7
Adsorption of Pathogenic Prion Protein to Quartz Sand XIN MA, ,‡ CRAIG H. BENSON, DEBBIE MCKENZIE, § JUDD M. AIKEN, § AND JOEL A. PEDERSEN* , ,‡, | Department of Soil Science, University of Wisconsin, Madison, Wisconsin 53706, Department of Civil and Environmental Engineering, University of Wisconsin, Madison, Wisconsin 53706, Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin 53706, and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706 Management responses to prion diseases of cattle, deer, and elk create a significant need for safe and effective disposal of infected carcasses and other materials. Furthermore, soil may contribute to the horizontal transmission of sheep scrapie and cervid chronic wasting disease by serving as an environmental reservoir for the infectious agent. As an initial step toward understanding prion mobility in porous materials such as soil and landfilled waste, the influence of pH and ionic strength (I) on pathogenic prion protein (PrP Sc ) properties (viz. aggregation state and œ-potential) and adsorption to quartz sand was investigated. The apparent average isoelectric point of PrP Sc aggregates was 4.6. PrP Sc aggregate size was largest between pH 4 and 6, and increased with increasing I at pH 7. Adsorption to quartz sand was maximal near the apparent isoelectric point of PrP Sc aggregates and decreased as pH either declined or increased. PrP Sc adsorption increased as suspension I increased, and reached an apparent plateau at I 0.1 M. While trends with pH and I in PrP Sc attachment to quartz surfaces were consistent with predictions based on Born-DLVO theory, non-DLVO forces appeared to contribute to adsorption at pH 7 and 9 (I ) 10 mM). Our findings suggest that disposal strategies that elevate pH (e.g., burial in lime or fly ash), may increase PrP Sc mobility. Similarly, PrP Sc mobility may increase as a landfill ages, due to increases in pH and decreases in I of the leachate. Introduction Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal, neurodegenerative disorders affecting a variety of mammalian species and include bovine spongiform encephalopathy (“mad cow” disease), chronic wasting disease (CWD) of deer, elk and moose, sheep scrapie, and Creutzfeldt-Jakob disease in humans (1). These protein- misfolding diseases are characterized by spongiform de- generation of the brain, accumulation of abnormal prion protein, personality and memory changes, loss of coordina- tion, and inevitably death (1). No cure exists for these diseases. The infectious agents in these diseases, referred to as prions (from proteinaceous-infectious particles), are apparently devoid of nucleic acid, and are composed primarily, if not exclusively, of a misfolded form of the prion protein, a normally benign cell-surface glycoprotein designated PrP C (1). The disease-associated form of the prion protein (PrP Sc ) is identical to PrP C in amino acid sequence and covalent post-translational modifications (1). PrP C and PrP Sc differ only in their conformation (i.e., folding): PrP C has a high R-helix content (42%) and negligible -sheet character (3%), while PrP Sc is rich in -sheet (43%) and exhibits diminished R-helix content (30%) relative to PrP C (2). Conversion of PrP C to PrP Sc represents the central event in prion disease propagation and confers distinct biophysical properties to the protein including dramatically increased protease re- sistance, marked detergent insolubility, and a propensity to aggregate (1). Prions exhibit extraordinary resistance to a variety of conditions that inactivate conventional pathogens including exposure to ionizing, ultraviolet, and microwave radiation; protease treatment; contact with most chemical disinfectants; boiling; autoclaving under conventional con- ditions; and dry heat at temperatures under 600 °C(3, 4). An environmental reservoir of prion infectivity appears to contribute to the transmission of CWD and scrapie (5). Several lines of evidence support the concept that soil comprises a component of this reservoir (5). Prions can persist in soil for g 3y(6) and can be introduced into soil when infected carcasses decompose and through shedding in saliva and, presumably, feces (7). The presence on the landscape of decomposed infected carcasses or residual excreta from infected animals is sufficient to transmit CWD to mule deer (8). Oral transmission of CWD via saliva from infected deer has been demonstrated (7), and shedding of prions in urine from animals with chronic kidney inflammation has been reported (9). Since herbivores consume soil both deliberately and incidentally (10, 11), ingestion of prion-contaminated soil may contribute to the natural CWD and scrapie transmission (5, 12). Montmorillonite-associated prions were recently demonstrated to retain infectivity despite extremely avid adsorption of PrP Sc to the clay particles (12). Mechanisms governing prion interaction with soil par- ticles remain poorly understood. Greater insight into these mechanisms will improve our ability to predict prion bioavailability and mobility in natural environments and engineered systems (e.g., landfills). For example, the acces- sibility of prions to grazing animals and other soil-ingesting species is expected to depend in part on retention of the infectious agent near the soil surface, while the risk posed by landfilling infectious materials depends on prion mobility in the waste, soils used for daily cover, and granular materials used for leachate collection (5). As an initial step toward understanding prion interaction with soil and landfill materials, batch sorption experiments were conducted to assess pH and ionic strength (I) influences on PrP Sc adsorption to quartz sand. Experimental results were interpreted using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid stability (13, 14). Materials and Methods Chemicals. Analytical grade 3-N-morpholinopropanesulfonic acid (MOPS), Tris-(hydroxymethyl)aminomethane HCl (Tris), and sodium acetate were obtained from Fisher Scientific (Hampton, NH). * Corresponding author phone: (608) 263-4971; fax: (608) 265- 2595; e-mail: [email protected]. Department of Soil Science. Department of Civil and Environmental Engineering. § Department of Comparative Biosciences. | Molecular and Environmental Toxicology Center. Environ. Sci. Technol. 2007, 41, 2324-2330 2324 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 7, 2007 10.1021/es062122i CCC: $37.00 2007 American Chemical Society Published on Web 03/06/2007

Adsorption of Pathogenic Prion Protein to Quartz Sand

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Page 1: Adsorption of Pathogenic Prion Protein to Quartz Sand

Adsorption of Pathogenic PrionProtein to Quartz SandX I N M A , † , ‡ C R A I G H . B E N S O N , ‡

D E B B I E M C K E N Z I E , §

J U D D M . A I K E N , § A N DJ O E L A . P E D E R S E N * , † , ‡ , |

Department of Soil Science, University of Wisconsin, Madison,Wisconsin 53706, Department of Civil and EnvironmentalEngineering, University of Wisconsin, Madison, Wisconsin53706, Department of Comparative Biosciences, University ofWisconsin, Madison, Wisconsin 53706, and Molecular andEnvironmental Toxicology Center, University of Wisconsin,Madison, Wisconsin 53706

Management responses to prion diseases of cattle, deer,and elk create a significant need for safe and effectivedisposal of infected carcasses and other materials.Furthermore, soil may contribute to the horizontal transmissionof sheep scrapie and cervid chronic wasting disease byserving as an environmental reservoir for the infectious agent.As an initial step toward understanding prion mobility inporous materials such as soil and landfilled waste, theinfluence of pH and ionic strength (I) on pathogenic prionprotein (PrPSc) properties (viz. aggregation state andú-potential) and adsorption to quartz sand was investigated.The apparent average isoelectric point of PrPSc aggregateswas 4.6. PrPSc aggregate size was largest between pH4 and 6, and increased with increasing I at pH 7. Adsorptionto quartz sand was maximal near the apparent isoelectricpoint of PrPSc aggregates and decreased as pH eitherdeclined or increased. PrPSc adsorption increasedas suspension I increased, and reached an apparentplateau at I ∼ 0.1 M. While trends with pH and I in PrPSc

attachment to quartz surfaces were consistent withpredictions based on Born-DLVO theory, non-DLVO forcesappeared to contribute to adsorption at pH 7 and 9 (I )10 mM). Our findings suggest that disposal strategies thatelevate pH (e.g., burial in lime or fly ash), may increasePrPSc mobility. Similarly, PrPSc mobility may increase as alandfill ages, due to increases in pH and decreases in Iof the leachate.

IntroductionTransmissible spongiform encephalopathies (TSEs), or priondiseases, are fatal, neurodegenerative disorders affecting avariety of mammalian species and include bovine spongiformencephalopathy (“mad cow” disease), chronic wastingdisease (CWD) of deer, elk and moose, sheep scrapie, andCreutzfeldt-Jakob disease in humans (1). These protein-misfolding diseases are characterized by spongiform de-generation of the brain, accumulation of abnormal prion

protein, personality and memory changes, loss of coordina-tion, and inevitably death (1). No cure exists for these diseases.The infectious agents in these diseases, referred to as prions(from proteinaceous-infectious particles), are apparentlydevoid of nucleic acid, and are composed primarily, if notexclusively, of a misfolded form of the prion protein, anormally benign cell-surface glycoprotein designated PrPC

(1). The disease-associated form of the prion protein (PrPSc)is identical to PrPC in amino acid sequence and covalentpost-translational modifications (1). PrPC and PrPSc differonly in their conformation (i.e., folding): PrPC has a highR-helix content (42%) and negligible â-sheet character (3%),while PrPSc is rich in â-sheet (43%) and exhibits diminishedR-helix content (30%) relative to PrPC (2). Conversion of PrPC

to PrPSc represents the central event in prion diseasepropagation and confers distinct biophysical properties tothe protein including dramatically increased protease re-sistance, marked detergent insolubility, and a propensity toaggregate (1). Prions exhibit extraordinary resistance to avariety of conditions that inactivate conventional pathogensincluding exposure to ionizing, ultraviolet, and microwaveradiation; protease treatment; contact with most chemicaldisinfectants; boiling; autoclaving under conventional con-ditions; and dry heat at temperatures under 600 °C (3, 4).

An environmental reservoir of prion infectivity appearsto contribute to the transmission of CWD and scrapie (5).Several lines of evidence support the concept that soilcomprises a component of this reservoir (5). Prions can persistin soil for g 3 y (6) and can be introduced into soil wheninfected carcasses decompose and through shedding in salivaand, presumably, feces (7). The presence on the landscapeof decomposed infected carcasses or residual excreta frominfected animals is sufficient to transmit CWD to mule deer(8). Oral transmission of CWD via saliva from infected deerhas been demonstrated (7), and shedding of prions in urinefrom animals with chronic kidney inflammation has beenreported (9). Since herbivores consume soil both deliberatelyand incidentally (10, 11), ingestion of prion-contaminatedsoil may contribute to the natural CWD and scrapietransmission (5, 12). Montmorillonite-associated prions wererecently demonstrated to retain infectivity despite extremelyavid adsorption of PrPSc to the clay particles (12).

Mechanisms governing prion interaction with soil par-ticles remain poorly understood. Greater insight into thesemechanisms will improve our ability to predict prionbioavailability and mobility in natural environments andengineered systems (e.g., landfills). For example, the acces-sibility of prions to grazing animals and other soil-ingestingspecies is expected to depend in part on retention of theinfectious agent near the soil surface, while the risk posedby landfilling infectious materials depends on prionmobility in the waste, soils used for daily cover, and granularmaterials used for leachate collection (5). As an initial steptoward understanding prion interaction with soil and landfillmaterials, batch sorption experiments were conducted toassess pH and ionic strength (I) influences on PrPSc adsorptionto quartz sand. Experimental results were interpreted usingthe Derjaguin-Landau-Verwey-Overbeek (DLVO) theoryof colloid stability (13, 14).

Materials and MethodsChemicals. Analytical grade 3-N-morpholinopropanesulfonicacid (MOPS), Tris-(hydroxymethyl)aminomethane HCl (Tris),and sodium acetate were obtained from Fisher Scientific(Hampton, NH).

* Corresponding author phone: (608) 263-4971; fax: (608) 265-2595; e-mail: [email protected].

† Department of Soil Science.‡ Department of Civil and Environmental Engineering.§ Department of Comparative Biosciences.| Molecular and Environmental Toxicology Center.

Environ. Sci. Technol. 2007, 41, 2324-2330

2324 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 7, 2007 10.1021/es062122i CCC: $37.00 2007 American Chemical SocietyPublished on Web 03/06/2007

Page 2: Adsorption of Pathogenic Prion Protein to Quartz Sand

PrPSc Source. Samples enriched in PrPSc were preparedfrom brains of hamsters clinically infected with the Hyperstrain of hamster-adapted transmissible mink encephal-opathy using the Bolton et al. (15) procedure modified byexcluding proteinase K digestion (16). The resulting prepara-tion (in 0.01 M Tris pH 7.4, 0.133 M NaCl) corresponded to4 g brain‚mL-1 (see Supporting Information for protein geland immunoblot of prion preparation) and contained ∼109

infectious units‚g-1 as determined by end-point titrationbioassay.

Characterization of PrPSc. Electrophoretic mobilities ofPrPSc aggregates were measured using a ZetaSizer Nano ZSinstrument (Malvern Instruments, Worcestershire, UK) overa range of pH and I conditions. Prion-enriched samples (2µL) were diluted to the equivalent of 8 mg brain‚mL-1 in750-µL solutions of desired pH and I, equilibrated for g10min, sonicated for 3 min, and transferred to folded capillarycells for electrophoretic mobility measurement. For I >0.05M, the Fast Field Reversal mode was used. Instrumentperformance was verified using manufacturer-providedpolymer microsphere transfer standards.

Changes in apparent PrPSc aggregate size as a function ofpH and I were examined by dynamic light scattering (DLS;ZetaSizer Nano ZS). After diluting PrPSc preparation to theequivalent of 32 mg brain‚mL-1 in solutions of desired pHand I, the suspension was equilibrated for g10 min, sonicatedfor 3 min, and transferred to a glass cuvette. Three measure-ments (10 runs per measurement) were acquired from eachof triplicate samples for each solution condition. Theautocorrelation function was analyzed by the method ofcumulants to obtain the moments of the PrPSc aggregate sizedistribution. This method is appropriate for particle suspen-sions with narrow to intermediate polydispersities (e.g., 0.2-0.4). The average polydispersity of PrPSc aggregates underthe solution conditions employed was 0.4. The intensity-average (Z-average) hydrodynamic radius (rh,z) was calculatedfrom measured diffusivities using the Stokes-Einstein equa-tion. The rh,z is a single, intensity-averaged value representingthe entire size distribution.

Quartz Sand Preparation and Characterization. IOTAquartz sand (Unimin Corporation, New Canaan, CT) wasfractionated by wet sedimentation/flotation to obtain the0.18-0.25 mm particle size fraction (17) (98% between 0.12and 0.25 mm, sieve analysis, ASTM D 422). Metal and organiccontaminants were removed from fractionated sand by 24-hsoaking in 12 N HCl, rinsing with distilled deionized water(ddH2O), and baking overnight at 800 °C (17, 18). Cleanedsand was stored in a vacuum desiccator and rehydrated byboiling for g1 h in ddH2O prior to use.

X-ray diffraction (Scintag PAD V diffractometer, Cupertino,CA) and X-ray fluorescence analyses (Bruker AXS Model 3400,Madison, WI) indicated that the cleaned sand contained99.5% quartz and 0.5% bytownite plagioclase feldspar (aver-age formula: Ca0.87Na0.13Al2Si2O8) by mass. The sand had a2.62 ( 0.11 mmol(+)‚kg-1 cation exchange capacity (com-pulsive exchange with Ba2+, ref 19), a 0.20 m2‚g-1 specificsurface area (Kr adsorption, Brunauer-Emmet-Tellermethod, Micrometrics Analytical Services, Norcross, GA), anda 2.65 specific gravity (ASTM D 854).

Batch Sorption Experiments. PrPSc adsorption to quartzsand particles was examined as a function of pH, I, and prionprotein concentration in batch sorption experiments. Solu-tion pH was maintained using 0.01 M acetate (pH 3-5), MOPS(pH 6-7), or Tris (pH 8-9); the desired I was achieved byNaCl addition.

Quartz sand (100 mg) was equilibrated with 6 mL ofsolutions of desired pH and I in 50-mL fluorinated ethylenepolypropylene (Teflon FEP) tubes for >15 h. An aliquot ofprion preparation [20 µL (equivalent to 0.08 g brain) for pHand I experiments; 5-50 µL (equivalent to 0.02-0.2 g brain)

for isotherm experiments] was sonicated for 3 min at 750 W(CV33 probe, Sonics and Materials GE750, Newtown, CT),then added to the sand. The mixture was then gently shaken(∼14 rpm) at 23-25 °C for 4 h. Previous experiments (12)indicated that adsorption was complete within this time. Allexperiments were conducted in triplicate.

To separate unbound from adsorbed PrPSc, the PrPSc-sandsuspension was settled under gravity through a 2-mL sucrosecushion (0.75 M) followed by 7-min centrifugation at 1500gthrough a 0.5-mL sucrose cushion (0.75 M). Supernatantsfrom both separations were combined. The solutions usedin adsorption experiments were used to prepare the sucrosecushions (12). The sedimented sand was washed four timeswith the same solution as used in the adsorption experiment,and the first three wash solutions were combined with thesupernatant. The final wash solution was analyzed separately(results combined with those of the supernatant during dataanalysis). After 3-min sonication, 100-µL aliquots from eachsupernatant were transferred into LoBind microcentrifugetubes. A 100-µL aliquot of 10 M urea (in 0.01 M Tris HCl, pH7.4) was added to each supernatant and final wash sampleto achieve a 5 M urea concentration. PrPSc in these sampleswas denatured by 10-min heating at 100 °C in 5 M urea.

Adsorbed PrPSc was desorbed from the sand using twosequential extractions. In the first step, PrPSc was extractedand denatured with 10 M urea at 100 °C for 10 min. Ureaextracts were analyzed by enzyme-linked immunosorbentassay (ELISA). The sand was then re-extracted with 10%sodium dodecylsulfate (SDS) in 0.1 M Tris pH 8.0, 7.5 mMEDTA, 0.1 M DTT, and 30% glycerol at 100 °C for 10 min (12).SDS extracts were analyzed by immunoblotting.

Enzyme-Linked Immunosorbent Assay. PrPSc in thesupernatant, sedimented sand wash, and primary extract ofthe sedimented sand was measured using a double-antibodysandwich ELISA (SPI-bio, Massy Cedex, France) distributedby Cayman Chemicals (Ann Arbor, MI). Following themanufacturer’s instructions, samples were diluted to e0.5M urea and analyzed with the colorimetric ELISA. Absorbanceat 410 nm was measured with a Dynatech model MRXmicroplate reader (Chantilly, VA). PrPSc was quantified against8-point calibration curves prepared by serial dilution of thestarting PrPSc preparation in the same solutions as used inadsorption experiments. Samples with absorbances outsidethe linear range of the standard curve were diluted andreanalyzed. Preliminary experiments using immunoblotanalysis indicated that capture and detection antibodyepitopes were not lost on detachment from the quartz grains,and that the pH and I of the solutions used in the adsorptionexperiments did not affect quantitation by ELISA.

Immunoblot Analysis. Immunoblot analysis was con-ducted as described previously (12). Briefly, samples werefractionated on 4-20% pre-cast polyacrylamide gels (Bio-Rad, Hercules, CA) under reducing conditions, transferredto PVDF membranes, and immunoblotted with anti-PrPmonoclonal antibody 3F4. Horseradish peroxidase-conju-gated goat anti-mouse immunoglobulin G (Bio-Rad) was usedfor detection.

Born-DLVO Calculations. The total energy of PrPSc

interaction with quartz surfaces (ΦT) was calculated bysumming the electrostatic double layer (ΦEDL), van der Waals(ΦVDW), and Born (ΦBorn) contributions (20):

Electrostatic double layer interaction energies werecalculated for constant potential surfaces by modeling thePrPSc-quartz interaction with sphere-plate geometry (21):

ΦT ) ΦEDL + ΦVDW + ΦBorn (1)

VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2325

Page 3: Adsorption of Pathogenic Prion Protein to Quartz Sand

where ε0 is the permittivity of free space, εr is the relativepermittivity of water, rp is the PrPSc aggregate radius, ψp andψc are the PrPSc aggregate and the quartz particle surfacepotentials, κ is the inverse Debye length, and h is theseparation distance between the PrPSc aggregate and thequartz surface. Potential energy calculations used rh,z for PrPSc

aggregate size. While rh,z are larger than hydrodynamic radii(rh) based on number distributions, calculating rh fromnumber distributions requires numerous assumptions aboutthe aggregate size distribution that may not be valid. Theú-potentials of PrPSc aggregates and quartz surfaces wereused as estimates for ψp and ψc. The magnitude of the κ

depends solely on the properties of the liquid and wascalculated for the aqueous 1:1 electrolyte solutions at 25 °Cusing the following equation (22):

The retarded van der Waals interaction energy wascalculated using the following expression (23):

where A denotes the Hamaker constant. The characteristicwavelength of the dielectric, λ, is typically assumed to be 100nm (24).

The Born repulsion energy was calculated using thefollowing expression (25):

where the Born radius (σB) was taken to be 0.5 nm (21). Thisstrong, very short-range repulsive force arises from theoverlap of electron clouds of atoms and determines thesmallest separation distance between two molecules (22).

The Hamaker constant (App) for PrPSc (p) is not currentlyavailable. Bovine fibrin was used as a surrogate. Like PrPSc,bovine fibrin is hydrophobic, tends to aggregate in aqueoussuspension (25), and has similar R-helix and â-sheet content[∼30% and ∼40% (26) as compared to ∼30% and ∼43% forPrPSc (2)]. An estimate of App (7.47 × 10-20 J) was obtainedfrom the Lifshitz-van der Waals component of the totalsurface tension (γp

LW ) 40.2 mJ‚m-2) of bovine fibrin usingthe following(25):

where lo denotes the minimum equilibration distance (1.568( 0.093 Å, ref 25).

The Hamaker constant (Apwq) for PrPSc interacting withquartz (q) across water (w) was calculated as 5.42 × 10-21 Jusing the following combining relation (22):

where Aww and Aqq are the Hamaker constants for water (4.62× 10-20 J, ref 25) and crystalline SiO2 (9.47 × 10-20 J, ref 27).

Results and DiscussionElectrokinetic Properties of PrPSc Aggregates and QuartzGrains. The ú-potential is the potential measured by elec-

trokinetic methods at the shear plane separating the thinlayer of liquid adhering to the particle surface from the bulksolution; ú-potentials are commonly used as surrogates forsurface potentials (25). The average ú-potential of PrPSc

aggregates was slightly positive at pH e 4 and becamenegative as pH increased (Figure 1a). PrPSc aggregatesexhibited an apparent average isoelectric point (pI) of 4.6,within the pI ranges reported previously (28, 29). The averageú-potential at pH 7 became less negative as I increased dueto electrostatic double layer compression (Figure 1b). Theú-potentials for PrPSc aggregates remained stable over 4 h.The ú-potentials for the sand were negative for all pH andI conditions (Figure 1a), as expected from the point of zerocharge for quartz (2.9, ref 30), and were comparable to thosereported previously for the same sand (18, 31).

Prion Protein Aggregate Size. Transmission electronmicroscopy demonstrated that the prion enrichment pro-cedure yielded the expected rod-like structures (data notshown). DLS was used to determine PrPSc aggregate hydro-dynamic radii (rh) in the prion-enriched preparation. Thehydrodynamic radius is equivalent to the radius of hardsphere with the same diffusion coefficient. PrPSc aggregaterh do not necessarily reflect the effective radii of individualrods as they may include contributions from aggregates ofrods.

ΦEDL ) πε0εrrp{2ψpψcln[1 + exp(- κh)

1 - exp(- κh)] + (ψp2 + ψc

2)ln

[1 - exp( -2κh)]} (2)

κ ) 0.304

x[NaCl](3)

ΦVDW ) -Arp

6h [1 + 14hλ ]-1

(4)

ΦBorn )AσB

6

7560[ 8rp + h

(2rp + h)7+

6rp - h

h7 ] (5)

App ) 24πlo2γp

LW (6)

Apwq ≈ (xApp - xAww)(xAqq - xAww) (7)

FIGURE 1. Intensity-averaged hydrodynamic radii of PrPSc ag-gregates and ú-potentials of PrPSc aggregates and quartz particlesas a function of (a) pH and (b) ionic strength. Solutions were bufferedwith 0.01 M acetate (pH 3-5), MOPS (pH 6-7), or Tris (pH 8-9).Desired ionic strength was achieved by addition of NaCl. In (a),ionic strength was maintained at 0.01 M; in (b) pH was maintainedat 7. Mean electrophoretic mobilities of PrPSc aggregates (determinedat 150 V and 25 °C from triplicate samples, 3 measurements persample, 25 runs per measurement) and streaming potentials of thequartz sand (Paar Physica Electro Kinetic Analyzer (Anton PaarUSA, Ashland VA) at the Particle Engineering Research Center,University of Florida) were converted to ú-potentials using theSmoluchowski equation. The ú-potential at 100 mM NaCl exhibitedlarge variation because the change in voltage with pressure duringstreaming potential measurement was near zero. Data points aremeans of at least 3 replicate samples; error bars represent 1 standarddeviation. Variations in rh,z among prion preparations were withinthe error of the measurement.

2326 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 7, 2007

Page 4: Adsorption of Pathogenic Prion Protein to Quartz Sand

PrPSc aggregate rh,z varied depending on solution condi-tions (Figure 1). As pH increased from 3.0 to 4.0, PrPSc

aggregate rh,z increased significantly (p ) 0.00145) (Figure1a). PrPSc aggregate size remained relatively constant betweenpH 4 and 6. Near the pI, electrostatic repulsion betweenPrPSc molecules is at a minimum, favoring aggregation. AspH increased to 8, PrPSc aggregate size declined; furtherincreases in pH did not change PrPSc aggregate rh,z. PrPSc

aggregate rh,z increased as I rose from 4.2 to 10 mM; furtherincreases in I did not affect rh,z (Figure 1b). Increasing Icompresses the electrostatic double layer allowing closerapproach of like-charged particles, favoring aggregation. Therh,z values were stable over a 4-h period.

Inspection of scattering intensity distributions revealedthat PrPSc aggregate size distributions were bimodal at pH7 and 9 (I ) 10 mM) with most of the scattering intensity (87to >99%) contributed by large aggregates; scattering intensitydistributions were unimodal for pH 4 (I ) 10 mM) and forI g 100 mM (data not shown). Although derivation ofhydrodynamic radii based on mass and especially numberdistributions from DLS data involves numerous assumptions,the mass and number distributions can be useful for makingcomparisons among samples. However, confidence in the rh

values based on mass and number distributions is less thanfor rh,z. Intensity, mass, and number distributions exhibitedsimilar trends with pH and I. Number distributions revealedthat at pH 7 and 9 under low I conditions, the majority ofPrPSc aggregates was substantially smaller than those re-sponsible for most of the scattering. For the dominant mode,rh estimated on a number basis declined as pH increased (I) 10 mM): 333 nm (pH 4) > 60 nm (pH 7) > 31 nm (pH 9).At pH 7, rh estimated from the number distribution increasedfrom 60 nm at I ) 10 mM to 429 and 443 nm at I ) 100 and500 mM.

Effect of pH and I on PrPSc Adsorption to QuartzParticles. PrPSc adsorption to quartz sand exhibited amaximum at pH 4 (Figure 2a), near the apparent pI (4.6) ofPrPSc aggregates. PrPSc adsorption declined at higher andlower pH. Similar pH-dependent behavior has been notedfor the sorption of other proteins to mineral surfaces (32).At the pI, electrostatic interactions of proteins with high-energy surfaces are at a minimum while “hydrophobic”attraction is maximal (22). Declines in protein adsorptionfor pH values on either side of the pI have been ascribed tolateral electrostatic repulsion of like-charged protein mol-ecules or conformational changes (unfolding) on the surface(33), increasing the area occupied by a single protein (34).The operation of different mechanisms above and belowprotein pI has also been proposed (32, 33). Below the pI, thepositively charged protein can unfold due to strong elec-trostatic attraction to the negatively charged surface; abovethe pI, both the protein and surface carry net negative chargeand electrostatic repulsion disfavors adsorption.

The effect of I on PrPSc adsorption to quartz sand wasexamined at pH 7 because near-neutral pH values arecommonly encountered in the environment. At pH 7, PrPSc

adsorption to quartz particles increased as I rose from 4.2 to10 mM (p ) 0.0447); further increases in I up to 300 mM didnot enhance adsorption (Figure 2b). At this pH, both thePrPSc aggregate and the quartz surface were negativelycharged. Double layer compression at higher I apparentlyreduced electrostatic repulsion between PrPSc and the quartzsurface leading to increased absorption.

Adsorption Isotherms. PrPSc adsorption to quartz sandwas examined as a function of protein concentration at pH4 and 7 (Figure 3). In the pH 7 experiments, low levels ofPrPSc were observed in SDS extracts from three samples (initialPrPSc concentration, C0 g 0.02 g brain equivalent‚mL-1); forthese samples, sorbed PrPSc concentrations were based onthe combined results from ELISA and immunoblot analysis.

The presence of PrPSc in SDS extracts at concentrations belowthe limit of immunoblotting detection cannot be excluded,but would be expected to decline as C0 decreased. To evaluate

FIGURE 2. Adsorption of PrPSc to quartz sand as a function of (a)pH (I ) 0.01 M) and (b) ionic strength (pH ) 7.01 ( 0.03, 0.01 MMOPS). Initial PrPSc concentration, C0 was equivalent to 0.08 gbrain per mL. Proton activity in (a) was maintained using 0.01 Macetate (pH 3-5), MOPS (pH 6-7), or Tris (pH 8-9); ionic strengthwas adjusted to 0.01 M with NaCl. Fraction of PrPSc adsorbedcalculated from CsMs/(CsMw + CwVw), where Cs and Cw are themeasured bound and unbound concentrations, Ms is adsorbent mass,and Vw is solution volume. Data points are means of triplicatemeasurements; error bars represent one standard deviation.

FIGURE 3. Isotherms for PrPSc adsorption to quartz sand at pH 4 and7. Solution pH was buffered to 4.04 or 7.07 with 0.01 M acetate orMOPS. Solid lines correspond to least-squares fits of the experi-mental data to a linear isotherm. For pH 4, Kd ) 8.8 ( 1.6 L‚kg-1

(p < 0.0001), R 2 ) 0.65; for pH 7, Kd ) 3.0 ( 0.4 L‚kg-1 (p < 0.0001),R 2 ) 0.82. Dashed lines are least-squares fits of the experimentaldata to a linearized form of the Freundlich isotherm (obscured inthe case of the pH 7 data). Freundlich equation parameters: for pH4, log KF ) 1.52 ( 0.46 (p ) 0.0044), n ) 0.88 ( 0.13 (p < 0.0001),R 2 ) 0.74; pH 7, log KF ) 0.10 ( 0.49 (p ) 0.85), n ) 1.10 ( 0.14(p < 0.0001), R 2 ) 0.80. Note KF value for pH 7 was not significant.The lack of an asymptotic approach to a plateau in the Cs vs Cw

plots suggests that the adsorption capacity of the quartz sand wasnot attained in these experiments.

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the effect of hypothetical and nondetectable PrPSc in SDSextracts, the maximum error in the sorbed PrPSc concentrationwas computed assuming that the concentration of nonde-tectable PrPSc in the extract equaled the limit of immuno-blotting detection (∼1.2 × 10-4 g brain equivalent). With asingle exception, this calculation indicated that nondetectablePrPSc introduces a maximum error in the sorbed concentra-tions of 0.9 to 9.7%. The exception was a maximum 29.0%error in samples with C0 ) 0.02 g brain in the pH 7experiments. Actual error in this and the other samples withno detectable PrPSc in the SDS extracts was likely smaller.

Experimental data were fit to a linear isotherm, Cs ) Kd‚Cw, where Cs and Cw are the concentrations sorbed to thequartz particles and remaining in suspension as measuredby ELISA (and immunoblotting for the three highest PrPSc

concentrations in the pH 7 experiment), and Kd is thedistribution coefficient (L‚kg-1). Least-squares fits to the linearmodel had R 2 ) 0.65 (pH 4) and 0.82 (pH 7). Scatter in thedata is attributed to heterogeneity in the PrPSc population,which is inherent structurally (e.g., degree of glycosylationof monomeric PrPSc, glycan composition), conformationally(e.g., both proteinase K-sensitive and -resistant PrPSc arepresent; ref 37) and in aggregation state. As expected fromobserved trend in PrPSc adsorption with pH (Figure 2), Kd

was larger at pH 4 (near the average apparent pI of the PrPSc

aggregates; Kd ) 8.8 ( 1.6 L‚kg-1) than at pH 7 (PrPSc

aggregates carrying net negative charge; Kd ) 3.0 ( 0.4 L‚kg-1).In general, least-squares fits of the experimental data to

the Freundlich, Langmuir, Langmuir-Freundlich, Temkin,and Redlich-Peterson equations either resulted in non-significant model parameters (p > 0.05) or failed to increaseR 2 (data not shown). The sole exception was a fit at pH 4 tothe linearized form of the Freundlich equation: log Cs )n‚log Cw + log KF, where KF is the adsorption capacity at aspecific Cw, and n provides a measure of the distribution ofadsorption energies. The adsorption isotherm at pH 4exhibited marked nonlinearity (n ) 0.88 ( 0.13, p < 0.001),suggesting a distribution of energies of interaction betweenPrPSc aggregates and quartz surfaces.

Interaction Energy Profiles. At pH 7 (I ) 10-500 mM)and 9 (I ) 10 mM), the sand and PrPSc aggregates carried netnegative charge (Figure 1). Thus, electrostatic repulsionshould inhibit PrPSc attachment to the quartz surfaces.Nevertheless, PrPSc adsorption was observed under unfavor-able electrostatic conditions. To obtain further insight intomechanisms controlling PrPSc adsorption to the quartz grains,interaction energies for PrPSc aggregates and quartz surfaceswere calculated as a function of separation distance bysumming electrostatic double layer and van der Waalsinteraction energies (DLVO theory) and the Born repulsionenergy (20).

Total Born-DLVO interaction energy profiles indicatedfavorable conditions for PrPSc attachment to quartz surfacesat pH 4 (I ) 10 mM) (Figure 4); no secondary minimum(Φ2°min) and no energy barrier (Φmax) to adsorption in theprimary minimum (Φ1°min) were predicted (Table 1). At pH7 and 9 (I ) 10 mM), substantial Φmax (110 kBT and 217 kBT)and shallow Φ2°min values (0.40 kBT and 0.19 kBT) werepredicted (Table 1). At pH 7, increasing I from 10 to 100 mMresults in disappearance of Φmax and deepening of Φ1°min

allowing adsorption in the primary minimum. While thepredicted trends with pH and I appear consistent with ourexperimental results (Figure 2), the shallow Φ2°min predictedfor pH 7 and 9 (I ) 10 mM) appear insufficient for attachment(i.e., Φ2°min <1 kBT), and the Φmax are sufficiently large topreclude adsorption in the primary minimum (25). Thesefindings suggest additional mechanisms contribute to PrPSc

attachment to quartz surfaces (25). PrPSc was not readilydesorbed by solutions having the same composition as thoseused in adsorption experiments. However, significant de-

sorption occurred with concentrated urea, suggesting thatLewis acid-base interactions (e.g., the cohesion of water, ref25) are important in PrPSc binding to the quartz surface.Additional factors potentially contributing to PrPSc adsorptionto quartz surfaces include steric forces (22) and locallyfavorable nanoscale interactions (25, 34, 35). Hydrodynamicforces imposed by agitation during experiments may havealso enhanced adsorption.

A limitation of this analysis is the reliance on rh,z for PrPSc

aggregate size. Sensitivity analyses were conducted toexamine the effect of uncertainty in the PrPSc aggregate sizeon the Born-DLVO calculations. Use of rh based on thenumber distribution in the DLVO calculations decreases thedepth of the secondary minimum and increases the heightof the energy barrier, but does not alter our interpretationof the data. For example, at pH 7 (I ) 10 mM), using rh

obtained from the number distribution (60 nm) Born-DLVOcalculations decreases Φ2°min from 0.40 to 0.10 kBT and lowersΦmax from 110 to 26 kBT. In both cases, the depth of Φ2°min

appears insufficient for attachment and Φmax is sufficientlylarge to prevent attachment in the primary minimum (25).Based on the sensitivity analysis, larger PrPSc aggregates weremore likely to be adsorbed, while smaller aggregates wereless prone to attach to quartz surfaces.

Environmental Implications. Our results demonstratethat PrPSc interaction with a common hydrophilic soil mineralis sensitive to unique properties of the protein and how theseproperties vary with solution conditions. Caution is thereforerequired when making inferences about the environmentalbehavior of prions from studies conducted using surrogatesfor PrPSc. A non-infectious, experimentally â-sheet-enrichedform of recombinant ovine PrP (recPrP) lacking glycosylation

FIGURE 4. Calculated Born-DLVO interaction energy profiles asa function of pH and ionic strength for PrPSc aggregates. Interactionenergies based on constant potential interaction and sphere-plategeometry using measured ú-potentials (Figure 1), Hamaker constant) 5.42 × 10-21 J (see text), and Z-average hydrodynamic radii forPrPSc aggregates (Figure 1). The shallow Φ2°min for pH 7 and 9 (I )0.1 M) not visible due to the scale of the y-axis. See Table 1 forvalues

TABLE 1. Estimated Born-DLVO Interaction Parameters forMean PrPSc Aggregatesa

pHI

(M)Φmax

(kBT)Φ2˚min

(kBT)h2˚min(nm)

4.0 0.01 nb na na7.0 0.01 110 0.40 24.07.0 0.10 nb na na9.0 0.01 217 0.19 27.6

a Abbreviations: h2°min, secondary minimum separation; I, ionicstrength; kB, Boltzmann constant; na, not applicable; nb, no calculatedenergy barrier to adsorption; Φmax, height of energy barrier; Φ2°min, depthof the secondary minimum. DLVO parameters calculated based onsphere-plate geometry assuming constant potential surfaces and usingZ-average hydrodynamic radii of PrPSc aggregates.

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has been used as a PrPSc surrogate in some environmentalfate studies (36). Glycosylation strongly influences PrPSc

surface charge (28) and is therefore expected to impactinteraction with soil particles. Nonglycosylated ovine recPrPexhibits isoelectric points between 9 and 10 as determinedby isoelectric focusing (37). In contrast, the apparent averagepI for PrPSc aggregates determined in the present study was4.6, and PrPSc adsorption to quartz surfaces was stronglypH-dependent with maximal sorption near the pI. While theaggregation state in our preparations likely differs from thatof PrPSc released into the environment, we expect PrPSc

obtained from infected animals to better reflect the surfaceproperties of the agent than does recPrP. The surfaceproperties of PrPSc from species of interest (e.g., cattle, deer,sheep) under environmentally relevant conditions (e.g.,decaying carcasses) warrants investigation. Although PrPSc

is clearly related to prion infectivity (1), infectivity varies withaggregate size (38). Advances in defining the precise natureof the prion and in methods to isolate the agent in purerform will facilitate environmental fate studies.

Although a simplified experimental system was used inthis study, a number of implications are apparent for landfilldisposal of prion-contaminated materials and for environ-mental TSE transmission. The potential for PrPSc migrationin porous media would likely be larger in more basic soilenvironments. Addition of backfill materials in landfills suchas fly ash and lime to degrade prions may enhance PrPSc

mobility due to diminished sorption at higher pH. Similarenhanced mobility may occur as leachate pH rises duringlandfill maturation. PrPSc mobility may also increase in landfillsettings where I diminishes, such as with leachate recircula-tion or as the waste ages. Fine textured and/or acidic surfacesoils would slow vertical migration of PrPSc. In natural systemsthis is expected to keep the agent near the soil surface andincrease the potential for ingestion by animals. Experimentsexamining PrPSc transport through soils and porous mediarepresentative of landfill waste will be required to confirmthese expectations.

AcknowledgmentsWe thank Allen Herbst, Peter Schramm, and ChristianBartholomay for assistance with animal bioassays, RichardRubenstein for the gift of 3F4, Cynthia Stiles for mineralogicalcharacterization, and Hsin Chiang for laboratory assistance.We thank Graheme Williams, Kevin Mattison, Gary Litton,Fran Kremer, and Susan Mooney for helpful discussions.This research was supported, in part, by grants from theDepartment of Defense (DAMD17-03-1-0369), U.S. Envi-ronmental Protection Agency (4C-R070-NAEX), NationalScience Foundation (BES-0547484), and National Cattlemen’sBeef Association. The content of this manuscript has notbeen subject to review by the sponsors. Endorsement by thesponsors is not implied and should not be assumed.

Supporting Information AvailableGel stained for protein and immunoblot of prion preparation.This material is available free of charge via the Internet athttp://pubs.acs.org.

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Received for review September 6, 2006. Revised manuscriptreceived January 12, 2007. Accepted January 29, 2007.

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