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