on PS particles in methanol/buffer and glycerol/buffer systems. They found that both the rate and the extent of Rnase A adsorption on PS were impaired by addition of methanol while glycerol caused the reverse trend. Electrostatic interactions also play a role in protein adsorption. The surface charge of the adsorbents or the proteins can be changed either by surface modification or by changing pH. For example, pH variations change the amount of surface charge and its sign (11, 12). In our work, the effect of electrostatic interactions on protein adsorption was studied by varying the pH from 5 to 9. We have focused our efforts on the determination of the thickness of the adsorbed layer by using DLS—but a few determinations of surface concentrations were performed. The quantity measured in DLS is the autocorrelation function that can be used to determine the diffusion coefficient of the scatters. The equivalent sphere diameter (ESD) or Stokes diameter of the scatters is obtained from the diffusivity, assuming than the scatters are spherical. The difference between the diameters of the scatters before (bare PS) and after (protein + PS) adsorption is equal to twice the thickness of the adsorbed layer. Although most proteins are nonspherical, previous studies have shown that DLS is a powerful tool for studying protein structures in solution (13–18).

through a Whatman 0.2-µm polyethersulfone membrane filter. The concentrations of cosolvents in the DLS experiments were 5% (w/w) methanol and 15% (w/w) glycerol. The absorbent used was monodisperse polystyrene (PS) latex from Duke Scientific Corp. with a particle average diameter of 102 ± 3 nm (10% solids) or 309 ± 7 nm (1% solids). Because a proprietary surfactant was added to the PS suspensions by the manufacturer to keep the particles from aggregating and this surfactant affects the adsorption of protein on the surface, PS suspensions were dialyzed for 48 h to remove the surfactant before they were diluted to the required concentration with phosphate buffer at the desired pH. After sonication for 35 min, 0.005% and 0.0035% (solid content) undialyzed and dialyzed bare PS suspensions in different cosolvents and at different pH were prepared for the measurements of the 102- and 309-nm PS bare particle sizes, respectively. b. Proteins The lyophilized IgG and HSA (fatty acid free) of reagent grade were from Sigma. The proteins were used without further purification. The protein solutions were prepared freshly before each experiment. The protein concentrations were measured by UV spectrophotometry at 280 nm (14). The extinction coefficients at 280 nm IgG and HSA, together with other relevant properties, are listed in Table 1. For the protein ESD measurements, 2 mg/ml IgG and 25 mg/ml HSA at different pH and in different cosolvents were prepared in the filtered buffer. The prepared protein solutions were filtered through an MSI 0.1-µm nylon membrane filter before each measurement. Direct UV spectrometry measurements showed that protein concentrations were nearly unchanged after filtration. For the protein adsorption experiments, HSA was dissolved in phosphate buffer containing 3 mM NaN3 at pH 5.5 and ionic strength 0.016. Stock solutions containing 0.5, 1.0, 2.0, and 6.0 mg/ml protein were prepared. IgG was dissolved in a 0.01 M sodium phosphate buffer containing 0.15 M NaCl and 0.1% NaN3 at pH 7.4. Stock solutions containing 0.5, 1.0, 2.0, and 6.0 mg/ml were prepared. All stock solutions were used within 3 h of their preparation.

MATERIALS AND METHODS

TABLE 1 Molecular Parameters of IgG and HSA

a. Solvents and Absorbent All the chemicals used in this work were of analytical grade. Nanopure water was used in all the experiments. The buffers at pH 4.8, 6.9, and 9.0 were made from 0.067 M KH2 PO4 and 0.067 M Na2 HPO4 using different volume proportions and then diluting with water to 2 mM ionic strength. The pH was adjusted to the desired value with 2 mM sodium hydroxide or hydrochloric acid. Before each experiment, the buffers were filtered through a Whatman 0.45-µm polysulfone membrane filter and then mixed with methanol and glycerol. Because the solutions used in DLS particle size measurements need to be very clean and dust-free, these filtered solutions were filtered again

Isoelectric point Mwa IgG

156–161k

Literature Measured 5.8–7.3

6.9–7.3

Molecular dimensions (nm)

NPPb E 280nm d

21.9 × 15.5 × 1.5e 0.585

13.8

23.5 × 4.4 × 4.4 HSA a

69k

4.9

4.8–5.0

8 × 6.9 × 3

1.008c

5.8

Mw is the protein molecule weight (57). NPP is the ratio of the sum of nonpolar residues to polar residues (58). c At pH 5.09. d Adsorption of 1% solution, 1 cm light path at 280 nm (14). e From SANS data. b

27

PROTEIN ADSORPTION ON POLYSTYRENE LATEX BY DLS

c. Isoelectric Focusing (IEF) Experiment The pI of IgG and HSA were determined by IEF performed on a Bio-Phoresis Horizontal Electrophoresis Cell using an analytical IEF polyacrylamide thin-layer (0.8-mm-thick) gel that covers a pH range from 3 to 10. The bands were detected by the Coomassie Blue G-250 method. The pI obtained for IgG and HSA were in the ranges 6.9–7.3 and 4.8–5.0, respectively. d. Potentiometric Titration Potentiometric titrations of the PS latex were performed by the method described by Parks and de Bruyn (15). Five milliliters 0.6 vol% undialyzed and dialyzed PS suspensions were titrated with 0.1 N HCl and 0.1 N NaOH. NaCl was used as the supporting electrolyte. A series of titrations were performed at ionic strengths 0.1, 0.001, and 0, and the pH range 3–10 was covered. e. Differential UV Spectroscopy Analysis HSA and IgG were tested for possible denaturation upon the addition of methanol. The pH and concentration of HSA and IgG solutions were 5.5, 3 mg/ml and 7.4, 0.75 mg/ml, respectively. The solutions were scanned over wavelengths ranging from 250 to 320 nm using a doubled-beam spectrophotometer at room temperature. The second-order derivative spectra were calculated automatically by the spectrometer with a sensitivity of 3 and plotted against wavelength. Due to its improved resolution and precision, it is possible to monitor partial denaturation and structural changes more closely by the differential UV spectra (16). f. Protein Adsorption DLS experiments. A 1.5-ml 0.01% v/v (102 nm PS) or 0.007% v/v (309 nm PS) latex suspension was prepared in pure buffer, 10% methanol, or 30% glycerol at pH 4.8, 6.8, or 9.0 before every experiment. Just before the adsorption experiment was started, the prepared PS latex suspension was placed in an ultrasonic bath for 35 min to break up particle aggregates caused by removing the surfactant. Then 1.5 ml 3 mg/ml IgG or 2 mg/ml HSA was added to the latex suspension to make a solution of 5% methanol, 15% glycerol, or pure buffer at the appropriate pH. So, the final dialyzed PS concentrations and surface areas were 0.005%, 29.4 cm2 /ml and 0.0035%, 6.80 cm2 /ml for 102 nm and 309 nm PS, respectively; the final protein concentrations were 1.5 mg/ml and 1 mg/ml for IgG and HSA, respectively; the ionic strength in the solutions was 2 mM. The mixtures were allowed to equilibrate for 2 h before the DLS measurements. Surface concentration experiments. Only microspheres 309 nm in diameter were used for these experiments since the smaller PS particles did not produce sediment even after prolonged centrifugation. Protein adsorption experiments were performed as follows. The latex suspension was diluted to 5% w/v with nanopure water. Then 0.5 ml of the polystyrene latex (surface area 0.492 m2 ) and 0.5 ml of the protein stock solution

were added to a microcentrifuge tube. The suspensions were equilibrated in a water bath at 30◦ C with intermittent shaking for 2 h. The microspheres were separated by centrifugation at 12,400 rpm for 60 min. The protein concentration before and after adsorption was determined spectrophotometrically, and the amount of protein adsorbed was calculated by the difference. g. DLS Measurements The DLS experiments were performed as soon as the adsorption experiments were finished. The thickness of the protein layer was obtained from the difference between the particle hydrodynamic radius before and after adsorption. The DLS experiments were performed on a fiber-optic quasi-elastic light scattering (FOQELS) instrument from Brookhaven Instruments Corp (Brookhaven). The FOQELS uses an 800-nm solid-state laser (70 mW) with a fixed scattering angle 2 = 155◦ and a digital autocorrelator. The delay time intervals are not linearly spaced, which allows broad distributions to be sampled properly. The sample was poured into a 4.5-ml rectangular plastic cuvette and was placed inside a constant temperature holder which is capable of controlling the temperature in the range 5–75◦ C in steps of 0.1◦ C. The temperature was fixed at 23.9◦ C for all measurements. Each sample was run between 5 and 7 times for about 2 min each time (2 min were sufficient to obtain enough counts). The method of cumulants was used to analyze the data. Particle size can be related to the diffusivity, D for simple common shapes like spheres, ellipsoids, cylinders, and random coils. For a uniform sphere, the Stokes–Einstein relation is D=

kB T , 3π ηDh

[1]

where kB is Boltzmann’s constant, T is the temperature, η is the viscosity of the liquid in which the particle is moving, and Dh is the hydrodynamic diameter. Equation [1] assumes that the particles are moving independently of one another. With sufficient laser power (translated into count rates for photon counting, we reached anywhere from 300 to 500 kcps in our measurements), the diffusion coefficient can be determined with excellent precision, subject to sample stability during the duration of the experiment. The single-mode operation of the receiving fiber provides for a higher signal/noise ratio due to the acceptance of only one coherence area and thus provides for high efficiency at any scattering angle. When a distribution of sizes is present, the effective diameter can be obtained by averaging the intensity-weighted diameters. In FOQELS, a multimodal size distribution (MSD) analysis is available through the nonnegatively constrained least-squares (NNLS) approach developed by Grabowski and Morrison (17). h. Scanning Electron Microscopy Analysis The PS latex was spread onto a cover glass with a thickness of 0.13–0.17 mm and the solvent was evaporated for 24 h at room temperature before the dried beads were coated with a

28

SONG AND FORCINITI

thin layer of carbon (about 10 nm). SEM pictures were taken by using a Hitachi S 570 Scanning Electron Microscopy with a Kevex Delta I EDS/WDS system and SESAME automation. The particle size was determined by averaging at least 10 measurements.

RESULTS AND DISCUSSION

Because the expected thickness of the protein layer is a few nm, it is extremely important to obtain reliable and precise values for the PS beads’ sizes. SEM and DLS measurements were done to determine the particle sizes and size distributions of 102- and 309-nm PS samples. DLS measurements yield hydrodynamic diameters of 310.4 ± 0.9 and 101.9 ± 0.5 whereas SEM yields an average size of 308 ± 7 nm (the size of the smaller beads was not measured by SEM). Our results are in good agreement with the specifications of the manufacturer, which were obtained by transmission electron microscopy (309 ± 7 and 102 ± 3). The samples’ polydispersity indexes (the variance divided by the square of the average) were very low (0.005), as they should be for monodisperse or nearly monodisperse samples. In addition, the difference between the particle sizes of undialyzed and dialyzed PS in different cosolvents was within the range of instrumental error. These results fulfilled our requirements for the measurement of the hydrodynamic thickness of protein layers. Protein adsorption at solid–liquid interfaces depends on the surface charge of the proteins and the absorbent. Since persul-

fate is used as the initiator by the manufacturer of the PS latex, the charge on the beads needs to be determined. This was done by potentiometric titration. Because the polymerization procedures are the same for 102- and 309-nm PS, only the 309-nm PS was titrated. The result for dialyzed PS is shown in Fig. 1. Three supporting electrolyte concentrations were used. A sharp equivalent point around pH 7 corresponding to the strongly acidic sulfate surface groups is observed—this is in good agreement with previous reports (18, 19). No other titrable surface groups were detected on the dialyzed PS latex. The intersection point of the three curves at three different electrolyte concentrations is equal to the pH at which the charge on the latex is zero (point of zero charge, PZC) (20, 21). Since the surface group on the PS sample is a sulfate group, the PZC of a dialyzed PS is around 3. So, under the experimental conditions used in this work the particles were always negatively charged. Because the manufacturer did not mention the surfactant type in the latex, a titration in water of undialyzed PS was performed. The shape of the titration curve for the undialyzed PS sample is obviously different from that of dialyzed PS (see Fig. 1). The smooth flex point around pH 9 corresponds to a weak acid. This also shows that dialysis eliminates most of the surfactant. The removal of most of the surfactant upon dialysis of the latex has been corroborated by FT-IR in our laboratory (data not shown). Because some organic solvents can denature the proteins, absorbance and second-derivative UV spectra were obtained for IgG and HSA to check for denaturation due to the addition

FIG. 1. Potentiometric titration curves of 309-nm polystyrene latex: (–x–) dialyzed PS in 0 M NaCl; (–∗–) dialyzed PS in 0.001 M NaCl; (–▲–) dialyzed PS in 0.1 M NaCl; (–■–) undialyzed PS in 0 M NaCl.

29

PROTEIN ADSORPTION ON POLYSTYRENE LATEX BY DLS

FIG. 2. Second-order derivative UV spectra of IgG in pure buffer and buffer/methanol cosolvent at pH 7.4: (–h–) IgG in pure buffer; (–◆–) IgG in 5% methanol.

of methanol. Since glycerol is a protein stabilizer that induces preferential hydration of protein, i.e., exclusion of the cosolvent molecules from the protein surface which creates a tendency of the protein to minimize its surface (22), no test was performed for water/glycerol systems. The denaturing power of alcohols increases with chain length (23). So, methanol is the mildest alcohol that can be used. The second-derivative UV spectra for IgG with and without methanol almost overlap each other, indicating that there was negligible or no protein denaturation up to 5% methanol (Fig. 2). The same result was obtained for HSA, and these results were also supported by FT-IR experiments (data not shown). The three-dimensional structure of HSA has been resolved and analyzed by X-ray crystallography (24). It is an asymmetric heart-shaped molecule with sides of 8 nm and a thickness of 3 nm that can be roughly approximated as an equilateral triangle with an altitude of 6.9 nm. The two heart “lobes” contain HSA’s two binding sites, which consist almost exclusively of hydrophobic side chains, while the outside of the molecule contains most of the polar groups. The tip of the heart is positively charged at physiological pH. Since its binding sites are so hydrophobic, they are potential adsorption sites on hydrophobic PS. It is widely accepted that IgG is a Y-shaped molecule composed of three covalently linked compact globular domains (two Fab fragments and one Fc fragment). It can be envisioned as an ellipsoid of dimensions 23.5 × 4.4 × 4.4 nm (5). By fitting small-angle neutron scattering (SANS) data (25) using the Debye simulation method described by Perkins (26), we found that IgG can

be modeled as a Y shape with a thickness of 1.5 nm, a height of 21.9 nm, and a maximum width of 15.5 nm. Some molecular parameters of these two proteins are shown in Table 1. The hydrodynamic diameters of HSA and IgG in different cosolvents measured by DLS are shown in Tables 2 and 3. The ESDs of HSA are in the range 5.2 to 6.7 nm, which agree very well with the size of 6.3 nm reported by Sontum and Christiansen (27) and of 5.9∼8.1 nm reported by Luik et al. (28). Compared to the three-dimensional sizes shown in Table 1, the ESD represents some average over the shape as expected (29). Although it is known that serum albumin has an extended conformation below pH 4.5, there have been some contradictory reports about the change of the hydrodynamic size of serum albumin in a relatively mild pH range. Raj and Flygare (30) and Sontum and Christiansen (27) believed that no conformational change takes place over the pH range 4.5∼10.5. In contrast, Luik et al. (28) indicated that serum albumin has its most compact configuration at pH 7.4, whereas Champagne (31) suggested that the most TABLE 2 Hydrodynamic Diameter of HSA (25 mg/ml) pH

5% methanol/water (nm)

15% glycerol/water (nm)

Pure buffer (nm)

4.8 6.9 9.0

5.4 ± 0.2 5.7 ± 0.3 6.0 ± 0.4

6.7 ± 0.3 5.6 ± 0.3 5.4 ± 0.2

6.4 ± 1.0 5.3 ± 0.3 6.5 ± 0.3

30

SONG AND FORCINITI

TABLE 3 Hydrodynamic Diameter of IgG (2 mg/ml) 5% methanol/water (nm)

15% glycerol/water (nm)

Pure buffer (nm)

pH

ESD

Dp a

ESD

Dp a

ESD

Dp a

4.8 6.9 9.0

18.6 ± 0.7 22.1 ± 0.3 22.9 ± 0.8

12.3 11.0 11.8

25.8 ± 0.8 25.2 ± 1.3 29.9 ± 1.1

12.1 11.2 12.3

20.2 ± 0.7 24.2 ± 0.4 25.5 ± 0.8

12.0 10.8 12.0

a

Dp is the hydrodynamic diameter value at the highest intensity peak obtained by MSD.

compact structure exists at its pI (∼4.6). Our results indicate that a relatively constant shape persists in the pH range covered in this work. Because different shapes may yield the same Stokes diameter, small conformational changes may be undetectable by hydrodynamic measurements. The observed sizes in glycerol and methanol are nearly the same as those in pure buffer; this indicates that the addition of cosolvents does not cause a measurable alteration of the shape of HSA. The IgG ESDs measured in this work are much larger than the previously reported values (13, 32, 33). For example, Singh et al. (13) obtained 10.4 nm at 25◦ C and 23.7 nm at 50◦ C. Singh et al. interpretated the size increase as heat aggregation induced by high temperatures. Joessang et al. (32) obtained an ESD of 11 nm for monomeric IgG and of 15 nm for dimeric IgG; Liang et al. reported a value of 12 nm (33). Because our samples were filtered carefully before the DLS measurements, the effect of dust should be ruled out. The large ESD values indicate that there were some IgG dimers or higher oligomers in the samples. This tendency to aggregate shown by IgG versus the relative

stability of HSA can be interpreted in terms of preferential hydration and hydration forces. Squire and Himmel (34) found that proteins with higher hydration had lower fractions of polar residues, which was contrary to the widely accepted hydration by hydrogen bonding mechanism for organic molecules. Higher hydration causes a repulsive force as a result of the overlap of the hydration layers. Since IgG has fewer nonpolar residues than does HSA (Table 1), we expect IgG to be less hydrated and, therefore, to have a stronger tendency to aggregate than HSA. Because aggregation of IgG in solution was observed, of the MSD analysis is needed to estimate the hydrodynamic size this molecule. An MSD plot of an IgG sample is given in Fig. 3. The MSD creates a plot of intensity vs particle diameter by an NNLS algorithm. The most important information obtained from multimodal size distribution analysis is the peak position. The highest intensity-weighted peak values (Dp ) obtained by MSD analysis are shown in Table 3. These Dp values range from 10.8 to 12.3 nm, which agrees well with the reported values (13, 32, 33) for this protein. It is worth mentioning here that in all the DLS measurements of IgG solutions there were always two peaks, a large peak at 11–12 nm and a much smaller peak anywhere from 33 to 100 nm. Although the second peak is much smaller than the Dp peak, it contributes heavily to the calculated hydrodynamic diameter because large particles contribute more than small ones to the average diffusion coefficient. This feature makes DLS a sensitive method for detecting aggregation. The position of the second peak (from 33 to 100 nm) corresponds to oligomers of IgG. Although the averaged ESD values of IgG do not reveal any trend with pH changes, the Dp values at pH 6.9 in different cosolvent mixtures indicate that IgG adopted a more

FIG. 3. Multimodal size distribution of 2.0 mg/ml IgG in pH 4.8 buffer.

PROTEIN ADSORPTION ON POLYSTYRENE LATEX BY DLS

compact shape near the pI. The structure of the IgG molecule has been found to be more flexible than that of the serum albumin (35). This might explain why no compacting was found near the pI of HSA. As with HSA, we did not observe any change in the size of IgG upon addition of glycerol or methanol. To calculate the protein layer thickness on the PS beads, we assumed that the proteins were well distributed on the PS beads surface, like a hard impermeable core with a shell of closepacked protein molecules. To validate this assumption, SEM pictures (Fig. 4) were taken and the particle sizes were measured. Only the photograph of 309-nm PS with and without adsorbed IgG at pH 4.8 is shown. The PS particles without a protein layer are rounded and the edges are nearly smooth (Fig. 4a). After IgG adsorbed on the PS latex, the measured average size of particles increased by about 41 nm, which corresponds to a 20.5-nm protein layer on the surface of the beads. Figure 4b shows that uneven edges appeared around the PS beads after the protein was adsorbed, but the beads still had an approximated spherical shape. Fair and Jamieson (5) found that the

31

Stokes size was insensitive to surface coverage, and other authors also suggested that a nearly constant hydrodynamic size could be measured upon randomly removing 50% of the molecules from the surface (36–39). Therefore, the assumption that the particle remains spherical after protein adsorption is well justified. The adsorbed layer thicknesses of HSA and IgG layers on dialyzed PS latex are shown in Figs. 5a and 5b. We would like to emphasize that although we used PS spheres 309 and 102 nm in diameter, all the protein thicknesses reported in this paper were obtained with the 102-nm spheres. The reason for this is that although we can reach a precision as good as 0.3%, the reproducibility (measurement of another sample prepared from scratch) is not better than 1%. This reproducibility is too high for the thickness that we expect to measure if the larger beads are used. In contrast, a reproducibility between 0.8 and 1% in the diameter of 102-nm PS microspheres still allows for the determination of protein thickness (a few nm) with confidence. Some previously reported data on the thickness of HSA and IgG layers on solid hydrophobic surfaces obtained by different

FIG. 4. SEM photograph of the 309-nm PS latex taken at 70000 × magnification: (a) bare PS particles: (b) PS particles with absorbed IgG layer at pH 4.8.

32

SONG AND FORCINITI

FIG. 4—Continued

methods together with some of the results of this work are shown in Tables 4 and 5. These results agree with our results very well. In a separate study (40) we have determined adsorption isotherms on PS latex for these two proteins with and without the addition of methanol and glycerol at pH values close to their isoelectric points. These studies show that the addition of 5% methanol has a negligible effect on the amount of HSA or IgG adsorbed on PS latex whereas the addition of 15% glycerol produces a considerable increase in the amount of HSA adsorbed but

TABLE 4 IgG Layer Thickness on Hydrophobic Surfaces HTh a (nm)

pH

Surface

Measurement method

Ref.

18 ± 2 17 ± 5 21 ± 1 21.6 20.2 ± 0.2

7.2 × 7.4 6.7 4.8

methylated silica methylated silica PS PS PS

ellipsometry ellipsometry DLS DLS DLS

(59) (60) (5) (44)

19.7 ± 0.6

9.0

PS

a

HTh , layer thickness.

this work this work

only a marginal increase in the amount of IgG adsorbed. These results are used below to explain the trends observed by DLS. The layer thickness of HSA reaches a maximum at pH 4.8 (Fig. 5a) under all solvent conditions. Near the pI, the adsorption of many proteins reaches its maximum (8, 41) because of

TABLE 5 HSA Layer Thickness on Hydrophobic Surfaces HTh a (nm)

pH

Surface

Measurement method

Ref.

4±2 3.4 ± 1.0

7.2 7.2

methylated silica PS PS

ellipsometry

(59)

DLS

(61)

3.0 ± 1.0b 4.2 ± 0.3b 8 6.2 ± 1.7 4.9 ± 0.3

7.2 7.4 7.4 7.4 4.8

PS PS PS

DLS DLS DLS

(5) (62) (63)

PS

DLS

2.9 ± 0.3 2.1 ± 0.5

9.0 6.9

this work this work this work

a b

HTh , layer thickness. Bovine serum albumin.

PROTEIN ADSORPTION ON POLYSTYRENE LATEX BY DLS

33

FIG. 5. Dependence of the thickness of the protein layer on PS on cosolvents and solution pH: (a) HSA; (b) IgG.

the minimum solubility of the protein in the bulk, the decreased electric adsorption barrier, and the decreased repulsion in the interfacial layer (42). This maximum in adsorption correlates with layer thickness since at the isoelectric point, lateral repulsions decrease and, therefore, a more extended configuration of the layer is possible. The thickness of the adsorbed layer at pH 6.9 and 9.0 decreases considerably. That is due to the increased electrostatic repulsion between different protein molecules which makes a flatter configuration energetically more favorable. The fact that at pH 9.0 the adsorbed layer is thicker than at pH 6.9 may be due to an expansion of the protein at this very high pH as a consequence of strong electrostatic repulsions between groups in the polypeptide chain (although the same expansion was not observed with the protein in solution in this pH range). The addition of 15% glycerol produces a considerable increase in the layer’s thickness at all pH values. In contrast, the thickness of the adsorbed layer from a methanol solution is significantly reduced. At pH far from the pI, the thickness even decreases to a negligible level. The limited information that we have about the amount of protein adsorbed indicates that it does not change upon addition of methanol but increases upon addition of glycerol (at the adsorption plateau, we found 1.1 mg/m2 (pure buffer), 1.3 mg/m2 (15% glycerol), 1.1 mg/m2 (5% methanol), and 0.92 mg/m2 (10% methanol)). These amounts are below the surface concentration at complete

coverage (4.43 and 3.85 mg/m2 for a tips-on molecule and a side-on molecule, respectively). Tilton et al. (6) observed an enhanced adsorption rate of Rnase A on PS in the presence of 33% glycerol and a reduction in the presence of 25 and 50% methanol solutions. However, they found that the adsorbed amount from a glycerol solution was indistinguishable from that of pure buffer at high bulk protein concentrations and that the adsorbed amount from a 33% glycerol solution at lower bulk concentrations was even lower than the amount in pure buffer solution. They interpreted this observation as post-adsorption reconfiguration of Rnase A molecules. We have found both an increase in the amount adsorbed and an increase in the thickness of the adsorbed layer upon addition of glycerol. As discussed before, hydrophobic interactions cause highly structured water to surround nonpolar groups. Because methanol is less polar than water with a smaller dipole moment and dielectric constant (43), the nonpolar side chains of protein molecules will prefer it to water. So, methanol decreases the polarity of the solvent and most likely replaces the structured water molecules from those nonpolar groups, disrupting the clathrate structure and decreasing the hydrophobic interactions. When the protein has the same charge as the surface (both negative), the effect of methanol is enough to preclude the protein from being adsorbed. Glycerol, on the contrary, increases the affinity of the nonpolar domains of the protein for the surface.

34

SONG AND FORCINITI

FIG. 5—Continued

Because Morrissey and Han noted that the adsorbed protein could keep the native conformation on PS (44), and because our second-order derivative UV spectrometry and DLS results also showed no significant conformational changes upon addition of the cosolvents, we could interpret the changes in the thickness of the adsorbed protein under different solution conditions by using different protein orientations on the solid surface (shown schematically in Fig. 6) which result from their interactions with the solid surface. For HSA on PS, the largest thickness of 6.4 nm at pH 4.8 in glycerol solution indicates an end-on orientation (Fig. 6, b2). This end-on orientation allows more protein to deposit on the surface, explaining the observed increase in the amount adsorbed mentioned above. The protein probably binds to the surface through its hydrophobic domain III. The thickness of 4.9 nm at pH 4.8 in pure buffer indicates an orientation between side-on (Fig. 6, b1) and end-on by the effect of increased electrostatic repulsion. We call it edge-on orientation (Fig. 6, b5). Under all the other conditions, HSA shows a side-on orientation, except that at pH 6.9 and 9.0 with methanol the adsorbed layer thickness drops to nearly zero. A complete different picture emerges from our studies of IgG. First, experiments below and above the pI of this protein yield the same thickness. So, the orientation of the protein at the surface does not depend on pH. Second, the addition of cosolvents (in particular methanol) has only a minor effect on both the layer

thickness and the amount adsorbed. (We found surface concentrations in the plateau region of 2.3 mg/m2 (buffer), 2.5 mg/m2 (15% glycerol), 2.2 (5% methanol), and 2.2 (10% methanol).) The thickness at pH 4.8 in pure buffer agrees very well with the SEM data presented before. At pH 4.8 IgG is positively charged whereas at 9.0 IgG is negatively charged. Since there is almost no difference in the observed thickness at those two pH values, the adsorption site is either neutral or does not change signs as the entire molecule moves from below to above its isoelectric point (45). Figure 5b shows that the orientation of IgG at the surface is not too sensitive to the addition of methanol, but there is an increase in the thickness upon addition of glycerol. Figure 6a illustrates some possible scenarios. Because the smaller thickness observed in this work is about 20 nm, structure a3 can be eliminated. Structure a5 is very unlikely, and a4 can also been ruled out (see Table 1). It has been known that IgG prefers an end-on adsorption orientation with its Fc part (composed mostly by CH3 and -CH2 - domains; see Fig. 6, a1) onto hydrophobic surfaces (46, 47). Therefore, in the presence of methanol or in pure buffer the predominant orientation of IgG on the latex corresponds to that shown in Fig. 6, a1. IgG is less hydrophobic than HSA; therefore methanol should affect the adsorption of IgG more than that of HSA. However, we observed that the decrease in hydrophobic interactions caused by the addition of 5% methanol is not enough to inhibit its adsorption as

PROTEIN ADSORPTION ON POLYSTYRENE LATEX BY DLS

35

FIG. 6. Diagrams of protein orientations on the PS bead surface: (a) possible orientations of IgG on the PS surface; (b) possible orientations of HSA on the PS surface.

in the case of HSA. This indicates that hydrophobic interactions may not be important or that the local hydrophobicity of IgG must be considered. We need to point out that making the solvent more hydrophobic is equivalent to making the surface more hydrophilic since protein adsorption (and orientation at the surface) should be the consequence of a balance of forces between the protein and the surface and between the protein and the solvent. Our results compare well with those reported by Buijs et al. (45), who found that IgG could adsorbed on hydrophilic latex by a side-on orientation while it mainly had an end-on orientation on hydrophobic surfaces. Although we did not observe any change in the dimensions of this protein upon addition of methanol (Table 2), it is still possible that the molecule is more extended in the presence of this solvent when it is against a surface. An equally plausible explanation is that our observation is the result of an expansion of the molecule because of an increase in electrostatic repulsions due to a decrease in dielectric constant upon addition of methanol. Because the adsorption of this protein on PS does not depend on pH and because the addition of methanol did not affect adsorption, neither hydrophobic nor Coulombic forces should play a major role in the adsorption of IgG, and therefore, its adsorption is dominated by short-ranged dispersion forces. Both thicknesses in aqueous glycerol solutions exceeded the molecular dimensions of IgG, indicating that protein aggrega-

tion takes place or that a multilayer has been formed on the latex surfaces. When studying the adsorption of IgG in narrow spaces, Vroman and Adams (48) found that IgG could deposit a multimolecular layer if enough IgG per surface area was available, and this layer could be replaced by another layer of IgG if sufficient IgG remained in the bulk solution. It is apparent that the possible aggregation of IgG on the surface or the formation of multilayers depends on the composition of the solvent and that this aggregation is favored by solvents that enhance hydrophobic interactions. The formation of more than one layer does not considerably increase the amount of protein adsorbed. It is possible that a random mixture of side-on and end-on orientations exists on the surface. We think an extension of the protein in the presence of glycerol should be discarded because: (1) glycerol is known to induce more compact structures and (2) it decreases the dielectric constant of the solvent increasing electrostatic repulsions between charged groups in the Faba and Fabb domains. In this work, measurements of IgG–PS at pH 6.8 were not possible because large aggregates were found. The hydrodynamic diameter measured 15 min after the adsorption began was over 1000 nm and increased to over 2000 nm after 2 h. Bare PS latex and HSA–PS complex did not aggregate under similar conditions. Flocculation can be the result of the screening effect caused by high ionic strength; this must be ruled out because the ionic strength used in this work was low. It has

36

SONG AND FORCINITI

been known that proteins have two different effects on the colloid stability of a PS suspension. When the surface coverage is low, the adsorbed protein decreases the stability of the suspension because, if the surface coverage is not adequate, the proteins directly interact with the bare surface of two or more beads (bridging effect). When the coverage is high enough, the proteins increase the stability of the suspension because of the steric repulsion by the coated protein molecules or the socalled steric stabilization effect (49). The flocculation of IgG with PS latex has been observed by Singer et al. (50) and Morrissey and Han (44). They concluded that the flocculation resulted from the bridging effect of protein molecules between incompletely covered PS particles. The stability of the HSA–PS complex has been shown by Tamai et al.’s study (51). Because we have shown that IgG aggregates more than HSA, we believe that the difference between the surface characteristics of these two proteins contributes much to the stability of the PS suspension. In addition, according to Van Der Scheer et al., increasing the effective collision radius of a coated PS particle will increase the flocculation rate of a colloidal system (49). Walles (52) believed that longer polymer chains caused flocculation more effectively than shorter ones because it is easier for longer chains to form a bridge between particles. Therefore, an alternative explanation for the coagulation of the latex by IgG but not by HSA can be found in the different lengths and flexibilities of these two proteins. Two alternative explanations can be offered to explain the fact that flocculation occurs only at pH 6.9. The first, and obvious one, is that flocculation is caused by the low solubility of IgG near its pI. The second, and more subtle, is that flocculation at the pI of IgG occurs because at this pH the protein is in a conformation that favors bridging. As discussed above, IgG adsorbed on the hydrophobic surfaces with its Fc part, leaving the flexible Fab fragments oriented toward the bulk solution. At a pH near the pI, the protein carries an almost zero net charge, allowing the Fab domains to get closer to each other, while the net charge at other pH values will cause these domains to repel one other (53, 54) (Fig. 7). Bagchi and Birnbaum (55) suggested that adsorbed IgG has an end-on “arm-collapsed” Y orientation at the pI and an end-on “arm-extended” T orientation at pH away from the pI. So at pH 6.9, the molecule will be longer and the occupied surface area per molecule will be smaller than at the other pH values. This leads to a smaller surface coverage which facilitates the formation of bridges between particles. The decreased electrostatic repulsion between protein molecules near their pI could also help the formation of the bridge. It is necessary to mention that a different mixing method for the protein and latex solutions caused flocculation in all the samples; something similar was observed by Roberts et al. (56). In the procedure that caused flocculation, 0.03 ml 1% PS suspension was added directly into 3 ml 2 mg/ml HSA or 1.5 mg/ml IgG solution to make the same final concentration of protein and latex concentration. The only difference between this and the method described in Materials and Methods is that in the latter a diluted PS suspension was added to a protein solution whereas

FIG. 7. Different conformations of the flexible Fab fragments of IgG changed by electrostatic repulsion at different pH: (a) without charge; (b) with charge repulsion.

in the former a small volume of a highly dense PS suspension was added to a protein solution. So, high local concentration of protein molecules or PS particles causes flocculation. It is very important to remember these differences when one studies protein adsorption on latex as an absorbent because totally different results can be obtained using different mixing methods. SUMMARY

We found that IgG has a more compact structure near its pI while the size of HSA did not change from pH 4.8 to 9.0; this indicates that IgG is more flexible than HSA. Without denaturing the proteins, the addition of 15% glycerol or 5% methanol changed the interactions between the proteins and the surface significantly. In a glycerol solution, multilayers or molecular aggregation at the surface of IgG was detected. Immediate flocculation of the colloidal system occurred for IgG at pH 6.9. Incorrect sample preparation procedures also caused flocculation of the protein-covered PS beads, indicating the importance of the sample preparation procedure. At the pI of HSA, protein adsorption reached its maximum. IgG showed an end-on orientation on the PS surface whereas the surface orientation of HSA was sensitive to the solvent properties. We can conclude that both hydrophobic and electrostatic interactions have important effects not only on the amount of protein adsorbed but also on protein orientation on the surface. Therefore, we do not think that statements like “hydrophobic interactions are the main driving force for adsorption” can be justified. IgG is at least one exception. ACKNOWLEDGMENTS We are grateful to the Whitaker Foundation for its financial support and to Dr. Stephen J. Perkins of the Royal Free Hospital School of Medicine, London, for kindly providing the Debye modeling program.

PROTEIN ADSORPTION ON POLYSTYRENE LATEX BY DLS

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