Fate of Prions in Soil: Adsorption Kinetics of Recombinant

Biomacromolecules 2005, 6, 3425-3432

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Fate of Prions in Soil: Adsorption Kinetics of Recombinant Unglycosylated Ovine Prion Protein onto Mica in Laminar Flow Conditions and Subsequent Desorption Elena N. Vasina,† Philippe De´ jardin,*,† Human Rezaei,‡ Jeanne Grosclaude,‡ and Herve´ Quiquampoix§ European Membrane Institute, UMR 5635 (CNRS, ENSCM, UMII), Universite´ Montpellier II, CC047, 2 Place Euge` ne Bataillon, F-34095 Montpellier Cedex 5, France, Virologie et Immunologie Mole´ culaires, INRA Domaine de Vilvert, F-78350 Jouy en Josas, France, and Rhizosphe` re et Symbiose, UMR 1222 (INRA-ENSAM), 2 Place Pierre Viala, F-34060 Montpellier Cedex 1, France Received July 13, 2005; Revised Manuscript Received September 2, 2005

Prions can be disseminated in soils. Their interaction with soil minerals is a key factor for the assessment of risks associated with the transport of their infectivity. We did not examine here the infectivity itself but the adsorption kinetics of an ovine recombinant prion protein (ovine PrPrec), as a noninfectious model protein, on muscovite mica, a phyllosilicate with surface properties analogous to soil clays, in conditions of laminar flow through a channel. The protein was labeled with 125I, and its adsorption examined between pH 4.0 and 9.0. At wall shear rate 100 s-1, we found the process to be controlled mainly by transport at the beginning of the adsorption process. Additional experiments at 1000 s-1 (pH 5 and 6) determined that the diffusion coefficient was in accordance with the hydrodynamic radius measured by size exclusion chromatography. The pseudo-plateau of the interfacial concentration with time was compatible with more than a monolayer and suggests the presence of aggregates. Desorption was not observed in the presence of buffer between pH 4 and 9 and sheep plasma, while the addition of alkaline detergent or 10-1 M NaOH allowed an almost complete removal from the interface. The ensemble of results suggests that the largely irreversible adsorption of the ovine PrPrec onto mica is mainly due to electrostatic attraction between the protein and the highly negatively charged mica surface. Possible consequences for the mode of dissemination of prion proteins in soils are indicated. 1. Introduction Prion proteins (PrP) are cell membrane glycosylphosphatidylinositol (GPI)-anchored proteins directly involved in transmissible spongiform encephalopathies (TSE) affecting humans and wild or domestic animals.1 A structurally modified, protease resistant, form of the cellular prion protein (PrPC) is the hallmark of the pathology (PrPSc)2, getting to a state which accumulates in tissues of TSE-infected individuals. Growing evidence supports the hypothesis that PrPSc is by itself the infectious agent. In vitro generation of infectious scrapie prions could be obtained by physical treatments3,4 and synthetic infectious prions could be obtained in vitro from recombinant mammalian prion protein expressed in procaryotes.5 A major concern in prion disease control and eradication arises from the possibility of the persistence of infectious material due to its adsorption on solid surfaces, either in surgical6,7 or environmental contexts. From a general point of view, a concentration process occurs when the proteins are adsorbed on a surface, favoring PrP precipitation, * Corresponding author. Tel: +33 467 14 91 21. Fax: +33 467 14 91 19. E-mail: [email protected]. † Universite ´ Montpellier II. ‡ INRA Domaine de Vilvert. § INRA-ENSAM.

aggregation, or formation of fibrils. So adsorption on any surface may represent an important step to transport infectious material, or material able to become infectious by the adsorption process or in later steps. Efficient and convenient decontamination processes for medical devices are needed8 taking into account the effect of extreme pH values (basic or acid) leading to PrP aggregation. Several investigations have shown that soil contamination may be involved in the horizontal transmission of scrapie in grazing herds.9-11 Experimental studies have shown that the ovine prion was still infectious after 3 years in soil.12 Soils offer a very large adsorption surface for proteins through their mineral and organomineral components.13 An adsorption/desorption cycle from soil solid surfaces might constitute a delayed risk for prion dissemination. As a whole, control of adsorption of prion protein on solid surfaces calls for further knowledge of the mechanisms involved in the interaction. To get insight into the molecular mechanisms of PrP adsorption on solid surfaces, we used a model system. In the present work, we examine the adsorption properties of ovine PrPrec onto mica, but not the infectious criteria. Recombinant ovine prion protein or PrPrec was taken as representative, at the structural point of view, of natural forms of PrP,14,15 except for the glycosyl parts. The protein is

10.1021/bm050492d CCC: $30.25 © 2005 American Chemical Society Published on Web 10/13/2005

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formed of an unstructured domain on the N-terminal side (23-90 in the ovine sequence) and a globular part on the C-terminal side. Since the N-terminal side is rich in lysine, arginine, and histidine groups, these residues may play an important role in adsorption on negative supports, whereas the globular C-terminal side bears both positive and negative residues. Given the global protein charge, the electrostatic interaction component for the couple PrPrec/mica should be attractive. Mica, a phyllosilicate, has similar physicochemical surface properties to soil clays, an important fraction of the total liquid-surface area of most soils at the microscopic level, and can be considered as a model of negatively charged mineral surfaces. The mica surface structure is very regular with an area per elementary charge (K+ in native muscovite mica) of 0.47 nm2, which corresponds to the charge density16 σ0 ) -0.35 C m-2. Adsorption studies should show if the interaction between protein and mica is strong enough to create a concentration “device”, which in addition can be transported through the landscape. Analysis of desorption should give clues about the possibility of release from the interface and the level of mobility at the interface. Mica presents also a well-defined surface chemistry and geometry, and therefore, it has been used as a flat substrate in atomic force microscopy (AFM) experiments to look at fibrils related to other analogous amyloid type diseases, including the neurodegenerative Alzheimer’s disease.17 For example, it has been used to examine the lithostatine photobril, which is involved in this disease,18 and to study the kinetics of spontaneous assembly of amyloid fibrils of wild-type β2-microglobulin.19 Similar studies could be considered with PrPrec oligomers. The common features of Alzheimer’s disease and TSE have recently been analyzed.17 Using the same experimental setup as one in a previous study on adsorption of R-chymotrypsin,20,21 one protein of about the same molar mass as the prion protein, onto mica, we continuously observed PrPrec adsorption kinetics on this material under well controlled hydrodynamic conditions. We focus in the present paper on the influence of pH on adsorption and on the stability of the adsorbed protein. 2. Materials and Methods 2.1. Protein and Buffer. A high yield one-step method for the purification (over 99% final purity) of the full-length recombinant sheep PrP was developed, based on the affinity of the conserved octapeptide repeats for transition-metal cations.22 In the present study, we used the full length A136, R154, Q171 variant which shows a β-sheeted structure in the unfolding intermediates to fibrils.23 The molar mass of the ovine PrPrec was 22 900 g/mol, including the unstructured N-terminal part. The equivalent sphere hydrodynamic radius determined by size exclusion chromatography was 3.5 nm. The crystal structure of the globular domain has recently been determined.24 It has approximate dimensions of 5.5 nm × 3.5 nm × 2.5 nm. If the molecule of mass m is adsorbed at the interface via its smallest dimensions a1 ) 3.5 nm and a2 ) 2.5 nm (“end-on” conformation), the ellipsoid model

Vasina et al.

Figure 1. Example of radioactivity decrease of the buffer solution flowing under the dialysis membrane to eliminate free iodide from the protein solution positioned above the membrane.

provides an estimation of the area occupied by one molecule as s ) πa1a2/4 ) 6.87 nm2. The compact hexagonal monolayer with a coverage fraction θ ) 0.907 gives an interfacial concentration (m/s) θ ) 0.50 µg cm-2, the square lattice model (coverage fraction 0.785) 0.43 µg cm-2. Comparing the area accounting for K+ site of muscovite mica (0.47 nm2) with the molecular size of the protein, one finds about 15 such sites per protein section area in the end-on conformation. For the adsorption studies, the protein was provided as lyophilized powder. It was radiolabeled with 125I (10-20 µL NaI alkaline solution; Amersham) by means of the Iodobeads (Pierce) technique. The labeling procedure involved 1.01.5 mL addition of the protein solution (MOPS 0.1 M, pH 7.2) to 0.5 mL of the NaI-Iodobeads solution in the same buffer, to obtain a protein concentration of 4-6 mg/mL. After labeling, the PrPrec solution was placed in a well (diameter 15 mm), with a membrane as base (MWCO 40006000 g/mol; pore size 1 nm; Cellu-Sep). Buffer (MOPS 20 mM pH 7.2) flowed under the membrane in a compartment with gentle mixing. The compartment was connected upward to a syringe-pump and downward to a beaker through Teflon tubing. The radioactivity level of the flowing dialysate was continuously recorded by positioning part of the downward Teflon tubing in the well of a γ-radioactivity detector (Figure 1). An additional control was performed by sampling (2 µL) the radioactivity of the protein solution above the membrane using Riastar equipment (Packard). The monitoring of removal of free iodide is necessary as perturbation of results by traces may occur, as observed especially on gold surfaces.25,26 The dialyzed solution was adjusted with the same buffer to a protein concentration of 1 mg/mL and stored at 4 °C as aliquots of 1 mL. We used the following buffers: Tris(hydroxymethyl)amino methane (TRIS) (Sigma) at pH 9.0 (Tris/Tris-HCl), MOPS (Sigma) at pH 6.0 (MOPS/MOPS hemisodium salt) and 7.2 (MOPS hemisodium salt), and citric acid (Sigma) at pHs 5.0 and 4.0 (Citric acid/trisodium citrate). The solutions were filtered on Millex-HV 0.45 µm (Millipore, ref SLHV025LS) to remove possible large aggregates. Concentration of stock solutions was determined by UV absorption at 280 nm, with an extinction coefficient of ) 2.52 cm2 mg-1. The protein solution concentration Cb for adsorption experiments was generally between 2.5 and 5.0 µg/mL (0.1-0.2 µM).

Adsorption of PrPrec onto Mica

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were passed through the cell. For the desorption step, several types of solutions (unlabeled protein, detergent, NaOH, urea, plasma, etc.) depending on each experiment were flowed through the cell. Continuous data acquisition began at the same time as buffer flow, and data treatment was performed using a procedure analogous to that employed for capillary geometry.28-31 All flows were managed by means of syringe pumps (Harvard Apparatus). The spacer between mica sheets was constituted of a few 125 µm thick paraffin films (Parafilm “M”, Chicago, IL) or by one 350 µm thick poly(ethyleneterephthalate) sheet (Goodfellow). We controlled the channel geometry after complete assembly of the flow cell by measuring “hydrodynamic” channel height bhyd as described elsewhere.20 The channel height bhyd was equal to the spacer thickness within a maximal variation of 3%. Figure 2. (a) View of the flow cell with the mica sheets constituting the two faces of the thin channel. (b) Schematic representation of the flow system.

3. Results and Discussion

Sheep plasma was prepared by adding 5 mL of deionized water to the lyophilized material (Sigma P4389) at 37 °C before flowing this solution through the cell using a syringe pump. 2.2. Detergents. The behavior of the adsorbed protein was examined in the presence of the cleaning agents chosen for the whole flow system. We used two types of commercial detergent, one slightly acid and the other very alkaline. Majola (Lysoform, Berlin, Germany) is a washing lotion for hands and skin with antibacterial action and contains no alkaline substances, according to the supplier. In our experiments, it was diluted to 1% concentration by weight (pH ) 5.0). RBS-50 (Chemical products SA, Bruxelles, Belgium) is an alkaline polyvalent detergent for washing machines and ultrasound baths. It contains phosphates and polyphosphates. We used it at concentration 10 wt % (pH 12.5). 2.3. Flow Cell. A flow cell, of dimensions compatible with insertion in a gamma radiation detector, 4 cm × 11 cm × 1 cm, consisted of two PMMA plates where between them two sheets of muscovite mica with a spacer between were pressed to form a rectangular channel. The channel width w was between 0.6 and 0.8 cm. Detailed description of the flow cell system has been published elsewhere.20 Figure 2, parts a and b, shows the flow cell and a scheme of the system used. The shape of the gamma counter (NaI, Tl) was especially designed to have a slit (thickness 14 mm) to insert such flow cells, instead of the common well. Mica (Class CLSS, Metafix, Montdidier, France) was freshly cleaved before experiments. In the natural mica KAl2(AlSi3O10)(OH)2, K+ binds neighboring sheets. Cleavage of mica produces two large, atomically flat surfaces, with half of the cavities on each surface occupied by K+. In aqueous solutions, the K+ ions can be exchanged by other cations.27 We used two lead shields to limit the detection to the central part (4 mm wide and 3 cm long) of the channel, to avoid recording of possible edge effects. Measurements were performed at the mean distance x ) 4 cm from the rectangular channel entrance. For the adsorption step, buffer, then the solution of the radiolabeled protein, and finally buffer again (T ) 25 °C)

The isoelectric point of the full length ovine PrPrec pI ) 9.77 was measured via isoelectric focusing and agrees with calculation (23-234pI ) 9.82; 23-124pI ) 10.0; 124-234pI ) 9.0). This means that, in the pH range of study, the protein had net positive charge, whereas mica was negative. Ionic strength is expected to be an important parameter in the determination of protein-protein and protein-surface interactions. Strong attractive forces, or screened repulsive forces, between proteins can lead to aggregation, which reveals itself by a decreased diffusion coefficient. In addition, for prion type proteins, there exists the auto-induced aggregation phenomenon by partial unfolding of the molecule. The possible occurrence of aggregation is examined via the quantitative analysis of the initial adsorption kinetics which provides an estimation of the diffusion coefficient (section 3.1). The analysis of the interfacial conformation is of interest but was outside the scope of the present work. It has recently been demonstrated using infrared absorption32 that the conformational change of a prion protein adsorbed on montmorillonite was significantly different from that observed in solution. Analogous studies for the amyloid β-peptide aggregation on surfaces were performed recently by circular dichroism.33 However, studying the stability of the adsorbed proteins in the presence of buffer (section 3.2) and other solutions (section 3.3) will give indirect information on the possible interfacial mobility. 3.1. Initial Adsorption Kinetic Constant. Contrary to the adsorption process in the absence of flow, where a concentration profile normal to the wall is developing away from the interface with time, the presence of convection gives a limit to this development by providing continuously solute molecules. Thus, a steady-state profile of concentration is reached. Practically, when the protein solution arrives in the channel, the steady-state profile (and adsorption rate) is established after some lag time if the solution concentration is low enough. The general time-independent expression can be written as (∂Γ / ∂ t) ) k(x) Cb ) ka C(x,0), where Cb is the bulk protein solution concentration; Γ is the interfacial protein concentration; t is the time; ka is the interfacial

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Figure 4. Initial adsorption rate of ovine PrPrec on mica as a function of bulk concentration. pH 6. MOPS 20 mM (ionic strength 1.2 mM). Slope k ) 1.0 × 10-4 cm s-1; x ) 4 cm; γ ) 100 s-1.

Figure 3. Parabolic velocity profile v(y) ) γ y(1 - y/b) with wall shear rate γ ) 100 s-1 in a channel of height b ) 350 µm. γ is the slope of the tangent to the velocity profile at the wall which is approximated there by v ≈ γy (dashed line). Flow occurs along the x direction.

adsorption kinetic constant; C(x,0) is the protein solution concentration at distance x from the channel entrance and at y ) 0, where y is the distance from the wall; k(x) is the initial steady-state kinetic constant of the overall process at x. In the case of the fully transport-controlled process (C(x,0) ) 0), the adsorption rate at distance x from the channel entrance depends only on transport phenomena, i.e., diffusion through the solute diffusion coefficient D, and convection through wall shear rate γ. Figure 3 illustrates the velocity profile in the channel and the wall shear rate. According to Le´veˆque34 its expression is kLev(x) ) 0.538 (D2γ / x)1/3. When the adsorption process is controlled by the interfacial reaction with kinetic constant ka, then k ) ka. The simplest approximation to describe the passage between the two limits is k-1 ) ka-1 + kLev-1. More complex expressions35 describe exactly the continuous passage from one limit to the other. We recently proposed a simple accurate approximation21 with the adapted plot of data to derive the adsorption constant and the solute diffusion coefficient. This kind of plot was used for these data. To obtain the initial adsorption rate with confidence, it is necessary to use low concentration solutions, as it is important that the surface occupation effect does not appear too soon and moreover that there is enough time available to observe the steady-state concentration profile or adsorption rate. If the concentration is too high, a major part of the surface will be occupied during the lag time and there is some risk that the correct steady-state will not be visible and, in addition, it would be too short-lived. For the solution concentrations used here, we assumed the linear variation of the adsorption rate with solution concentration, which was verified in one case (Figure 4).

Figure 5. Adsorption kinetics of ovine PrPrec on mica followed by successive flows of buffer, then different solutions. The unlabeled solutions is of the same protein concentration as the labeled one. Soap Majola for hand washing (pH 5) and RBS (pH 12.5) for labware cleaning are commercial products. (a) Tris buffer 20 mM; pH 9.0; Cb ) 3.7 µg/ mL; (b) Mops buffer 20 mM, pH 6.0; Cb ) 2.4 µg/ mL (c) citric buffer 20 mM, pH 4.0; Cb ) 5.0 µg/ mL. Straight lines illustrate the initial adsorption rates and emphasize the subsequent rate increase before its final slow-down.

Figure 5 provides examples of PrPrec adsorption and desorption kinetics on mica. The initial linear part Γ(t) gives a rather precise approximation to the initial adsorption rate


Figure 6. Experimental initial adsorption kinetic constant k(x) as a function of 1.859 k(x/γ)1/3, equivalent of (k/kLev)D2/3, where x ) 4 cm is the distance to the channel entrance, γ is the wall shear rate. Experiments performed at the same wall shear rate are on a straight line passing through the origin. At pH 5 (citric 58 mM ionic strength; Ο) and 6 (Mops 1.2 mM ionic strength; 0), the increase of wall shear rate does not show, within experimental error, any curvature toward the ordinate axis: intercept with this axis (which would be obtained from data at much higher wall shear rates) cannot be determined as ka is very large. Dashed line is the example with D ) 7.0 × 10-7 cm2 s-1 and ka ) 5.0 × 10-3 cm s-1. Only D2/3 can be estimated from the mean value of the abscissas (dash-dotted line), which is close to the expectable intercept with abscissa axis.

dΓ/dt. The initial adsorption constant k is defined by Cb-1 (dΓ/dt)tf0, where Cb is the protein bulk concentration. The representation of experimental initial adsorption kinetic constant k as a function of 1.859k(x/γ)1/3 (Figure 6), where x is the distance from the channel entrance and γ the wall shear rate, comes out from a representation of k/ka as a function of k/kLev and is made explicit in a previous work.21 When it is possible to have data describing the passage from a transport-controlled process to an interface-controlled one by varying the convection conditions, these data appear, in this type of plot, as a curve with negative curvature, the ordinate and abscissa intercepts being ka and D2/3, respectively, where D is the protein diffusion coefficient. We did not focus on this kind of study in the present work, because most experiments occurred at the same wall shear rate, about 100 s-1. As all of the recordings concern the adsorption rate at fixed distance x from the channel entrance, all of the points obtained for the same wall shear rate are on a single straight line passing through origin. Two experiments performed at 1000 s-1 (pH 5.0 and 6.0) show (Figure 6) that increasing the wall shear rate does not give any significant curvature toward the ordinate axis. These two points suggest rather a variation parallel to this axis. That means that the adsorption kinetic constant is too large to be determined. For example, the theoretical variation with ka ) 5.0 × 10-3 cm s-1 is provided in Figure 6. The very high interfacial adsorption kinetic constant ka may also be related to the observation of the adsorption rate increases (Figure 5). This type of phenomenon occurs when the adsorption constant is so high


that the surface becomes saturated upward of the point of examination very quickly. Saturating the surface upward, at distances smaller than x, suppresses the concentration depletion at the interface there and increases the adsorption rate downward by the propagation of the depletion reduction along the flow axis. Illustration of this effect by numerical simulations can be found elsewhere.20 Only the diffusion coefficient can be estimated from the mean value of abscissas from Figure 6 (Table 1; pH 5.0 and pH 6.0): The two values 6.7 and 7.2 × 10-7 cm2 s-1, although not determined with a good precision, are anyway around the value 7.0 × 10-7 cm2 s-1 deduced from the Stokes-Einstein relation D ) kBT/6πηr (kB ) 1.381 × 10-23 J K-1; T ) 298 K; η ) 9.0 × 10-4 Pa s) with the equivalent sphere radius r ) 3.5 nm determined by size exclusion chromatography.36 The relatively large hydrodynamic size, compared to the dimensions of the globular part, probably reflects the contribution of the “free” N-terminal part, in accordance with previous analysis for the full-length hamster prion protein.37 Our measurements showed that no obvious large protein aggregation occurred, in this investigated range of Cb (2.5 µg mL-1 ) 0.1 µM) and pH. The ionic strength is important in the description of the electrostatic interactions. This parameter however varies from one buffer to another (Table 1), and therefore, analysis of a pH effect alone is difficult. We note however that, in the range of wall shear rate examined in the present work, no strong effect of the ionic strength and pH was observed (Figure 7). The adsorption process was anyway mainly transport-controlled. With D ) 7.0 × 10-7 cm2 s-1, the upper theoretical transport-controlled Le´veˆque limit is kLev ) 1.24 × 10-4 cm s-1 at x ) 4 cm and wall shear rate γ ) 100 s-1. Experimental values of k were dispersed around this value. 3.2. Interfacial Concentration. We did not study in detail the amount adsorbed when the adsorption rate was near zero (pseudoplateau of interfacial concentration as a function of time). Not all experiments were carried on until the pseudoplateau situation. However, few determinations are given in Table 1. The number of determinations was too low to allow a precise study on the influence of pH and/or ionic strength. Therefore, no definitive conclusions about the absolute value can be drawn, but relative variations in the desorption process by several agents, as examined in the following paragraph, remain valuable. However, given the initially transportcontrolled process, hence a high interfacial adsorption constant ka, it is clear that the affinity is high since we found that, in the presence of quite low bulk concentrations Cb (0.1-0.2 µM), the maximal interfacial concentration was always larger than the monolayer value (0.50 µg cm-2). This would suggest the presence of some aggregates at the interface. The high affinity of the ovine PrPrec for mica was also confirmed by the difficulty to desorb the protein in the presence of buffer immediately after flowing the protein solution. The stability of the protein in the adsorbed state in the presence of other several media is discussed below. 3.3. Desorption. Figure 5 provides examples of the very low desorption, if any, by flowing buffers between pH 4.0 and 9.0. Flowing unlabeled solutions of ovine PrPrec (same concentration) at pH 6.0 has a very tiny capacity to displace

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Table 1. Initial Adsorption Kinetic Constant k for Adsorption of Ovine PrPrec on Mica at Different pHsa buffer

pH

g [s-1]

k ( std dev [cm s-1] × 104

D ( std dev [cm2 s-1] × 107

citric 20 mM citric 20 mM

4.0 5.0 5.0 6.0

Tris 20 mM

9.0

1.15 1.3 ( 0.1 2.6 1.56 1.04 ( 0.6 2.8 0.9 ( 0.3

n.d. 7.2 ( 0.3

citric 0.4 mM Mops 20 mM

100 100 1000 100 100 1000 100

ionic strength [mM] 27 58

κ-1 [nm]

rκ

Γp-plateau [µg cm-2]

1.85 1.26

0.81 1.2

0.56 0.94

n.d. 6.7 ( 1.4

1.2 1.2

8.9 8.8

0.17 0.17

0.70

n.d.

1.8

7.1

0.21

1.08

a Diffusion coefficient D estimated when experiments at 100 s-1 and 1000 s-1 were performed. n.d. ) not determined. Electrostatic characteristics of the buffer solutions: ionic strength and Debye screening length κ-1. Ratio rκ of the radius of the molecule (assuming r ) 1.5 nm) over the Debye screening length. Γp-plateau is the interfacial concentration when an almost constant value was reached during the flow of the protein solution.

Figure 7. Initial adsorption kinetic constant of ovine PrPrec onto mica as a function of pH and ionic strength (wall shear rate 100 s-1). See Table 1 for definition of the different buffers.

the initially adsorbed proteins. It is very likely that this power will decline further if the residence time at the interface before the flow of the unlabeled solution were to be increased. Neither was removal of the proteins obtained with the commercial hand-washing soap Majola (pH 5), whereas an almost complete removal (Figure 5b,c) occurred using a usual detergent solution recommended for cleaning lab-ware, commercial RBS 10%, which is very alkaline (pH 12.5) at this concentration. Using 1 M urea or nonionic detergent (triblock copolymer poly(ethylene oxide) (PEO)-polypropyleneoxide (PPO)-PEO; 8400 g/mol at 0.3% by mass) did not remove the protein from the interface. pH is probably an important parameter since similar removal was observed with NaOH 10-1 M (Figure 5a,c: 0.6% and 2% left, respectively). However, the presence of detergents in addition to alkaline conditions helps to achieve the complete cleaning of the interface (Figure 5c). Other examples are provided in Figure 8. The very alkaline conditions seem to be an important parameter for protein removal since using the more dilute NaOH 10-2 M is not so efficient (Figure 8a). Even a concentrated mixture of proteins, such as sheep plasma, could not remove the adsorbed ovine PrPrec from the interface (Figure 8b). Figure 8c illustrates that there is a stabilization process at the interface, as incubation overnight led to a

Figure 8. Illustration of the stability of the adsorbed ovine PrPrec: (a) right after adsorption in Mops 20 mM at pH 6, 10-2M NaOH was not efficient enough to remove all the protein from the interface. (b) After adsorption in Mops 20 mM at pH 6, neither buffer, nor sheep plasma were able to remove protein from the interface. (c) after adsorption at pH 9.0 in TRIS and incubation overnight in this buffer; 10-1 M NaOH did not remove all the adsorbed protein, suggesting a stabilization process. Alkaline RBS detergent was efficient, after rinsing with hand-washing soap (pH 5).

significant proportion that resisted desorption after washing with NaOH 10-1 M. The complete removal was obtained with RBS detergent after Majola soap 1%. Thus, the very alkaline pH is not the only condition for a full removal of the proteins from the interface: detergents and/or phosphates present in RBS are necessary, as it was already suggested


previously (Figure 5c) 1 h after adsorption at pH 4.0. There are probably also hydrophobic attractive interactions, which the detergents help to break. Moreover, although a huge proportion of the adsorbed protein population was removed with RBS alone, we found that a complete removal required a pretreatment with the slightly acid Majola soap. Therefore, the process of complete removal from the interface is not a simple one. However, the alkaline conditions could suggest the importance of the ionic interactions between protein and mica in the removal process. At large pH values, both protein and mica are negative and therefore have an electrostatic global repulsive component in their interactions. Under such conditions however we cannot exclude the possibility of conformational changes of the protein. The importance of the electrostatic interactions was recently emphasized in the interpretation of the binding of Syrian hamster recombinant prion protein to negative lipid membranes.38 It was proposed that the attractive component at neutral pH between the positive protein and the negative surface was decreased by the presence of NaCl. 4. Conclusion The adsorbed layer of ovine PrPrec was very stable in the presence of buffer at any pH between 4.0 and 9.0. At pH 6.0, even in the presence of an unlabeled protein solution or of plasma, almost no removal of the initially adsorbed protein occurred. The data considered together suggest strongly that the molecules which are adsorbed never leave the surface even after a very short residence time at the interface. This suggests that the mobility of the proteins at the interface was very low. Negative surfaces such as mica are able to concentrate protein, and possibly to change protein conformation. However, in the context of the hypothesis that mobility is required for the propagation of a self-induced conformation change, mica probably does not constitute a strong “pathogen” device as do the raft domains in lipid membranes,39 unless the difficulty for desorption of the protein results from a very rapid aggregation on the surface. Assuming similarity of behavior between the model protein PrPrec and the natural protein, the counterpart of this property is that a large dissemination of the prion can occur by transport on soil mineral solid surfaces, and, if infectivity is intrinsically associated to the modified protein, a potential infectivity might appear if desorption is triggered somewhere. However, within the interpretation based on the global charge of the protein to suggest an adsorption process mainly due to electrostatic interactions, at least in the first steps of adsorption, it should be kept in mind that the negatively charged glycosyl groups of natural prions could decrease significantly the isoelectric point 40,41 and hence the attractive electrostatic attractions between the prion and the mineral. An environmental consequence of these conclusions is that prions liberated in soil rich in phyllosilicates, such as clay soils, would be strongly adsorbed. This factor increases the risks of reinfestation of ruminants grazing pastures or of contamination of surface water by mineral particles transported by erosion. The vertical contamination of groundwater by colloid-facilitated transport in gravitational water perco-


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Fate of Prions in Soil: Adsorption Kinetics of Recombinant

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