Molecular Interactions Between Proteins and Synthetic Membrane Polymer Films Frederic Pimet,+Eric Perez,? and Georges Belfort*

Langmuir 1995,11, 1229-1235 1229

Molecular Interactions between Proteins and Synthetic Membrane Polymer Films Frederic Pimet,+Eric Perez,? and Georges Belfort*

Howard P. Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180-3590 Received August 27, 1993. In Final Form: December 5, 1994@

To help understand the effects of protein adsorption on membrane filtration performance, we have measured the molecular interactions between cellulose acetate films and two proteins with different properties (ribonucleaseA and human serum albumin) with a surface force apparatus. Comparison of forces between two protein layers with those between a protein layer and a cellulose acetate (CAI film shows that, at high pH, both proteins retained their native conformation on interacting with the CA film while at the isoelectric point (PI) or below the tertiary structure of proteins was disturbed. These measurements provide the first molecular evidence that disruption of protein tertiary structure could be responsiblefor the reduced permeation flows observed during membrane filtration of protein solutionsand suggest that operating at high pH values away from the PI of proteins will reduce such fouling.

Introduction the solution-membrane interface.14-18 One of the most During the filtration of protein-containing solutions with studied model protein systems for understanding mem- pressure-driven membrane processes, the protein mol- brane polarization and fouling has been BSA solutions. ecules oRen adsorb onto and into the membranes. Besides For example, fluxes are higher when the pH is away from this adsorption process, exposure to fluid shear stresses the isoelectric point (PI) of the pr0tein,2~~~~J~J~when little can also denature the protein molecules rendering des- salt is present,1° and when the surface chemistry of the orption difficult if not impossible. Adsorption of protein membrane is hydr0phi1ic.l~ molecules in the membrane pores will affect the mean In order to help understand these effects, direct pore size and pore-size distribution available for the flow measurements of the intermolecular forces between and, hence, the permeation and retention characteristics selected model proteins, such as ribonuclease A (RNase of the membrane.’ A) and human serum albumin (HSA), and a widely used Much work has been reported on membrane filtration synthetic membrane material, cellulose acetate (CA),have with solutions containing macromolecules such as proteins been measured. ARer describing the procedures used with [bovine serum albumin (BSA), ovalbumin and lactoglo- the surface force apparatus, details are presented on the bulins], poly(viny1 alcohol),polyethylene glycol, dextrans, proteins and the celluloseacetate material. The properties and others. The decline of flux with time or total of CA films and those of two adsorbed HSA layers are first integrated volume permeated was initially explained as characterized by force measurements. Then, results membrane compaction. However, it soon became clear consisting of the direct measurements of the intermo- that different rates of flux decline occurred with different lecular forces between HSA and CA, and RNase A and CA solutions under similar experimental conditions. are presented. Several phenomena have been proposed to explain this. They include (1)protein adsorption, aggregation, and gel (8) Nabetani, H.; Nakajima, M.; Watanabe, A.; Nokao, S.; Kimura, formation, due to the effect of pH and ionic strength on S. Effects of osmotic pressure and adsorption on ultrafiltration of the solute-solute and solute-membrane interaction^,^-'^ ovalbumin AIChE J. 1990,36 (6), 907-915. (9) Blatt, W. F.; Dravid, A.; Michaels, A. S.; Nelson, L. Membrane and (2) reduced driving force caused by the effect of the Science & Technology; Flinn, J. E., Ed.; Industrial Biological Waste osmotic pressure due to the buildup of macromolecules at Treatment Processes; Plenum Press: New York, 1970. (10) Fane, A. G.; Fell, J. D.; Waters, A. G. Ultrafiltration of protein solutions through partially permeable membranes-the effect of adsorp- * Corresponding author. tion and solution environment J. Membr. Sci. 1983, 16, 211-224. Laboratoire de Physique Statistique, Ecole Normale Superieure, (11) Nakao, S.; Nomura, T.; Kimura, S.; Characteristics of macro- 24 rue Lhomond, 75005 Paris, France. molecular gel layer formed on ultrafiltration tubular membrane AIChE *Abstract published in Advance ACS Abstracts, February 15, J. 1979,25,615-622. 1995. (12) Nakao. S.;Kimura, S. Analysis of solutes rejection in ultrafil- (1) Belfort, G.; Pimbley, J. M.; Greiner pi.; Chung, K-Y. Diagnosis of tration J. Chem. Engr. Jpn 14 (1),-1981, 32-37. membrane fouling using a rotating annular filter. 1. Cell culture media. (13) Tirmizi, N. P., Study of UF of macromoleculars solutions Ph.D. J. Membr. Sci. 1993, 77, 1-22. thesis, Department of Chemical Engineering, RPI, Troy, NY, 1990. (2) Fane, A. G.; Fell, C. J.; Suzuki, A. The effect of pH and ionic (14) Goldsmith, R. L. Macromolecular ultrafiltration with mi- environment on the ultrafiltration of protein solutions with retentive croporous membranes Znd. Eng. Chem. Fundam. 1971,10,113-120. membranes J. Membr. Sci. 1983,16, 195-210. (15)Leung, W. F.; Probstein, R. F. Low polarization in laminar (3) Mattiasson, E. Role of macromolecular adsorption in fouling of ultrafiltration of macromolecular solutions Id.Eng. Chem. Fundam. ultrafiltration membranes J. Membr. Sci. 1983, 16, 23-36. 1979,18 (3), 274-278. (4) Nyst”, M., Laatikainen, M., Turku, M.; Jarvinen, P. Resistance (16) Viker, V. L.; Colton, C. K.; Smith, K. The osmotic pressure of to fouling accomplished by modification on ultrafiltration membranes concentrated protein solutions: effects of concentration and pH in saline Prog. Colloid Polym. Sei. 1990, 52, 321-329. solutions of bovine semm albumins J. Colloid Interface Sci. 1981, 79 (5) McDonogh, R. M.; Bauser, H.;Stroh, N.; Chmiel, H. Concentration (2), 548-566. polarization and adsorption effects in cross-flow ultrafiltration of proteins (17) Jonsson, G. Boundary layer ofphenomena duringultrafiltration Desalination 1990, 79, 217-231. of dextran and whey protein solutions Desalination 1984,51, 61-77. (6) Robertson, B. C.; Zydney, A. L. Protein adsorption in asymmetric (18) Wijmans, J. G.; Nakao, S.; Smolders, C. A. Flux limitation in ultrafiltration membranes with highly constricted pores J. Colloid ultrafiltration: osmotic pressure model and gel-layer model J. Membr. Interface Sci. 1990,134 (2), 563-575. Sci. 1984,20, 115-124. (7) Meireles, M.; Aimar, P.; Sanchez, V. Albumin denaturation during (19) Hannemaaijer, J. H.; Robertsen, T.; van der Boomgaard, Th.; ultrafiltration: effecta of operating conditions and consequences on Olieman, C.; Both, P.; Schmidt, D. G. Characterization of cleaned and membrane fouling Biotechnol. Bioengr. 1991,38,528-534. fouled ultrafiltration membranes Desalination 1988, 68, 93-108. 0743-746319512411-1229$09.00/0 0 1995 American Chemical Society 1230 Langmuir, Vol. 11, No. 4, 1995 Pincet et al. Materials and Methods Surface Force Apparatus (SFA). The technique involves the direct measurements of the intermolecular forces between 2.5 I two separate layers; one, an adsorbed protein layer, and the other, Y 1...... 1...... 1...... ' ...... a film of cellulose acetate or adsorbed protein. Each layer is e hi deposited onto a smooth mica half-cylinder. The forces are 2 a obtained as a function of separation distance, which is measured ...... with an interferometry technique.20s21 It is the same method as 1 1.5 k that used to elucidate the intermolecularforces and conformation 1.5 -(d of adsorbed molecules between mica surfaces covered with ...... 8 surfactant,22-24 polymers,25-30 lipid mono- and bilayers,31-35 --+.) le -1 I proteins,36-@and glycolipids.44 The mica surfaces were immersed 2 E c4 (20)Israelachvili, J. N.; Adams, G. E. Measurementaofforcesbetween 3 0.5 0.5 \ two mica surfaces in aqueous electrolyte solutions in the range 1-100 0 5 10 15 20 25 nm J. Chem. SOC.Faraday Trans. 1 1978, 74,975-1001. (21)Israelachvili, J. N. Measurements of forces between surfaces Adsorption time, t (h) immersed in aqueous electrolyte solutions Faraday Discuss. Chem. Soc. 1978,65,20-24. Figure 1. Amount ofhydrated RNase A adsorbed and fractional (22)Israelachvili, J. N.;Pashley, R. M. The hydrophobic interaction surface coverage on mica as a function of time. The solution is long-range, decaying exponentially with distance Nature 1982,300, contained a RNase A concentration of 0.1 mg/mL in M 341 -343. KC1, pH 5, and at 20 f 0.5 "C. (23)Israelachvili, J. N.; Pashley, R. M. Measurement of the hydrophobic interactions between two hydrophobic surfaces in aqueous solution J. Coll Intf. Sci 1984,98, 500-514. in aqueous electrolyte solution within a small Teflon bath. A (24)Pashley, R.M.; McGuiggan, P. M.; Ninham, B. W.; Evans, D. F. M KCl solution was used in all experiments unless otherwise Attractive forces between uncharged hydrophobic surfaces: direct stated. During each experiment, the KCl concentration and the measurements in aqueous solution Science 229, 1088-1089. pH value were kept constant. The pH value was, however, varied (25)Israelachvili, J. N.; Tirell, M.; Klein, J.; Almong, Y. Forces from one experiment to the next. A lo+ M KCl concentration between two layers of adsorbed polystyrene immersed in cyclohexane corresponds to a 3 nm Debye decay length. We therefore below and above the e temperature Macromolecules l9f34, 17, 204- 209. considered 3 nm as a reference for characterizing the measured (26)Klein, J. Forces between mica surfaces bearing layers of adsorbed forces. If the observed decay length (A) was much larger than polystyrene in cyclohexane Nature (London), 1980,288,248-250. 3 nm, then long-range steric interactions were likely. Overlap- (27)Klein, J.; Luckham, P. F. Long-range attractive forces between ping denatured proteins were mainly responsible for these steric two mica surfaces in an aqueous polymer solution Nature (London), forces, while electrostatic interactions were shorter-ranged. 1984,308,836-837. Alternately, if A was close to 3 nm, then electrostatic forces (28)Klein, J.; Luckham, P. F. Interactions between proteins and synthetic polypeptides adsorbed at solid-liquid interfaces Colloid Surf. dominated. At pH 2 and 10-2 M KC1 concentration, the electrolyte 1984,10,65-76. concentration was 2 x M. This corresponded to a Debye (29)Claesson, P.; Golander, C. Direct measurements of steric decay length of 2.2 nm rather than the 3 nm. The room interactions between mica surfaces covered with electrostaticallybound temperature was controlled at 20 f 0.5 "C, and all the solutions low molecular weight polyethylene oxide J. Coll. Zntf. Sci. 1987, 117, were in thermal equilibrium with room temperature for at least 366-374. 6 h before injection in the Teflon bath. (30)Klein, J. Long-ranged surface forces: The structure and dynamics of polymers at interfaces Proc. Appl. Chem. 1992, 64, 1577-1584. Proteins. Two proteins with different properties were (31)Marra, J. Controlled deposition of lipid monolayers and bilayers chosen: ribonuclease A (RNase A) and human serum albumin onto mica and direct force measurements between galactolipid layers (HSA). RNase A is a small stable protein while HSA is large and in aqueous solutions J. Coll. Intf. Sci. 1985, 107, 446-458. relatively easy to denature. RNase A was purchased from (32)Marra, J.; Israelachvili, J. N. Direct measurements of forces Pharmacia Biotech. (RNase A I "A"). The dimensions of a between phosphatidylcholine and phosphatidylethanolamine bilayers hydrated folded RNase A molecule are 2.8 x 3.4 x 4.4 nm and in aqueous electrolyte solutions Biochemistry 1986,24,4608. (33)Chen, Y. L.; Helm, C. A.; Israelachvili, J. N. Measurements of its molecular weight is 13 690 Da. Since the pH was the main the elasticproperties ofsurfactant and lipid monolayershngmuir 1991, variable used in this study, it is important to note that the PI 7.2694-2699. of RNase A is 9.2. Previously unpublished adsorption measure- (34)Helm, C. A.;Knell, W.; Israelachvili,J. N. Proc. Nat'l. Acad. Sci. menta by Cheng Sheng Lee of our laboratory between hydrated 1991,88,8169-8173. RNase A and mica at a solution concentration of 0.1 mg/mL in (35)Leckband, D. E.;Israelachvili, J. N.; Schmitt, F. J.; Knell, W. KCl, pH and 20 "C are summarized in Figure Long-range attraction and molecular rearrangements in reador-ligand M 5, at f 0.5 interactions Science 1992,255,1419-1421. 1.& The method used to obtain these results was based on a (36)Perez, E.; Proust, J. Forces between mica surfaces covered with measurement of the variation of refractive index in the SFA adsorbed mucin across aqueous solutions J. Coll. Intf. Sci. 1987,118, between the two mica surfaces as a function of adsorption time 182-1991. and separation distance together with the appropriatecalibration (37)Lee, C. S.;Belfort, G. Changingactivity ofribonucleaseAduring curves. The total estimated error in determining the adsorbed adsorption: A molecular explanation Proc. Nat'l. Acad. Sci. 1989,86, amounts was *lo%. AIthough the amount adsorbed increases 8392-8396. (38)Belfort, G.;Lee, C. S. Attractive and repulsive interactions with time, the fractional coverage appears to remain constant between and within adsorbed ribonuclease A layers Proc. Nat'l. Acad. at about 100%at and aRer 1 h adsorption. The reason for this, Sci. 1991,88,9146-9150. as confirmed by the SFA measurement^,^' is that the RNase A (39)Blomberg, E.; Claesson, P. M.; Wlander, C. Adsorbed layers of molecules change their orientation within a monolayer coueruge human serum albumin investigated by the surface force technique J. on the surface with time from side on to end on, thus allowing Disp. Sci., Technol. 12, 1991,(2) 179-200. more protein to adsorb and resulting in an increased protein (40)Malmsten, M.; Blomberg,E.; Claesson, P.; Carlsted, I.; Ljusegren, I. Mucin layers on hydrophobic surfaces, studies with ellipsometry and density at the mica interface with time. surface force measurements J. Coll. Zntf. Sci. 1992,151,579-590. HSA was purchased from Sigma (HSA#3782). Hydrated HSA (41)Fitzpatrick, H.;Luckham, P. F.; Eriksen, S.;Hammond, K. Bovine is heart-shaped, i.e. it can be approximated to a solid equilateral serum albumin adsorption to mica surfaces Colloid 6 Surfaces 1992, triangle with sides of about 8.6 nm, a base length of about 8.1 64, 43-49. nm, and an average depth of 3.6 nm.46 Its molecular weight is (42)Kekicheff, P.; Ducker, W. A.; Ninham, B. W.; Pileni, M. P. Multilayer adsorption of cytochrome C on mica around isoelectric pH 68 500 Da and its PI is 4.8. Another difference between the Langmuir 1990,6,1704-1708. (43)Leckband, D.; Israelachvili, J. N. Molecular basis of protein (45)Lee, C. S. Direct measurements ofintermolecular forces between function as determined by direct force measurements Microb. Technol. ribonuclease A layers sorbed onto mica. Thesis submitted in partial 1993,15,450-459. fulfillment of Ph.D., Department of Chemical Engineering, Rensselaer (44)Luckham, P.; Wood, J.; Swart, R. The surface properties of Polytechnic Institute, Troy, NY,1988. gangliosides 11. Direct measurements of the interaction between bilayers (46)He,XM.; Carter,D. C.Atomicstructureandchemistryofhuman deposited onto mica surfaces J. Coll. Zntf. Sci. 1993,156, 173-183. serum albumin Nature 1992,358,209-215. Molecular Forces between Proteins and Polymer Films Langmuir, Vol. 11, No. 4, 1995 1231 .

Gloss cross-eylinder CH~OAC OAc covered with mica and Glass cross-cylinder then cellulose acetate covered with mica '-31 film

Cellulose acetate [Ac = -COCH3] Figure 3. Chemical structure of cellulose acetate (CA).

I 1 pressured onto the newly formed CA fdm on the mica immediately Teflon bath 2 Protein solution after dipping. The local smoothness of the film was satisfactory Figure 2. Schematic showing the two cross-mica cylinders for for SFA measurements since, by changing somewhat the position (a) one surface covered with the protein solution and for (b) of the contact surface, the thickness of the film did not vary both surfaces covered with the protein. In part a this ensured significantly. Unfortunately, the reproducibility of this approach that protein was not adsorbed onto the upper surface. (with respect to thickness) was relatively poor; the film thickness could vary from 20 to 200 nm This large variation in thickness made interpolation of the distance measurements difficult. properties ofthese two proteins is their adiabatic compressibility However, since the refractive index of CA (1.4 f 0.1,5measure- values in water at 25 "C. This parameter is qualitatively used ments) was close to that ofwater, we could delineate the results to distinguish between rigid (Bll = 1.12 x 10-l1 N/m2 for RNase with a three-layer model (mica-CMwater-mica). Several A) and 5exible (B, = 10.5 x 10-l1N/m2 for bovine serum albumin) properties of CA membranes were relevant for this work. First, proteins.*T Blomberg et al. (Figure 1 of ref 39) have published under most conditions, the surface charge was negative due to adsorption isotherm data for HSA. From this figure of Blomberg the presence of disassociated carboxyl groups. At pH values et al. and the data in Figure 1shown here, it is clear that, for lower than 4.5, the surface potential can switch to a positive solution protein concentrations of 5 x mg/mL used in this value.49 Vos et aL50 measured the hydrolysis rate of Cellulose study, adsorbed monolayers of the same dimension as the native acetate (39.8%acetyl content) at different temperatures (23-95 proteins lying side on are expected. "C)asafunctionofpH(2.2-10). Theserateswereat aminimum Three types of experiments were conducted. In the first type at the PI (around 4.5). Second, CA films can be hydrolyzed to of experiment, one mica surface was covered with cellulose acetate cellulose at high pH values. At pH values lower than 9, the (CA) and the other was left bare. In the second type of experiment, hydrolysis rate was lower than S-I.~For this range of pH both mica surfaces were covered by adsorbed HSA. A similar (2-91, the films were not significantly hydrolyzed during the experiment for RNase A has been reported in the literat~re.~~~~~experiment. However, some measurements were conducted at In the third type of experiment, one mica surface was covered pH 11. For these cases, the hydrolysis process must be accounted with CA and the other covered with protein. The first step of the for. adsorption procedure was to inject the protein solution (always 5 x 10-3 mg/mL) into the SFA, the protein being in its native ReSultS state ( Figure 2). Since the adsorption rate depended on the particular protein, the time to reach saturation was protein Forces between a Cellulose Acetate (CAI Film and dependent. For each protein, however, the adsorption time was Mica. Compression of CA films resulted in compaction kept constant for all experiments. It has already been shown of the membrane pore structure. The membrane decom- that, with RNase A, an adsorption time of about 20 h provided pressed slowly and needed several hours to return to its full and dense coverage of the mica sheet^.^^^^^ For HSA, the problem was more complicated since it tended to aggregate. We original thickness. This, in turn, resulted in a hysteresis conducted several experiments with HSA adsorbed onto both effect in the normalized force-distance curves and surfaces. The results showed that if HSA adsorbed for 3 h or complicated the measurements. Because of membrane more, a strong repulsive force appeared when the mica surfaces compaction, the force measurements during compression were far apart. This suggestedthat aggregation ofHSAoccurred did not show the real interactions between the materials between the mica sheets. We therefore chose to restrict adsorp- being studied. Thus, only force measurements during tion to 2 h and considered that this time was sufficient for decompression were used. These measurements were monolayer coverage of HSA. mer adsorption was completed, highly reproducible. To handle this problem, we chose to the protein solution was flushed three times with M KCl compress each membrane down to a constant minimum solution. Then, the final protein-free KC1 solution M)was injected into the SFA. The pH of these KC1 solutions was always thickness for all the runs in a given experiment so that kept the same as the pH of the protein solution. For each protein, the minimum distance to the mica was always the same experiments were conducted at three different pH values: above, (at normalized forces, > 10 mN/m). Then, all the decom- below, and at the PI. Note that, though mica is a negatively pression curves could be superimposed. Here, only the charged material under the conditions used here, proteins, decompression curves will be shown. because they are amphoteric and contain hydrophobic patches, The interactions between CA and mica have been can adsorb even above their PI (Le. when the net charge is conducted at three different pH values, 2,5, and 11. The negative). results are given in Figure 4. The curves are very similar, Cellulose Acetate. As one of the most widely used synthetic and for the purpose of comparison with the forces between membrane materials for pressure-driven membrane processes, cellulose acetate (CA) is an appropriate choice for this study. Its proteins and CA films, they can be considered identical chemical structure results in a relatively hydrophilic surface at all pH values. Thus, the membrane behaved like a (Figure 3). For this study, cellulose acetate with a degree of smooth solid wall. Besides the wall-like stericforce,hardly substitution of 2.5 (40.3% acetyl content) was used. It was any other forces were observed except for very weak prepared by dipping the mica directly into a pure acetone solution electrostatic forces at long distances ('2 nm) that were of cellulose acetate (10-4 wt %) as per Riley et al.48 In order to possibly due to the low charge density on the CA film. The reduce the thickness of the adsorbed film, nitrogen gas was (49)Heyde, M. E.;Peters, C. R.; Anderson, J. E. Factors influencing (47)Gekko, R; Hasegawa,Y. Compressibility-structure relationship reverse osmosis rejection of inorganic solutes from aqueous solution J. of globular proteins Biochemistry 1986,25,6563-6571. COX Intf. Sci. 1975,50, 461-487. (48)Riley, R. L.; Landsdale, H. K.; Lyons, C. R.; Merten, U. (50)Vos, K. D.;Bums, Jr., F. 0.; Riley, R. L. Kinetic study of the Preparation of ultrathin reverse osmosis membranes and attainment hydrolysis of cellulose acetate in the pH range of 2-10 J. Appl. Polym. of theoretical salt rejection J. Appl. Polym. Sei. 1967,11,2143-2158. Sci. 1966,10,825-832. 1232 Langmuir, Vol. 11, No. 4, 1995 Pincet et al.

10:

b b h 00 E A 0 % 1: 'A 'A

0.1:

0901 I 0.01 1,.I .I,,,\ ;, , , : i 0246810 0 20 40 60 80 100 Distance (nm) distance (nm) Figure 4. Normalized force versus separation distance between Figure 5. Normalized force versus separation distance between a deposited film of cellulose acetate (on mica) and mica in two adsorbed human serum albumin protein layers in a M KC1 solution at pH 2 (open diamonds),pH 5 (filled triangles), M KC1 solution at pH 2 (open diamonds), pH 4.8(filled circles), and pH 11 (open squares). The distance is that between the and pH 9 (filled triangles).The lines are the theoretical Debye compressed cellulose acetate film and mica. The line is the decay lengths (full line for pH 4.8 and 9, dashed line for pH 2). theoretical Debye decay length (3 nm). Only decompression The differencebetween the measured slope and the Debye decay results are shown. length is due to the steric interactions.The distance was that between the two mica surfaces. Compressions and decompres- amplitude of these electrostatic forces was relatively small sions were superimposable. since, as will be observed later, forces about 10 times Table 1. Summary of HSA-€€SA Interaction Results in stronger than this were measured between proteins and M KC1 the CA surface. Thus, besides the wall-like effect, there were no other observable steric interactions between CA theoretical thickness of and mica. The expected Debye decay length (3 nm) was Debye decay measured decay adsorbed length (nm) length (nm) layer (nm) observed at pH 11 for long distances in M KC1. This PH suggests that the CA film was very smooth and few, if 2 2.2 5.6 4.0 any, polymeric chains extended out from the surface. Thus, 4.8 (PI) 3.0 21.6 10.2 a dense CA membrane exhibited highly reproducible and 9 3.0 3.7 4.0 simple behavior. Moreover, as already mentioned, mem- small, was significant, suggesting that weak steric in- brane hydrolysis could have changed the nature of the teractions were involved. At such low pH values, proteins surface at pH 11. These curves show that, at room are known to denature,5l or form a molten globular state.52 temperature and on the time scale of the experiment (5-8 When denatured or even partially denatured, the gyration h), hydrolysis is not significant. To conkthis, a CA radius of a protein is increased. This denaturation of the film was stored in a KC1 solution at pH 11 for 20 h at 20 proteins could explain the observed steric interactions. "C. This time is close to the time scale on which hydrolysis For this case, at large normalized forces, a thickness of was relevant.50 The normalized force-distance results about 8 nm is equal to about two adsorbed hydrated HSA before and after the 20 h exposure to high pH were layers (one flat-on monolayer on each mica surface; see markedly different (not shown). For the latter experiment, Figure 5). the decay length was 10 times higher than that measured Above the PI (Figure 5, filled triangles), the force- in the former experiment. Clearly, strong electrostatic distance profile showed a decay length in agreement with interactions appeared after a sufficient time, suggesting DLVO theory (3nm). The thickness of each of the adsorbed that the hydrolysis resulted in a partial degradation of layers was 4.0 nm, which suggests that the hydrated the polymer. These broken polymer chains were able to molecules were lying side on at the interface in their native extend into the solution. An alternative explanation could state. be delamination of the membrane. However, the results At the PI, however, steric forces were significant. The in Figure 4 show that for the conditions of the force difference between the results at the PI and at other pH experiments, the membrane is not significantly hydrolyzed values is interesting. The behavior at the PI was confirmed even at pH 11. by a measurement of the interactions between HSA and Forces between '.boHSAAdsorbed Layers. Three a bare mica surface (not shown) in which a long-range pH values were chosen: 2, 4.8, and 9. In all cases, the steric repulsion was also measured. The long range-force interactions were repulsive and approximately exponen- suggests that a fraction of HSA molecules were partially tial. The thicknesses of the adsorbed HSA layers were unfolded. The thickness of each of the adsorbed layers obtained by measuring the distance between the two mica was much larger than that measured above or below the surfaces when the normalized force was 4-5 mN/m at PI. low and high pH values ( Figure 5 and Table 1). Steric Interactions between HSAlayers have also been studied interactions could also be estimated by subtracting the by Blomberg et al.39 However, their protocol was some- theoretical DLVO profile from the measured force- distance profiles. (51)F'incet, F.; Perez, E.; Belfort, G. Do denatured proteins behave like polymers? Macromolecules 1994,27,(12) 3424-3425. Below the PI, the difference between the measured and (52)Alonso,D.O.V.;Dill,K.A.;Stigter,D. Thethreestatesofglobular the theoretical (DLVO) force-distance profile, although proteins: Acid denaturation Biopolymers 1991,31, 1631-1649. Molecular Forces between Proteins and Polymer Films Langmuir, Vol. 11, No. 4, 1995 1233

3Y B b LL 34 \

0.01 0 20 40 60 80 100 0 20 40 60 80 100 Distance (nm) distance (nm) Figure 7. Normalized force versus separation distance between Figure 6. Normalized force versus separation distance between a deposited film of cellulose acetate and an adsorbed ribonu- a deposited film of cellulose acetate and an adsorbed human clease A protein layer in a M KC1 solution at pH 5 (filled serum albumin protein layer in a M KC1 solution at pH circles), pH 9.2 (open diamonds), and pH 11 (filled triangles). 2 (filled circles), pH 4.8 (open diamonds), and pH 9 (filled The full lines are from fits to the data with the values given triangles). The full lines represent the curves obtained with in Table 3. The distance was that between the precompacted the values given in Table 2. The distance was that between the cellulose acetate film and a compressed layer of ribonuclease precompacted cellulose acetate film and a compressed layer of A. Only decompression is shown. human serum albumin. Only decompression is shown. Table 2. Summary of HSA-CA Interaction Results in M KCl what different from that used in this study. They force at conducted their adsorption and SFA measurements at the knee decay length (nm) pH 5.5 and M NaCl, while we varied pH values (2, PH (mN/m) short-range long-range 4.8, and 9) and used M KC1. All our measurements 2 0.5 7.4 60 were with 5 x mg of HSNmL, while they varied HSA 4.8 (PIof HSNCA) 1 4.7 21 concentration(O.OOl,O.Ol,and 1 mgofHSA/mL). Another 9 1.2 0.6 12 important difference,however, is that they conducted their direct force measurements in the protein solution and Table 3. Summary of RNase A-CA Interaction Results in varied the protein concentration while in our experiments M KCl the protein solution was replaced with a pure KC1 solution force at the knee decay length (nm) (with the same ionic strength as the protein solution) at different pH values. This may explain why the results PH (mN/m) short-range long-range presented here are slightly different from theirs. As 4.8 (PIof CA) 7 3.9 14 mentioned above, we obtained an adsorbed layer thickness 9.2 (PIof RNase A) <0.01 1.2 11 (0.01 1.1 of 4.0 nm, while they report 2.3 nm for the thickness of a layer at the PI at mg of HSNmL. They explain Table 4. Type of Interactions Observed for Each their result by suggesting that the adsorbed protein has Experiment, at, above, and Below the Isoelectric pH of been compressed below its free solution dimension. the Proteins“ Forces between Protein Layers and a CA Film. PH < PI pH = PI PH ’PI For the two proteins, HSA and RNase A, only repulsive e- st e- st e- st forces were measured. For HSA, the absence of attraction CA-mica 0 0 0 o+ 0 was confirmed by a measurement of the adsorption HSA-HSA + +O ++ 0 isotherms (not shown) at 20 & 0.5 “C. From UV absorption HSA-CA 0 +o + +(?I + spectroscopy of the solution, no detectable adsorption was RNaseA-CA 0 +o 00 0 measured between HSA and the CA membranes over e- represents electrostaticinteractions. st represents long-range several hours. To compare the results at the different pH steric interactions. + means that the indicated interaction was values, three parameters were used to characterize the involved in the experiment. 0 means that the indicated interaction repulsion: in the semilog plots the data followed two lines was not involved in the experiment. of different slopes; and the force at which the change in involving one layer of native RNase. For HSA-CA slope occurred is called the “force at the knee”. interactions, long-range steric forces were always ob- The normalized force between adsorbed HSA and the served. CA film is given in Figure 6 and Table 2. We found that the measured decay lengths decreased with increasing Discussion pH values. Similar behavior was observed for the Only repulsive forces were observed between CA films interaction between RNase A and a CA film ( Figure 7 and protein layers. These repulsive forces are likely due and Table 3). The main difference between the force- to steric interactions. For each experiment, the kind of distance results for the two proteins is that, at high pH force involved is shown in Table 4. The CA-mica results values (9.2and ll), the force between RNase A and a CA indicated that the CA membrane was stable at all pH film was dominated by short-range steric interactions values and that polymer chains did not extend from its 1234 Langmuir, Vol. 11, No. 4, 1995 Pincet et al. surface into the solution. Therefore, any steric forces have Table 6. Type of Charge on Each Materiala to be attributed to the proteins and not to the CA film. PH HSA CA RNase A This point is crucial, as steric interactions were then caused by the denatured protein molecules. 2 + + NS 4.8 0 0 + At low pH, for the HSA-HSA, HSA-CA, and RNase 9.2 - - 0 A-CA experiments, steric interactions were observed, 11 NS - indicating that at least some of the proteins were a The PI values for the HSA, CA, and RNase A are approximately denatured. No steric forces and thus no denaturation 4.8, 4-5, and 9.2, respectively. Positively, negatively, and net was observed when forces between two adsorbed RNase neutrally charged surfaces are denoted by +, -, and 0, respectively. Alayers were measured at the same low pH.38 This implies Doublely charged materials are strongly charged. The cases that that the CA film caused RNase A denaturation during were not studied are denoted by NS. the experimental process. It should also be noted that, even without the CA film, some HSA molecules were more easily. Thus, attraction was likely present for HSA, denatured well below the PI.^^ Unfolding of the proteins but because a net repulsive force was observed, steric could have been enhanced by the compression-decom- interactions were dominant. pression of the two mica-covered surfaces. It may even have been possible that a few protein molecules could Conclusion have anchored into the surface pores of the membrane. The main conclusions of this study are (i) at low pH If the fraction of protein molecules involved in bridging values HSA and RNase A molecules are denatured (i.e. was low, the expected attraction during separation of the would induce membrane fouling); (ii) at high pH values, surfaces would have been too small to detect. This could the proteins were less denatured than at lower pH values explain why attractive forces were not observed for these (i.e. would be less likely to foul); and (iii) HSA was more measurements. Also, at these low pH values, proteins easily denatured and interacted more strongly with CA are known to denat~re.~~-~~Therefore, in a pressure- membranes than RNase A. Previous work has shown driven membrane process, as soon as the adsorbed proteins that (i) proteins were susceptible to denaturation at low are exposed to low pH and fluid shear stresses, they pH values62-s6and (ii)RNase A was more conformationaly denature and block the pores. stable than HSA during adsorption on polystyrene latex, At high pH values, however, hardly any steric forces i.e., HSA was denatured more easily at interfaces and were measured for RNase A, suggesting that the proteins was more sensitive to pH changes than RNase A.59 It is were not denatured. For HSA, the protein still denatured also at the PI that more intense fouling occurs with but less so than at lower pH values. Apparently, at these hydrophobic membrane~.~J~We believe that these results pH values and ionic strengths, the presence of a CA film explain the observations of Fane et al.,2who obtained for did not significantly affect the protein structure. There- a 0.1% BSA solution with no salt and with 0.2 M NaCl fore, the CA film did not interact with the protein during higher fluxes at pH values above the PI of BSA (at pH the force measurement in the same way as at low pH. values of 10) [see their Figure 71. Thus, these measure- Adsorption of the protein on CA was not observed, ments provide the first molecular evidence that disruption suggesting that significant membrane fouling would not of the protein tertiary structure could be responsible for be expected at these high pH values. the reduced permeation flows observed during membrane At the PI of the protein, the HSA-CA force-distance filtration and suggest that operating at high pH values diagram showed long-range steric interactions while the away from the PI of the proteins will reduce such fouling. RNase A-CA interactions were mainly short-range. Also, we suggest that protein stability (i.e. as measured Again these proteidprotein measurements showed that by the adiabatic compressibility)and membrane polymer HSA was less stable at the PI than RN~s~A.~*This result composition are important factors in membrane fouling. is consistent with their respective adiabatic compress- For a more complete understanding of the effect of ibility values (cf. above). So, in the case of HSA, membrane protein adsorption on membrane filtration, force mea- fouling is expected by denaturation. To summarize the surements with hydrophobic materials such as polysulfone results with HSA, RNase A, and CA, membrane fouling and consideration of shear forces and their effect on protein will most likely be due to protein denaturation and could stability would also have to be considered. be reduced bv oDeratinrz at high DH. Drovided that the I I- membrane aid irotein ire stabe at this high pH. Also, Appendix A denaturation is less likely to happen with hard proteins such as RNase A than with soft proteins such as HSA.57p58 Validity of the Derjaguin Approximation during Why did the observed electrostatic interactions not Deformation Of the and Compaction Of the induce attractive forces? By considering the type of Acetate Film in the Surface Forces electrostatic charges on each material (Table 5),it appears Apparatus* compaction, a likely geometry Of the that for all the pH values both surfaces had the same type compacted is shown in Figure This of charge. fls0,in the presence of CA membrane, HSA tion suggests that the measured force was that between was much less stable than RNase A and tended to denature two parallel surfaces in zone A (zone B gave negli@ble contributions because the uncompacted CA film was sofi (53)Goto, Y.; Fink, A. L. Phase diagram for acidic conformational and the distances were large). %e forces can then be states of apoxyoglobin J. Mol. Biol. 1990,214, 803-805. reanalyzed by assuming this configuration: the area over (54)Anderson, D. E.; Becktel, W. J.; Dahlquist, F. W. pH induced which the surfaces are parallel can be calculated from the denaturation of proteins: A single salt bridge contributes 3-5 kcdmol thickness of the uncompacted and compacted CA films to the free energy of folding of T4 lysozyme Biochemistry 1990,29 (9), 2403-2408. and the radius of curvature of the surfaces. If this process (55)Puett, D. The equilibrium unfolding parameters of horse and is followed, the ensuingproblem arises: the force-distance sperm whale myoglobin J. Biol. Chem. 1973,248 (13),4623-4634. curves obtained in this way were not reproducible (56)Creighton, T.E. Proteins: Structures and Molecular Properties, W.H. Freeman and Co.: New York, 1984. anymore, while, with the Derjaguin's approximation, they (57)Kharakoz, D. P.; . Sarvazyan, A. P Hydiational and intrinsic compressibilities of globular proteins BiopoZymers 1993,33, 11-26. (59)Hesselink, F. Th. In AdsoTption from Solution ut the Solid1 (58)Gekko, K;Yamagami, K.Flexibility offood proteins as revealed LiquidZnterface;Parfitt, G.D., Rochester, C. H., Eds.; Academic Press, by compressibility J. Agric. Food Chem. 1991,39,57-62. New York, 1983; Chapter 8. Molecular Forces between Proteins and Polymer Films Langmuir, Vol. 11, No. 4, 1995 1235

zoneB zoneA zoneB 1 I

Figure 8. Hypothetical diagram of the compaction of cellulose acetate film in the SFA. were highlyreproducible over the whole set of experiments. This reproducibility cannot be fortuitous and a possible reason why Derjaguin's approximation remains valid is presented below. Figure 9, Surface deformation due to glue during compaction The CA films were precompacted under large forces of the cellulose acetate film. (i.e. >20 mN/m, which is larger than those shown in Figures 4-7). Under these conditions, the surfaces were the forces interacted, the geometry remained crossed substantially deformed: Attard and ParkerGogive the cylindrical, albeit with a thinner CA film than prior to relation between the flattening of the surfaces, the force, compaction, and the Derjaguin's approximation remained and the elasticity of the surfaces [which was due to the valid. glue: El(1- n2)= 0.36 J m-3, (Loubet, J. L., private Acknowledgment. We acknowledge the support of communication), where E is Young's modulus and n is the U. S. Department of Energy, Basic Chemical Science Poisson's ratio]. In our case, the surfaces were deformed Division (Grant No. DE-FG02-90ER14114). Bob L. Riley by about 30 nm (due to the glue), and the geometries of kindly supplied the cellulose acetate polymer. We thank the surhces before, during and after compaction were as JeMey A. Koehler and Brian Frank for discussions and shown in Figure 9. Thus, for the surface area over which Cheng Sheng Lee for measuring the RNase A adsorption kinetics shown in Figure 1. (60) Attard, P.; Parker, J. L. Deformation and adhesion of elastic bodies in contact Phys. Rev. A 1992,46 (12),7959-7971. LA9305280