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Protein-stabilized magnetic fluids
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Journal of Magnetism and Magnetic Materials 320 (2008) 634–641 www.elsevier.com/locate/jmmm
Protein-stabilized magnetic fluids S.J.H. Soenena, M. Hodeniusb, T. Schmitz-Rodeb, M. De Cuypera, a
Interdisciplinary Research Center, Katholieke Universiteit Leuven—Campus Kortrijk, University Campus, B-8500 Kortrijk, Belgium b Helmholtz Institute, Applied Medical Engineering, RWTH Aachen University, Aachen, Germany Received 5 February 2007; received in revised form 25 June 2007 Available online 2 August 2007
Abstract The adsorption of bovine serum albumin (BSA) and egg yolk phosvitin on magnetic fluid particles was investigated. Incubation mixtures were prepared by mixing an alkaline suspension of tetramethylammonium-coated magnetite cores with protein solutions at various protein/Fe3O4 ratios, followed by dialysis against a 5 mM TES buffer (pH 7.0), after which separation of bound and non-bound protein by high-gradient magnetophoresis was executed. Both the kinetic profiles as well as the isotherms of adsorption strongly differed for both proteins. In case of the spherical BSA, initially, abundant adsorption occurred, then it decreased and—at high protein concentrations—it slowly raised again. In contrast, with the highly phosphorylated phosvitin, binding slowly started and the extent of protein adsorption remained unchanged both as a function of time and phosvitin concentration. Competition binding studies, using binary protein mixtures composed of equal weight amounts of BSA and phosvitin, showed that binding of the latter protein is ‘unrealistically’ high. Based on the geometry of the two proteins, putative pictures on their orientation on the particle’s surface in the various experimental conditions were deduced. r 2007 Elsevier B.V. All rights reserved. PACS: 82.65.Y; 87.15; 68.45; 82.70.D; 83.70.H; 75.50.M; 75.50; 75.50.K Keywords: Bovine serum albumin; High-gradient magnetophoresis; Magnetite; Magnetic fluid; Phosvitin; Protein adsorption
1. Introduction The interest in water-based magnetic fluids in selected bioengineering and biomedical systems has been growing exponentially in the last decades [1–8]. To make the applications successful, in most cases, the magnetizable colloids have to be kept in solution by stabilizing them, sterically and/or electrostatically, with a coating consisting of ions [9] or polymers, either synthetic ones or from biological origin (e.g., dendrimers, dextrans, etc.) [6]. Within the polymer repertoire, it is surprising that proteins have been sparingly used to achieve this goal [10–12], in spite of the fact that, in general, proteins are known to bind to solid surfaces in an almost irreversible way [13]. In the present work, we further elaborated on this issue with the ultimate goal to create a long term, stable Corresponding author. Tel.: +32 56 24 62 21.
E-mail address: [email protected] (M. De Cuyper). 0304-8853/$ - see front matter r 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2007.07.027
magnetic fluid. The magnetic fluid from which we started consisted of Fe3O4 nanocolloids, originally stabilized with the low polarizing tetramethylammonium cations (TMA+), onto which the adsorption of bovine serum albumin (BSA) and phosvitin is investigated. The former protein is an ‘ordinary’ compact globular protein, very often used as a representative model for proteins in general; the second one is exceptional in having well over half of its 217 constituent amino acids residues as either phosphoserine or phosphothreonine [14,15]. As compared to most other proteins, it reveals as an extremely flexible, unordered polyelectrolyte which carries a very high negative charge (ca. 180 eV) at neutral pH [16]. Also, phosvitin has been described to bind ferric ions very strongly and extensively [17]. Upon dialysing the magnetic fluid suspension in the presence of BSA and/or phosvitin, it is shown that the covering TMA cations were replaced by the proteins. Information on the adsorption features of these proteins was obtained by merging theoretical data on particle and
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protein sizes, and experimentally derived adsorption kinetics and isotherms. Furthermore, qualitative data on the binding of both proteins was obtained by treating the protein-coated magnetic fluids with the surfactant sodium dodecylsulfate, and from competition binding experiments in which both proteins were added simultaneously to the magnetic fluid particles. 2. Materials and methods 2.1. Proteins BSA (globulin-free) and phosvitin from egg yolk were obtained from Sigma. Purity of the proteins was 499% as checked by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) using phosphorylase B (MW 94 kDa), BSA (MW 69 kDa), ovalbumin (MW 43 kDa), soybean trypsin inhibitor (MW 20.1) and a-lactalbumin (MW 14.4 kDa) as standards. Gels were fixed overnight in 45% methanol and 10% acetic acid. They were then stained for 6 h with 0.05% Coomassie blue in 20 mM AlCl3, 25% isopropylalcohol, 10% acetic acid and destained with 10% acetic acid, 5% ethanol. The inclusion of AlCl3 in the staining solution was necessary for the visualization of the phosphoprotein bands [18]. To quantify BSA, the Coomassie Plus Protein Assay Reagent (Pierce, Rockford, USA) was used. Phosvitin does not react with anionic dyes such as Coomassie blue [19] but the Folin–Ciocalteu’s phenol reagent was found to be applicable. However, the latter method proceeds in alkaline medium, thereby precipitating the Fe(II) and Fe(III) ions derived from the magnetite cores. Therefore, the phosvitin concentration was measured by a phosphate analysis following the method of Vaskovsky, as described in Ref. [20]. Based on a eM,275 nm value of 17,800 M1 cm1 [21], we found that the phosvitin preparation used in this work had a phosphate content of 7.75% by weight.
Fig. 1. Typical transmission electron micrograph of TMA+-stabilized Fe3O4 nanoparticles.
tubing packed with steel wool and positioned between the poles of an electromagnet in operation. The magnetic particles were captured on the steel wool and the nonadsorbed proteins were collected in the filtrate (eluate fraction). Then, after deactivation of the magnet, the trapped particles were collected by eluting them from the magnetic filter with buffer flowing over the filter at high speed (retentate fraction). 2.4. Adsorption kinetics
Magnetite nanoparticles were produced by precipitation of a solution of Fe(II) (80 mL; 1 M) and Fe(III) chloride (20 mL; 2 M) in 0.7 M ammonia. The particles were then magnetically decantated and subsequently peptized with 100 mL 1 M tetramethylammonium hydroxide [9]. The stock solution of magnetic fluid had a magnetite content of 9.30 mg Fe3O4 mL1; the pH was 12.3. The average core diameter was 1470.3 nm, as deduced from the transmission electron micrograph, presented in Fig. 1.
To follow the time course of protein binding onto the Fe3O4 particles, the desired amounts of proteins were mixed with a constant amount of particles and the incubation mixtures were immediately dialyzed (Dialysis Tubing Visking, size 1–8/32 in, Medicell International, London, UK; molecular weight cut-off: 10,000) at 4 1C against 5 mM N-Tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid (TES) buffer, pH 7.0. At regular times, a small portion of the incubation mixture was withdrawn from the dialysis bag and subjected to the aforementioned high-gradient magnetophoresis. Both the retentates and eluates were analyzed for iron and for protein (in case of BSA) or phosphate content (in case of phosvitin).
2.3. High-gradient magnetophoresis
2.5. Adsorption isotherms
Separation of proteins bound to the magnetite particles from those remaining in solution was done by highgradient magnetophoresis. Details on the experimental setup can be found in our previous papers [20]. In short, the various incubation media were pumped through a
To construct relevant adsorption isotherms, the content of various vials, each containing an identical amount of Fe3O4 particles but increasing amounts of proteins, was dialyzed at 4 1C for 24 h against the aforementioned TES buffer. Again, bound and non-bound proteins were
2.2. Magnetite particles
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separated by high-gradient magnetophoresis, and retentates and eluates were characterized as described above. The experimental data were fitted and linearized according to the Langmuir adsorption model (Eq. (1)): c 1 1 ¼ cþ , (1) GL GoL KL where GL is the number of gram of protein adsorbed per gram of Fe3O4, G0L is the value at saturation, c is the protein equilibrium concentration in (mg mL1) and KL is the association constant. 2.6. Analytical methods BSA and phosvitin concentrations were measured as described above. For total iron concentration determination, the particles were first dissoluted by digestion for 15 min in a mixture of concentrated HCl–HNO3 (3/1; v/v) at elevated temperature (60 1C), then stained with Tiron and the resulting Fe3+–Tiron complex was measured spectrophotometrically at 480 nm [20]. The average diameter of the iron oxide cores was measured by transmission electron microscopy (Zeiss 10C A). To this end, the aqueous dispersion of the magnetic fluid was drop-cast onto a carbon copper grid which was air-dried at room temperature before loading into the microscope. Chemicals used for the buffer and magnetite preparation were all of analytical grade and used without further purification. They were bought either from Sigma (Bornem, Belgium) or from Merck (VWR, Belgium). 3. Results and discussion 3.1. Kinetics of BSA adsorption on Fe3O4 nanocolloids Upon dialysing 3 mL of an eight-fold diluted Fe3O4– TMA magnetic fluid stock solution at 4 1C against 1 L of 5 mM TES, pH 7.0 the pH slowly dropped from about pH 12.0 to neutral pH within a time span of about 6 h (Fig. 2p). Concomitantly, since TMA+ ions were only weakly physisorbed, their stabilizing role perished, resulting in particle precipitation in the dialysis bag. Thus, the TMA+-coated Fe3O4 nanocolloid suspension could be considered as a rather unstable, dilution sensitive magnetic fluid. In case the dialysis step was executed in the presence of BSA (final concentration either 2.29 or 34.35 mg mL1, corresponding to a BSA/Fe3O4 weight ratio of 1.97 and 29.56, respectively), a similar decrease in pH of the medium is observed but, now, due to protein adsorption the magnetic fluid particles remain perfectly in solution. Changes in the BSA/Fe3O4 weight ratio as a function of time were depicted in Fig. 2. Within the first 200 min time interval the protein/magnetite weight ratio rised above 1.1 but then gradually dropped to about 0.40. Since the isoelectric point of BSA equals 4.7 [22] and the point-ofzero charge of magnetite is about 6.0 [23], meaning that
Fig. 2. Adsorption kinetics of BSA onto Fe3O4 nanocolloids, originally stabilized with TMA+ ions. The (J) symbols illustrate the time course of protein binding starting from a BSA/Fe3O4 weight ratio of 1.97; the (K) symbols represent protein binding at a protein/magnetite ratio of 29.56. The encircled capitals refer to the adsorption pattern as illustrated in Fig. 3. Measurements were done at 4 1C. From theoretical calculations (see text), it can be shown that if BSA entirely adsorbs in a side-on direction the BSA/magnetite ratio equals 0.29 (lower dotted line); in a complete endon orientation the value is 0.69 (upper dotted line). From the right hand side y-axis, changes in pH (p symbol) of the magnetic fluid suspension as a function of dialysis time can be derived. Dialysis occurred against 5 mM TES buffer, pH 7.0 at 4 1C.
both entities are anionic, at first sight, the high amount of sorbed BSA may be surprising. Recently, however, Brusatori [24] (see also Refs. [25–27]) attributed the effect of protein adsorption on surfaces with similar charge to the existence of small clusters of cationic charged groups at the protein’s surface (e.g., side groups of Lys or Arg residues), so that protein-surface electrostatic attractions rather than repulsions are operating. Phenomenologically, it is fairly well established that once the initial protein–surface contact has been made, protein rearrangements at the surface occur, thereby banishing some pre-adsorbed molecules [28,29]. In the present work, we did not focus on the overall mechanism underlying this process. Nevertheless, a realistic scenario (Fig. 3) is that following impingement of BSA molecules onto the surface, each individual protein molecule, partly unfolded by the high pH conditions [30], was first tethered by only a few contact points at a very small particle surface area (Fig. 3A). In this way, a significant amount of protein was bound (protein/ magnetite mass ratio 41.0; Fig. 2). Then, conformational changes, co-triggered by the decrease in pH [30] and by a time-dependent spreading of the molecule on the surface [31], did occur. Intra alia, we indeed found by fluorescence measurements that the emission spectrum of BSA (excitation wavelength at 276 nm) gradually raised as the pH was decreased from pH 12.0 to 7.0 (not shown). Undoubtedly, in the final positioning of the protein molecules on top of Fe3O4 nanocolloids, strong chemisorption forces between the iron oxide surface and –COOH groups (e.g., from Glu or Asp amino acids) played a key role. The occurrence of the latter binding type is well documented in literature [32].
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Fig. 3. Schematic representation of the consecutive adsorption events during incubation of BSA with the magnetic fluid particles (BSA/Fe3O4 ¼ 29.56) as shown in Fig. 2K.
After these initial binding events, the time course of the subsequent adsorption process depended on the BSA concentration applied to generate BSA-coated particles. At the lower BSA/Fe3O4 ratio of 1.97 (Fig. 2J), no further change in the particle’s protein content occurred; in contrast, at the higher protein concentration (Fe3O4/ BSA ¼ 29.56; Fig. 2K) it progressively raised, indicating multilayer adsorption. 3.2. Kinetics of phosvitin adsorption on Fe3O4 nanocolloids Data derived from kinetic setups consisting of either 0.85 or 16.90 mg phosvitin mL1 and 1.162 mg Fe3O4 mL1 (final concentrations) (Fig. 4) indicated that, in contrast to the situation with BSA, phosvitin binding got more slowly started, most probably due to the extremely high number of negative charges on the protein (see Section 1). Thus, here overall electrostatic repulsions were dominating. As the pH was lowered during dialysis, the adsorption gradually increased. Eventually, after about 5 h, a plateau value of 0.19 and 0.25 phosvitin/magnetite weight ratio was found for the low and high starting phosvitin concentrations, respectively. Based on theoretical data given below, it seemed that in the latter case many phosvitins landed within a very short time span on the particle so that, as opposed to the lower concentration conditions, full spreading of the molecule on the surface was partly blocked by the presence of sufficiently close neighbors. 3.3. Adsorption isotherms for BSA and phosvitin binding on Fe3O4 More detailed information on BSA and phosvitin binding on the Fe3O4 surface could be obtained by constructing adsorption isotherms which show the amount of protein bound as a function of the concentration of protein which remains free in solution after the adsorption has reached a steady state. In practice, BSA binding was studied starting from 1 mL of the magnetic fluid stock solution which was incubated with BSA (ranging from 0 to 7 mL of a BSA stock solution at a concentration of 59.0 mg mL1) and the final volume of each mixture was adjusted to 8 mL with TES buffer. After dialysis for 24 h at 4 1C and magnetic separation (see Section 2) the adsorption isotherm was constructed
Fig. 4. Adsorption kinetics of phosvitin onto Fe3O4 particles, originally stabilized with TMA+. The phosvitin/Fe3O4 mass ratio in the incubation mixture equalled 0.73 (n–n) and 14.54 (m–m). Incubations were done at 4 1C. The horizontal dotted line indicates the calculated level of phosvitin adsorption if the protein molecules were uniquely attached to the surface in a horizontal side-on direction (see text).
(Fig. 5A). It clearly showed two distinct plateaus: in the lower free protein concentration range (o25 mg mL1), protein immobilization levelled off at a 0.45 BSA/Fe3O4 weight ratio; at higher excess protein concentrations, a plateau value of about 0.90 was found (Fig. 5AK). As will be discussed below, the appearance of this second saturation plateau (ratio ¼ 0.90), can be rationalized by assuming the adsorption of a second protein layer on top of the first one [13]. Exactly what happens to the three-dimensional structure of a protein when it adsorbs onto a solid surface often remains an enigma despite the wide interest and practical importance of the phenomenon. However, based on the geometry of both the adsorbent and adsorbate, useful information on this issue was gathered to further understand the adsorption process. On the one hand, a 14 nm diameter magnetite particle, as used in the present work, exposes a surface area of 61575 A˚2. On the other hand, Peters [33] reported that the shape of a BSA molecule is approximated by an ellipsoid 141 A˚ long with a maximum transverse diameter of 42 A˚. In case protein unfolding did not occur during the adsorption process, it can be calculated that the smallest cross-sectional area of a BSA
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Fig. 5. Adsorption isotherms for binding of BSA (A) and phosvitin (B) on top of a nanometer-sized magnetite core in 5 mM TES buffer, pH 7.0. The closed symbols represent the data in the absence of surfactant; the open symbols illustrate the extent of protein adsorption after treating the protein-coated particles with SDS (final concentration 5%).
molecule (which is relevant for an ‘end-on’ vertical disposition on the solid surface) equals 1384 A˚2 and, thus, about 44 BSA molecules are needed to completely occupy the zone directly adjacent to the surface of a bare Fe3O4 particle. Since the molecular weight of BSA equals 69,000 Da and the density of magnetite is 5.1 g cm3 [Handbook of Chemistry and Physics 1975–1976], a theoretical BSA/Fe3O4 weight ratio of 0.69 can be calculated in case of a complete ‘end-on’ orientation of the protein. Alternatively, if BSA faces the surface in a horizontal, ‘side-on’ direction, the relevant cross section area of an individual BSA molecule equals 4651 A˚2, resulting in the binding of only 13 BSA molecules per particle, corresponding to a value of 0.20 for the BSA/ Fe3O4 weight ratio. Since the experimentally measured ratio (0.45; see the first plateau in the adsorption isotherm of Fig. 5AK) was in between the theoretical values found for an orientation in a flat and a vertical direction, in reality some intermediate orientation was most probably adopted (Fig. 3B). Of course, in spite of the fact that BSA is considered to be a so-called ‘hard’ protein, the occurrence of a partial, entropic driven protein unfolding cannot be entirely ruled out [25,34]. To get a qualitative, albeit indirect indication of the binding strength by which protein binding occurred, the various protein-coated particles were treated for 24 h with
SDS (final concentration 5%) and then magnetically fractionated. Fig. 5AJ shows that the ‘second plateau’ BSA molecules (BSA/Fe3O440.45) were removed, whereas the molecules initially adsorbed were reluctant to the rinsing process. Thus, the prevailing protein–protein interaction forces in the second layer on top of the first one were much weaker. At high protein concentrations, similar protein arrangements were reported, for instance, by Fang and Szleifer [13] and Lee and Ruckenstein [35]. Quantitative data, proving the existence of two categories of adsorbed proteins, was provided by fitting the data of Fig. 5 by Langmuir adsorption mathematics. Below a protein concentration of 25 mg mL1 the linearized Langmuir isotherm pointed to a uniform protein binding behavior with an association constant of 1.94 mL mg1 (corresponding to 0.029 mM1) (r2 ¼ 0.996), whereas at higher free protein concentrations the data points clearly deviated from the straight line (Fig. 6K). Fitting the binding data for the remaining proteins after SDS treatment led to a similar association constant of 1.47 mL mg1 (corresponding to 0.022 mM1) (r2 ¼ 0.996) (Fig. 6J). In a similar way, we also studied the adsorption behavior of phosvitin at 4 1C in 5 mM TES buffer, pH 7.0 (final Fe3O4 concentration ¼ 1.16 mg mL1; final phosvitin concentration between 0 and 45.15 mg mL1) (Fig. 5B). At a phosvitin coverage below 0.10 mg mg1 Fe3O4, apparently, the coating was incomplete since the magnetic fluid particles precipitated; at free phosvitin concentrations above 10 mg mL1 a plateau at 0.28 mg protein mg1 iron oxide was found (Fig. 5Bm). Attempts to extract the adsorbed protein with 5% SDS failed (Fig. 5Bn) testifying a strong anchoring. This observation is in line with earlier observations which proved that the phosphorylated form of phosvitin has a high affinity for Fe3+ ions [17] and that
Fig. 6. Linearized form of the Langmuir adsorption isotherms, derived from the data in Fig. 5. The (K) symbols illustrate protein binding at a BSA concentration o25 mg mL1; the (J) symbols represent protein binding in the protein-coated particles after treatment with 5% SDS. The solid and dashed lines are linear approximations of the (K) and (J) data, respectively.
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strong chemisorption exists between iron oxide surfaces and phosphate containing molecules [20,36,37]. As compared to BSA, phosvitin (MWE40 kDa; Petersen and Cox [38]) is a more elongated molecule with a prolate ellipsoidal shape; the axial ratios being between 20 and 25. On the basis of these data, Joubert and Cook [39] assumed that the dimensions of the molecule are about 14 A˚ by 280 A˚. This means that in case of an ‘end-on’ (relevant cross section area ¼ 154 A˚2) and ‘side-on’ adsorption (relevant cross section area 3077 A˚2) 399 and 20 molecules, respectively, are anchored at maximum on a 14 nm diameter magnetite particle, as used in the present work. These values correspond to a theoretical phosvitin/Fe3O4 weight ratio of 3.63 and 0.18, respectively. Since the latter approaches the experimental value of 0.19 in the lowprotein concentration regime (cf. plateau value in Fig. 5Bm), it can be concluded that phosvitin almost uniquely adsorbed in a flat, extended form in the concentration range studied. The slightly higher plateau value measured at a higher phosvitin concentration (Fig. 5Bm) then suggests that short fragments of the polypeptide chain are pointing away from the surface. 3.4. Kinetics of competitive adsorption of BSA and phosvitin on magnetite cores Protein adsorption from a binary mixture may not be considered as the sum of the binding behavior of the individual proteins and, indeed, it is the intricate result of a combination of a number of contributions such as the individual rate of adsorption, surface availability, charge and charge distribution of the adsorbing proteins, protein conformational flexibility, intermolecular protein interactions, protein–surface binding strength, etc. [13,26]. Consequently, it was not straightforward to obtain information concerning the affinities, not even relative values, of proteins for surfaces from competitive adsorption experiments. Nevertheless, the shape of the adsorption kinetic profiles could shed some light on the consecutive steps in the binding process. In the present experimental setup, mixtures of equal mass amounts of BSA and phosvitin in 5 mM TES buffer (pH 7.0) were confronted with the iron oxide colloids (final concentration: 1.16 mg Fe3O4 mL1); the total protein/ Fe3O4 weight ratios studied were 1.29 and 12.93. Fig. 7 shows that in both cases initially adsorption of BSA was favored over that of phosvitin, which was in line with the adsorption kinetics observed with single proteins as shown in Figs. 2 and 4. In the lower protein-to-magnetite regime (Fig. 7A) the BSA content then dropped to a value of 0.17 mg protein mg1 Fe3O4. This plateau value was identical to the one measured for anchored phosvitin. Taking into account that the entire particle surface was no longer occupied by a single protein type, these values suggested that, as in the case of the single protein situation (Fig. 2), BSA was adsorbed in a hybrid ‘end-on’–‘side-on’
Fig. 7. Kinetic profiles for protein adsorption onto magnetic cores, starting from a binary protein mixture containing equal weight amounts of BSA and phosvitin. The total protein/Fe3O4 weight ratios used were 1.29 (A) and 12.93 (B). The profiles for BSA anchoring are shown by the circle symbols; those for phosvitin binding by the triangles.
orientation, whereas phosvitin molecules mainly adopted a flat position, represented schematically in Fig. 8A (compare with the relevant values for a horizontal and vertical binding mode given above). At the higher protein-to-magnetite ratio (Fig. 7B) the amount of BSA similarly levelled off to 0.20 mg BSA mg1 Fe3O4 but, surprisingly, at the same time considerably more phosvitin was adsorbed than in conditions in which it was the sole protein present in the adsorption cocktail (compare the phosvitin saturation levels shown in Figs. 5Bm and 7Bm). Possibly, with the binary protein mixture, unoccupied magnetite surface patches between the pre-adsorbed ‘‘hard’’ ellipsoidal BSA molecules were occupied by multiple phosvitin molecules, which due to their flexible structure could easily enter into these free regions. Due to spatial constraints, however, we hypothesize that each individual phosvitin entity can only stick on the surface by a reduced number of attached segments and, hence, the remaining protein fragments dangle in the solution as loops and tails interacting with water molecules [33] as represented schematically in Fig. 8B. The question as to whether the final values in the kinetic profiles represented a kinetic as opposed to a
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vesicles [20,41]. Thus, it is believed that the binding profiles represent a kinetically controlled adsorption process, rather than a thermodynamical controlled one. However, often the time needed to reach ‘true’ equilibrium conditions in adsorption experiments can be very long—days, weeks, months or even longer [42,43]. 4. Conclusions The immobilization of biomolecules to solid phase materials is of uppermost relevancy in quite a lot of biotechnological and biomedical applications. In the present work, the anchoring of a highly phosphate-loaded, flexible protein on a magnetic fluid core was compared with the adsorption behavior of a ‘classical’ globular protein. The results show that the former protein type was strongly chemisorbed on the Fe3O4 surface thereby producing a highly charged magnetic fluid formulation exhibiting improved colloidal stability with respect to long-term storage. As such, the present study paves the way for the use of these magnetizable nanocolloids in many practical applications such as the design of biocompatible materials, drug carriers and biosensors among many others. Acknowledgments
Fig. 8. Proposed model for the adsorption of BSA (cloudy ellipses) and phosvitin (solid gray figures) on Fe3O4 particles at pH 7.0. The proteins are added as a binary mixture containing equal amounts (on a mass base) of both protein types. In panel (A), the mode of protein adsorption is depicted, starting from a total protein/Fe3O4 weight ratio of 1.29; in panel (B), the ratio equals 12.93. The pictures represent the situation where protein binding has come to a (pseudo) steady state.
thermodynamic equilibrium still remains open. Elwing and Go¨lander [40] found that, even for proteins present in equal amounts in a binary mixture and which, moreover, adsorbed at the same rate, eventually, at equilibrium, protein adsorption does not necessarily occur in a 50/50 ratio. Indeed, when a protein initially adsorbs on a solid surface it forms multiple contact points. After contact is made, the bonds between protein segments and surface sites are continuously broken and reformed. Proteins bearing groups with a higher affinity can interfere with this arrangement. Ultimately, the latter (in casu the highly flexible phosvitin) will be anchored by preference. In our experimental model system, this is a realistic scenario since phosphate-iron oxide chemisorptive forces which are dominating with the highly phosphorylated ‘‘soft’’ phosvitin, indeed, are stronger than –COOH-iron oxide interactions, occurring with the ‘‘hard’’ BSA molecule. A similar difference in adsorptive capacity of –COOH and phosphate groups for magnetite surfaces was previously exploited by us to construct so-called magnetoliposomes, starting from a lauric acid-stabilized magnetic liquid and phospholipid
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[30] G.D. Fullerton, K.M. Kanal, I.L. Cameron, Cell Biol. Int. 30 (2006) 86. [31] P.R. van Tassel, L. Guemouri, J.J. Ramsden, et al., J. Colloid Interface Sci. 207 (1998) 317. [32] L. Shen, P.E. Laibinis, T.A. Hatton, Langmuir 15 (1999) 447. [33] T.J. Peters Jr., Adv. Protein Chem. 37 (1985) 161. [34] C.M. Roth, J.E. Sader, A.M. Lenhoff, J. Colloid Interface Sci. 203 (1998) 218. [35] S.H. Lee, E. Ruckenstein, J. Colloid Interface Sci. 125 (1988) 365. [36] J. Giri, S.G. Thakurta, J. Bellare, et al., J. Magn. Magn. Mater. 293 (2005) 62. [37] Y. Sahoo, H. Pizem, T. Fried, et al., Langmuir 17 (2001) 7907. [38] L.C. Peterssen, R.P. Cox, Biochem. J. 192 (1980) 687. [39] F.J. Joubert, W.H. Cook, Can. J. Biochem. Physiol. 36 (1958) 399. [40] H. Elwing, C.-G. Go¨lander, Adv. Colloid Interface Sci. 32 (1990) 317. [41] M. De Cuyper, M. Joniau, Eur. Biophys. J. 15 (1988) 311. [42] F. Fang, I. Szleifer, Biophys. J. 80 (2001) 2568. [43] T.G.M. van de Ven, Adv. Colloid Interface Sci. 48 (1994) 121.
Journal of Magnetism and Magnetic Materials 320 (2008) 634–641 www.elsevier.com/locate/jmmm
Protein-stabilized magnetic fluids S.J.H. Soenena, M. Hodeniusb, T. Schmitz-Rodeb, M. De Cuypera, a
Interdisciplinary Research Center, Katholieke Universiteit Leuven—Campus Kortrijk, University Campus, B-8500 Kortrijk, Belgium b Helmholtz Institute, Applied Medical Engineering, RWTH Aachen University, Aachen, Germany Received 5 February 2007; received in revised form 25 June 2007 Available online 2 August 2007
Abstract The adsorption of bovine serum albumin (BSA) and egg yolk phosvitin on magnetic fluid particles was investigated. Incubation mixtures were prepared by mixing an alkaline suspension of tetramethylammonium-coated magnetite cores with protein solutions at various protein/Fe3O4 ratios, followed by dialysis against a 5 mM TES buffer (pH 7.0), after which separation of bound and non-bound protein by high-gradient magnetophoresis was executed. Both the kinetic profiles as well as the isotherms of adsorption strongly differed for both proteins. In case of the spherical BSA, initially, abundant adsorption occurred, then it decreased and—at high protein concentrations—it slowly raised again. In contrast, with the highly phosphorylated phosvitin, binding slowly started and the extent of protein adsorption remained unchanged both as a function of time and phosvitin concentration. Competition binding studies, using binary protein mixtures composed of equal weight amounts of BSA and phosvitin, showed that binding of the latter protein is ‘unrealistically’ high. Based on the geometry of the two proteins, putative pictures on their orientation on the particle’s surface in the various experimental conditions were deduced. r 2007 Elsevier B.V. All rights reserved. PACS: 82.65.Y; 87.15; 68.45; 82.70.D; 83.70.H; 75.50.M; 75.50; 75.50.K Keywords: Bovine serum albumin; High-gradient magnetophoresis; Magnetite; Magnetic fluid; Phosvitin; Protein adsorption
1. Introduction The interest in water-based magnetic fluids in selected bioengineering and biomedical systems has been growing exponentially in the last decades [1–8]. To make the applications successful, in most cases, the magnetizable colloids have to be kept in solution by stabilizing them, sterically and/or electrostatically, with a coating consisting of ions [9] or polymers, either synthetic ones or from biological origin (e.g., dendrimers, dextrans, etc.) [6]. Within the polymer repertoire, it is surprising that proteins have been sparingly used to achieve this goal [10–12], in spite of the fact that, in general, proteins are known to bind to solid surfaces in an almost irreversible way [13]. In the present work, we further elaborated on this issue with the ultimate goal to create a long term, stable Corresponding author. Tel.: +32 56 24 62 21.
E-mail address: [email protected] (M. De Cuyper). 0304-8853/$ - see front matter r 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2007.07.027
magnetic fluid. The magnetic fluid from which we started consisted of Fe3O4 nanocolloids, originally stabilized with the low polarizing tetramethylammonium cations (TMA+), onto which the adsorption of bovine serum albumin (BSA) and phosvitin is investigated. The former protein is an ‘ordinary’ compact globular protein, very often used as a representative model for proteins in general; the second one is exceptional in having well over half of its 217 constituent amino acids residues as either phosphoserine or phosphothreonine [14,15]. As compared to most other proteins, it reveals as an extremely flexible, unordered polyelectrolyte which carries a very high negative charge (ca. 180 eV) at neutral pH [16]. Also, phosvitin has been described to bind ferric ions very strongly and extensively [17]. Upon dialysing the magnetic fluid suspension in the presence of BSA and/or phosvitin, it is shown that the covering TMA cations were replaced by the proteins. Information on the adsorption features of these proteins was obtained by merging theoretical data on particle and
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protein sizes, and experimentally derived adsorption kinetics and isotherms. Furthermore, qualitative data on the binding of both proteins was obtained by treating the protein-coated magnetic fluids with the surfactant sodium dodecylsulfate, and from competition binding experiments in which both proteins were added simultaneously to the magnetic fluid particles. 2. Materials and methods 2.1. Proteins BSA (globulin-free) and phosvitin from egg yolk were obtained from Sigma. Purity of the proteins was 499% as checked by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) using phosphorylase B (MW 94 kDa), BSA (MW 69 kDa), ovalbumin (MW 43 kDa), soybean trypsin inhibitor (MW 20.1) and a-lactalbumin (MW 14.4 kDa) as standards. Gels were fixed overnight in 45% methanol and 10% acetic acid. They were then stained for 6 h with 0.05% Coomassie blue in 20 mM AlCl3, 25% isopropylalcohol, 10% acetic acid and destained with 10% acetic acid, 5% ethanol. The inclusion of AlCl3 in the staining solution was necessary for the visualization of the phosphoprotein bands [18]. To quantify BSA, the Coomassie Plus Protein Assay Reagent (Pierce, Rockford, USA) was used. Phosvitin does not react with anionic dyes such as Coomassie blue [19] but the Folin–Ciocalteu’s phenol reagent was found to be applicable. However, the latter method proceeds in alkaline medium, thereby precipitating the Fe(II) and Fe(III) ions derived from the magnetite cores. Therefore, the phosvitin concentration was measured by a phosphate analysis following the method of Vaskovsky, as described in Ref. [20]. Based on a eM,275 nm value of 17,800 M1 cm1 [21], we found that the phosvitin preparation used in this work had a phosphate content of 7.75% by weight.
Fig. 1. Typical transmission electron micrograph of TMA+-stabilized Fe3O4 nanoparticles.
tubing packed with steel wool and positioned between the poles of an electromagnet in operation. The magnetic particles were captured on the steel wool and the nonadsorbed proteins were collected in the filtrate (eluate fraction). Then, after deactivation of the magnet, the trapped particles were collected by eluting them from the magnetic filter with buffer flowing over the filter at high speed (retentate fraction). 2.4. Adsorption kinetics
Magnetite nanoparticles were produced by precipitation of a solution of Fe(II) (80 mL; 1 M) and Fe(III) chloride (20 mL; 2 M) in 0.7 M ammonia. The particles were then magnetically decantated and subsequently peptized with 100 mL 1 M tetramethylammonium hydroxide [9]. The stock solution of magnetic fluid had a magnetite content of 9.30 mg Fe3O4 mL1; the pH was 12.3. The average core diameter was 1470.3 nm, as deduced from the transmission electron micrograph, presented in Fig. 1.
To follow the time course of protein binding onto the Fe3O4 particles, the desired amounts of proteins were mixed with a constant amount of particles and the incubation mixtures were immediately dialyzed (Dialysis Tubing Visking, size 1–8/32 in, Medicell International, London, UK; molecular weight cut-off: 10,000) at 4 1C against 5 mM N-Tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid (TES) buffer, pH 7.0. At regular times, a small portion of the incubation mixture was withdrawn from the dialysis bag and subjected to the aforementioned high-gradient magnetophoresis. Both the retentates and eluates were analyzed for iron and for protein (in case of BSA) or phosphate content (in case of phosvitin).
2.3. High-gradient magnetophoresis
2.5. Adsorption isotherms
Separation of proteins bound to the magnetite particles from those remaining in solution was done by highgradient magnetophoresis. Details on the experimental setup can be found in our previous papers [20]. In short, the various incubation media were pumped through a
To construct relevant adsorption isotherms, the content of various vials, each containing an identical amount of Fe3O4 particles but increasing amounts of proteins, was dialyzed at 4 1C for 24 h against the aforementioned TES buffer. Again, bound and non-bound proteins were
2.2. Magnetite particles
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separated by high-gradient magnetophoresis, and retentates and eluates were characterized as described above. The experimental data were fitted and linearized according to the Langmuir adsorption model (Eq. (1)): c 1 1 ¼ cþ , (1) GL GoL KL where GL is the number of gram of protein adsorbed per gram of Fe3O4, G0L is the value at saturation, c is the protein equilibrium concentration in (mg mL1) and KL is the association constant. 2.6. Analytical methods BSA and phosvitin concentrations were measured as described above. For total iron concentration determination, the particles were first dissoluted by digestion for 15 min in a mixture of concentrated HCl–HNO3 (3/1; v/v) at elevated temperature (60 1C), then stained with Tiron and the resulting Fe3+–Tiron complex was measured spectrophotometrically at 480 nm [20]. The average diameter of the iron oxide cores was measured by transmission electron microscopy (Zeiss 10C A). To this end, the aqueous dispersion of the magnetic fluid was drop-cast onto a carbon copper grid which was air-dried at room temperature before loading into the microscope. Chemicals used for the buffer and magnetite preparation were all of analytical grade and used without further purification. They were bought either from Sigma (Bornem, Belgium) or from Merck (VWR, Belgium). 3. Results and discussion 3.1. Kinetics of BSA adsorption on Fe3O4 nanocolloids Upon dialysing 3 mL of an eight-fold diluted Fe3O4– TMA magnetic fluid stock solution at 4 1C against 1 L of 5 mM TES, pH 7.0 the pH slowly dropped from about pH 12.0 to neutral pH within a time span of about 6 h (Fig. 2p). Concomitantly, since TMA+ ions were only weakly physisorbed, their stabilizing role perished, resulting in particle precipitation in the dialysis bag. Thus, the TMA+-coated Fe3O4 nanocolloid suspension could be considered as a rather unstable, dilution sensitive magnetic fluid. In case the dialysis step was executed in the presence of BSA (final concentration either 2.29 or 34.35 mg mL1, corresponding to a BSA/Fe3O4 weight ratio of 1.97 and 29.56, respectively), a similar decrease in pH of the medium is observed but, now, due to protein adsorption the magnetic fluid particles remain perfectly in solution. Changes in the BSA/Fe3O4 weight ratio as a function of time were depicted in Fig. 2. Within the first 200 min time interval the protein/magnetite weight ratio rised above 1.1 but then gradually dropped to about 0.40. Since the isoelectric point of BSA equals 4.7 [22] and the point-ofzero charge of magnetite is about 6.0 [23], meaning that
Fig. 2. Adsorption kinetics of BSA onto Fe3O4 nanocolloids, originally stabilized with TMA+ ions. The (J) symbols illustrate the time course of protein binding starting from a BSA/Fe3O4 weight ratio of 1.97; the (K) symbols represent protein binding at a protein/magnetite ratio of 29.56. The encircled capitals refer to the adsorption pattern as illustrated in Fig. 3. Measurements were done at 4 1C. From theoretical calculations (see text), it can be shown that if BSA entirely adsorbs in a side-on direction the BSA/magnetite ratio equals 0.29 (lower dotted line); in a complete endon orientation the value is 0.69 (upper dotted line). From the right hand side y-axis, changes in pH (p symbol) of the magnetic fluid suspension as a function of dialysis time can be derived. Dialysis occurred against 5 mM TES buffer, pH 7.0 at 4 1C.
both entities are anionic, at first sight, the high amount of sorbed BSA may be surprising. Recently, however, Brusatori [24] (see also Refs. [25–27]) attributed the effect of protein adsorption on surfaces with similar charge to the existence of small clusters of cationic charged groups at the protein’s surface (e.g., side groups of Lys or Arg residues), so that protein-surface electrostatic attractions rather than repulsions are operating. Phenomenologically, it is fairly well established that once the initial protein–surface contact has been made, protein rearrangements at the surface occur, thereby banishing some pre-adsorbed molecules [28,29]. In the present work, we did not focus on the overall mechanism underlying this process. Nevertheless, a realistic scenario (Fig. 3) is that following impingement of BSA molecules onto the surface, each individual protein molecule, partly unfolded by the high pH conditions [30], was first tethered by only a few contact points at a very small particle surface area (Fig. 3A). In this way, a significant amount of protein was bound (protein/ magnetite mass ratio 41.0; Fig. 2). Then, conformational changes, co-triggered by the decrease in pH [30] and by a time-dependent spreading of the molecule on the surface [31], did occur. Intra alia, we indeed found by fluorescence measurements that the emission spectrum of BSA (excitation wavelength at 276 nm) gradually raised as the pH was decreased from pH 12.0 to 7.0 (not shown). Undoubtedly, in the final positioning of the protein molecules on top of Fe3O4 nanocolloids, strong chemisorption forces between the iron oxide surface and –COOH groups (e.g., from Glu or Asp amino acids) played a key role. The occurrence of the latter binding type is well documented in literature [32].
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Fig. 3. Schematic representation of the consecutive adsorption events during incubation of BSA with the magnetic fluid particles (BSA/Fe3O4 ¼ 29.56) as shown in Fig. 2K.
After these initial binding events, the time course of the subsequent adsorption process depended on the BSA concentration applied to generate BSA-coated particles. At the lower BSA/Fe3O4 ratio of 1.97 (Fig. 2J), no further change in the particle’s protein content occurred; in contrast, at the higher protein concentration (Fe3O4/ BSA ¼ 29.56; Fig. 2K) it progressively raised, indicating multilayer adsorption. 3.2. Kinetics of phosvitin adsorption on Fe3O4 nanocolloids Data derived from kinetic setups consisting of either 0.85 or 16.90 mg phosvitin mL1 and 1.162 mg Fe3O4 mL1 (final concentrations) (Fig. 4) indicated that, in contrast to the situation with BSA, phosvitin binding got more slowly started, most probably due to the extremely high number of negative charges on the protein (see Section 1). Thus, here overall electrostatic repulsions were dominating. As the pH was lowered during dialysis, the adsorption gradually increased. Eventually, after about 5 h, a plateau value of 0.19 and 0.25 phosvitin/magnetite weight ratio was found for the low and high starting phosvitin concentrations, respectively. Based on theoretical data given below, it seemed that in the latter case many phosvitins landed within a very short time span on the particle so that, as opposed to the lower concentration conditions, full spreading of the molecule on the surface was partly blocked by the presence of sufficiently close neighbors. 3.3. Adsorption isotherms for BSA and phosvitin binding on Fe3O4 More detailed information on BSA and phosvitin binding on the Fe3O4 surface could be obtained by constructing adsorption isotherms which show the amount of protein bound as a function of the concentration of protein which remains free in solution after the adsorption has reached a steady state. In practice, BSA binding was studied starting from 1 mL of the magnetic fluid stock solution which was incubated with BSA (ranging from 0 to 7 mL of a BSA stock solution at a concentration of 59.0 mg mL1) and the final volume of each mixture was adjusted to 8 mL with TES buffer. After dialysis for 24 h at 4 1C and magnetic separation (see Section 2) the adsorption isotherm was constructed
Fig. 4. Adsorption kinetics of phosvitin onto Fe3O4 particles, originally stabilized with TMA+. The phosvitin/Fe3O4 mass ratio in the incubation mixture equalled 0.73 (n–n) and 14.54 (m–m). Incubations were done at 4 1C. The horizontal dotted line indicates the calculated level of phosvitin adsorption if the protein molecules were uniquely attached to the surface in a horizontal side-on direction (see text).
(Fig. 5A). It clearly showed two distinct plateaus: in the lower free protein concentration range (o25 mg mL1), protein immobilization levelled off at a 0.45 BSA/Fe3O4 weight ratio; at higher excess protein concentrations, a plateau value of about 0.90 was found (Fig. 5AK). As will be discussed below, the appearance of this second saturation plateau (ratio ¼ 0.90), can be rationalized by assuming the adsorption of a second protein layer on top of the first one [13]. Exactly what happens to the three-dimensional structure of a protein when it adsorbs onto a solid surface often remains an enigma despite the wide interest and practical importance of the phenomenon. However, based on the geometry of both the adsorbent and adsorbate, useful information on this issue was gathered to further understand the adsorption process. On the one hand, a 14 nm diameter magnetite particle, as used in the present work, exposes a surface area of 61575 A˚2. On the other hand, Peters [33] reported that the shape of a BSA molecule is approximated by an ellipsoid 141 A˚ long with a maximum transverse diameter of 42 A˚. In case protein unfolding did not occur during the adsorption process, it can be calculated that the smallest cross-sectional area of a BSA
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Fig. 5. Adsorption isotherms for binding of BSA (A) and phosvitin (B) on top of a nanometer-sized magnetite core in 5 mM TES buffer, pH 7.0. The closed symbols represent the data in the absence of surfactant; the open symbols illustrate the extent of protein adsorption after treating the protein-coated particles with SDS (final concentration 5%).
molecule (which is relevant for an ‘end-on’ vertical disposition on the solid surface) equals 1384 A˚2 and, thus, about 44 BSA molecules are needed to completely occupy the zone directly adjacent to the surface of a bare Fe3O4 particle. Since the molecular weight of BSA equals 69,000 Da and the density of magnetite is 5.1 g cm3 [Handbook of Chemistry and Physics 1975–1976], a theoretical BSA/Fe3O4 weight ratio of 0.69 can be calculated in case of a complete ‘end-on’ orientation of the protein. Alternatively, if BSA faces the surface in a horizontal, ‘side-on’ direction, the relevant cross section area of an individual BSA molecule equals 4651 A˚2, resulting in the binding of only 13 BSA molecules per particle, corresponding to a value of 0.20 for the BSA/ Fe3O4 weight ratio. Since the experimentally measured ratio (0.45; see the first plateau in the adsorption isotherm of Fig. 5AK) was in between the theoretical values found for an orientation in a flat and a vertical direction, in reality some intermediate orientation was most probably adopted (Fig. 3B). Of course, in spite of the fact that BSA is considered to be a so-called ‘hard’ protein, the occurrence of a partial, entropic driven protein unfolding cannot be entirely ruled out [25,34]. To get a qualitative, albeit indirect indication of the binding strength by which protein binding occurred, the various protein-coated particles were treated for 24 h with
SDS (final concentration 5%) and then magnetically fractionated. Fig. 5AJ shows that the ‘second plateau’ BSA molecules (BSA/Fe3O440.45) were removed, whereas the molecules initially adsorbed were reluctant to the rinsing process. Thus, the prevailing protein–protein interaction forces in the second layer on top of the first one were much weaker. At high protein concentrations, similar protein arrangements were reported, for instance, by Fang and Szleifer [13] and Lee and Ruckenstein [35]. Quantitative data, proving the existence of two categories of adsorbed proteins, was provided by fitting the data of Fig. 5 by Langmuir adsorption mathematics. Below a protein concentration of 25 mg mL1 the linearized Langmuir isotherm pointed to a uniform protein binding behavior with an association constant of 1.94 mL mg1 (corresponding to 0.029 mM1) (r2 ¼ 0.996), whereas at higher free protein concentrations the data points clearly deviated from the straight line (Fig. 6K). Fitting the binding data for the remaining proteins after SDS treatment led to a similar association constant of 1.47 mL mg1 (corresponding to 0.022 mM1) (r2 ¼ 0.996) (Fig. 6J). In a similar way, we also studied the adsorption behavior of phosvitin at 4 1C in 5 mM TES buffer, pH 7.0 (final Fe3O4 concentration ¼ 1.16 mg mL1; final phosvitin concentration between 0 and 45.15 mg mL1) (Fig. 5B). At a phosvitin coverage below 0.10 mg mg1 Fe3O4, apparently, the coating was incomplete since the magnetic fluid particles precipitated; at free phosvitin concentrations above 10 mg mL1 a plateau at 0.28 mg protein mg1 iron oxide was found (Fig. 5Bm). Attempts to extract the adsorbed protein with 5% SDS failed (Fig. 5Bn) testifying a strong anchoring. This observation is in line with earlier observations which proved that the phosphorylated form of phosvitin has a high affinity for Fe3+ ions [17] and that
Fig. 6. Linearized form of the Langmuir adsorption isotherms, derived from the data in Fig. 5. The (K) symbols illustrate protein binding at a BSA concentration o25 mg mL1; the (J) symbols represent protein binding in the protein-coated particles after treatment with 5% SDS. The solid and dashed lines are linear approximations of the (K) and (J) data, respectively.
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strong chemisorption exists between iron oxide surfaces and phosphate containing molecules [20,36,37]. As compared to BSA, phosvitin (MWE40 kDa; Petersen and Cox [38]) is a more elongated molecule with a prolate ellipsoidal shape; the axial ratios being between 20 and 25. On the basis of these data, Joubert and Cook [39] assumed that the dimensions of the molecule are about 14 A˚ by 280 A˚. This means that in case of an ‘end-on’ (relevant cross section area ¼ 154 A˚2) and ‘side-on’ adsorption (relevant cross section area 3077 A˚2) 399 and 20 molecules, respectively, are anchored at maximum on a 14 nm diameter magnetite particle, as used in the present work. These values correspond to a theoretical phosvitin/Fe3O4 weight ratio of 3.63 and 0.18, respectively. Since the latter approaches the experimental value of 0.19 in the lowprotein concentration regime (cf. plateau value in Fig. 5Bm), it can be concluded that phosvitin almost uniquely adsorbed in a flat, extended form in the concentration range studied. The slightly higher plateau value measured at a higher phosvitin concentration (Fig. 5Bm) then suggests that short fragments of the polypeptide chain are pointing away from the surface. 3.4. Kinetics of competitive adsorption of BSA and phosvitin on magnetite cores Protein adsorption from a binary mixture may not be considered as the sum of the binding behavior of the individual proteins and, indeed, it is the intricate result of a combination of a number of contributions such as the individual rate of adsorption, surface availability, charge and charge distribution of the adsorbing proteins, protein conformational flexibility, intermolecular protein interactions, protein–surface binding strength, etc. [13,26]. Consequently, it was not straightforward to obtain information concerning the affinities, not even relative values, of proteins for surfaces from competitive adsorption experiments. Nevertheless, the shape of the adsorption kinetic profiles could shed some light on the consecutive steps in the binding process. In the present experimental setup, mixtures of equal mass amounts of BSA and phosvitin in 5 mM TES buffer (pH 7.0) were confronted with the iron oxide colloids (final concentration: 1.16 mg Fe3O4 mL1); the total protein/ Fe3O4 weight ratios studied were 1.29 and 12.93. Fig. 7 shows that in both cases initially adsorption of BSA was favored over that of phosvitin, which was in line with the adsorption kinetics observed with single proteins as shown in Figs. 2 and 4. In the lower protein-to-magnetite regime (Fig. 7A) the BSA content then dropped to a value of 0.17 mg protein mg1 Fe3O4. This plateau value was identical to the one measured for anchored phosvitin. Taking into account that the entire particle surface was no longer occupied by a single protein type, these values suggested that, as in the case of the single protein situation (Fig. 2), BSA was adsorbed in a hybrid ‘end-on’–‘side-on’
Fig. 7. Kinetic profiles for protein adsorption onto magnetic cores, starting from a binary protein mixture containing equal weight amounts of BSA and phosvitin. The total protein/Fe3O4 weight ratios used were 1.29 (A) and 12.93 (B). The profiles for BSA anchoring are shown by the circle symbols; those for phosvitin binding by the triangles.
orientation, whereas phosvitin molecules mainly adopted a flat position, represented schematically in Fig. 8A (compare with the relevant values for a horizontal and vertical binding mode given above). At the higher protein-to-magnetite ratio (Fig. 7B) the amount of BSA similarly levelled off to 0.20 mg BSA mg1 Fe3O4 but, surprisingly, at the same time considerably more phosvitin was adsorbed than in conditions in which it was the sole protein present in the adsorption cocktail (compare the phosvitin saturation levels shown in Figs. 5Bm and 7Bm). Possibly, with the binary protein mixture, unoccupied magnetite surface patches between the pre-adsorbed ‘‘hard’’ ellipsoidal BSA molecules were occupied by multiple phosvitin molecules, which due to their flexible structure could easily enter into these free regions. Due to spatial constraints, however, we hypothesize that each individual phosvitin entity can only stick on the surface by a reduced number of attached segments and, hence, the remaining protein fragments dangle in the solution as loops and tails interacting with water molecules [33] as represented schematically in Fig. 8B. The question as to whether the final values in the kinetic profiles represented a kinetic as opposed to a
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vesicles [20,41]. Thus, it is believed that the binding profiles represent a kinetically controlled adsorption process, rather than a thermodynamical controlled one. However, often the time needed to reach ‘true’ equilibrium conditions in adsorption experiments can be very long—days, weeks, months or even longer [42,43]. 4. Conclusions The immobilization of biomolecules to solid phase materials is of uppermost relevancy in quite a lot of biotechnological and biomedical applications. In the present work, the anchoring of a highly phosphate-loaded, flexible protein on a magnetic fluid core was compared with the adsorption behavior of a ‘classical’ globular protein. The results show that the former protein type was strongly chemisorbed on the Fe3O4 surface thereby producing a highly charged magnetic fluid formulation exhibiting improved colloidal stability with respect to long-term storage. As such, the present study paves the way for the use of these magnetizable nanocolloids in many practical applications such as the design of biocompatible materials, drug carriers and biosensors among many others. Acknowledgments
Fig. 8. Proposed model for the adsorption of BSA (cloudy ellipses) and phosvitin (solid gray figures) on Fe3O4 particles at pH 7.0. The proteins are added as a binary mixture containing equal amounts (on a mass base) of both protein types. In panel (A), the mode of protein adsorption is depicted, starting from a total protein/Fe3O4 weight ratio of 1.29; in panel (B), the ratio equals 12.93. The pictures represent the situation where protein binding has come to a (pseudo) steady state.
thermodynamic equilibrium still remains open. Elwing and Go¨lander [40] found that, even for proteins present in equal amounts in a binary mixture and which, moreover, adsorbed at the same rate, eventually, at equilibrium, protein adsorption does not necessarily occur in a 50/50 ratio. Indeed, when a protein initially adsorbs on a solid surface it forms multiple contact points. After contact is made, the bonds between protein segments and surface sites are continuously broken and reformed. Proteins bearing groups with a higher affinity can interfere with this arrangement. Ultimately, the latter (in casu the highly flexible phosvitin) will be anchored by preference. In our experimental model system, this is a realistic scenario since phosphate-iron oxide chemisorptive forces which are dominating with the highly phosphorylated ‘‘soft’’ phosvitin, indeed, are stronger than –COOH-iron oxide interactions, occurring with the ‘‘hard’’ BSA molecule. A similar difference in adsorptive capacity of –COOH and phosphate groups for magnetite surfaces was previously exploited by us to construct so-called magnetoliposomes, starting from a lauric acid-stabilized magnetic liquid and phospholipid
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