Adsorption of Insecticidal Cry1Ab Protein to Humic Substances. 1

Article pubs.acs.org/est

Adsorption of Insecticidal Cry1Ab Protein to Humic Substances. 1. Experimental Approach and Mechanistic Aspects Michael Sander,* Jeanne E. Tomaszewski, Michael Madliger, and René P. Schwarzenbach Institute of Biogeochemistry and Pollutant Dynamics (IBP), Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland S Supporting Information *

ABSTRACT: Adsorption is a key process affecting the fate of insecticidal Cry proteins (Bt toxins), produced by genetically modified Bt crops, in soils. However, the mechanisms of adsorption to soil organic matter (SOM) remain poorly understood. This work assesses the forces driving the adsorption of Cry1Ab to Leonardite humic acid (LHA), used as a model for SOM. We studied the effects of solution pH and ionic strength (I) on adsorption using a quartz crystal microbalance with dissipation monitoring and optical waveguide lightmode spectroscopy. Initial Cry1Ab adsorption rates were close to diffusionlimited and resulted in extensive adsorption, even at pH >6, at which LHA and Cry1Ab carry negative net charges. Adsorption increased with decreasing I at pH >6, indicating Cry1Ab−LHA patch-controlled electrostatic attraction via positively charged domains of Cry1Ab. Upon rinsing, only a fraction of Cry1Ab desorbed, suggesting a range of interaction energies of Cry1Ab with LHA. Different interaction energies likely resulted from nonuniformity in the LHA surface polarity, with higher Cry1Ab affinities to more apolar LHA regions due to the hydrophobic effect. Contributions from the hydrophobic effect were substantiated by comparison of the adsorption of Cry1Ab and the reference proteins albumin and lysozyme to LHA and to apolar and polar model surfaces.

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INTRODUCTION In many countries, agricultural production heavily relies on the use of genetically modified (GM) crops.1 About 40% of these plants are Bt crops that carry the trait of pest insect resistance by expressing one or more Bacillus thuringiensis gene sequences that code for insecticidal Cry proteins.1 Several of these proteins are commercially important, including Cry1Ab expressed by Bt maize. Cry proteins enter agricultural soils from Bt crops by several pathways,2−4 including via decaying plant material, and may lead to potential ecological impacts, including adverse effects on nontarget soil-dwelling organisms. These concerns were previously addressed in effect studies and by monitoring Cry protein dissipation in soils.4−10 However, adsorption, a key process that affects protein transport, persistence, and bioavailability, remains only partially understood at a mechanistic level. Several recent studies investigated the mechanism of Cry protein adsorption to mineral surfaces.11−16 Our group recently showed that the nonuniform surface charge distribution of Cry1Ab protein resulted in patch-controlled electrostatic attraction (PCEA) to SiO2, as evidenced from increasing adsorption of net negatively charged Cry1Ab at pH >6 to likecharged SiO2 when decreasing ionic strength (I) from 50 to 10 mM.11−13 At I = 100 mM, when PCEA was effectively screened, Cry1Ab did not adsorb to SiO2, suggesting only small contributions to Cry1Ab−SiO2 interactions from van der Waals (vdW) interactions, H-bonding interactions, the hydro© 2012 American Chemical Society

phobic effect, and conformational changes of adsorbed Cry1Ab. Conversely, Helassa et al. reported adsorption of Cry1Aa, a Cry protein similar to Cry1Ab, to montmorillonite even at high I = 350 mM,16 which was ascribed to the hydrophobic effect contributing to Cry1Aa adsorption to the relatively apolar siloxane surface of the montmorillonite. Compared to mineral surfaces, the adsorption of Cry proteins, as well as other proteins, to soil organic matter (SOM) has been much less investigated, most likely due to experimental limitations. A major component of SOM are humic substances (HS), which include humin and humic and fulvic acids.17 HS are chemically heterogeneous and amphiphilic, as they contain both apolar moieties and polar (i.e., H-bond donating and accepting) moieties that include ionogenic carboxylic and phenolic groups. Consistent with HS amphiphilicity, recent studies18−21 provide evidence that both electrostatics and the hydrophobic effect favorably contribute to protein−HS interactions. While a quantitative understanding of the hydrophobic effect remains elusive,22 it is known to drive protein folding in solution and protein adsorption to apolar surfaces.22−24 The hydrophobic effect results from energetically more favorable intermolecular interactions of water molecules with each other relative to the Received: Revised: Accepted: Published: 9923

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Figure 1. Sequential adsorption of poly-L-lysine (PLL), Leonardite humic acid (LHA), and Cry1Ab protein to SiO2 sensors and waveguides measured in duplicate by (a) quartz-crystal microbalance with dissipation monitoring (QCM-D) and (b) optical waveguide lightmode spectroscopy (OWLS). The schematics depict the different adsorption states during LHA film formation and subsequent protein adsorption. Rinsing was initiated at times indicated by R with adsorbate-free solutions, while maintaining constant pH 7 and ionic strength I = 50 mM. Differences in the flow cell geometries and volumetric flow rates between QCM-D and OWLS experiments resulted in different times at which Cry1Ab adsorption plateaued during the adsorption steps.

lyophilized) were from Sigma and Fluka, respectively. Table S1 of the Supporting Information provides key physicochemical properties of the proteins. Further details on the Cry1Ab purification and on the preparation of protein solutions are given elsewhere.11 Sorbents and Chemicals. Purified Leonardite humic acid standard (LHA) was from the International Humic Substance Society (St Paul, MN), and poly(acrylic acid) (PAA; M = 450 kDa) was from Aldrich. Poly-L-lysine (PLL; average M = 70−150 kDa), used to prepare adlayers, was from Fluka. All chemicals used were analytical grade (see the Supporting Information). Adsorption Experiments. These experiments were conducted at least in duplicate at pH 5 (3 mM acetic acid), pH 6 (3 mM 2-(N-morpholino)ethanesulfonic acid), pH 7 and 8 (3 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at total ionic strengths of I = 50 and 10 mM (adjusted by NaCl) and at 20.0 ± 0.1 °C. A peristaltic pump connected to the outlets of the QCM-D and OWLS flow cells was used to pump solutions over the sensors/waveguides, thoroughly cleaned before use,11 at volumetric flow rates of 20 and 50 μL min−1, respectively. QCM-D Measurements. These measurements were conducted on a Q-Sense E4 system (Q-Sense AB, Gothemburg, Sweden) equipped with flow-through cells, each holding a piezoelectric quartz crystal sensor. Adsorption to the sensor surface results in shifts in the resonance frequency (f) (5 MHz) and its overtones (n) and in the energy dissipation (D).29 The total sensed mass of thin and rigid adlayers, ΔmQCM‑D (ng cm−2), is proportional to the decrease in the resonance frequency, Δf n (Hz), according to the Sauerbrey relation29

interactions of water molecules with apolar (i.e., non-Hbonding) surfaces. Direct contact between apolar patches on a protein and HS surface can therefore be expected to be energetically favorable compared to states in which these surfaces are fully exposed to water. Previous qualitative work demonstrated fast, extensive, and largely irreversible adsorption of Cry proteins to HA;25,26 however, the relative contributions from electrostatic interactions, the hydrophobic effect, and other possible forces to total adsorption remained unidentified. In the work presented in this and a companion paper,27 we have systematically investigated the adsorption of Cry1Ab to various humic and fulvic acids representing a wide range of SOM characteristics. To this end, we have extended two in situ surface techniques, quartz crystal microbalance with dissipation monitoring (QCM-D) and optical waveguide lightmode spectroscopy (OWLS), used in our earlier work for mineral surfaces,11,12 to be also applicable to organic matter surfaces. In this paper, which provides the mechanistic basis for the companion paper,27 we discuss methodological aspects, and address the driving forces that govern Cry1Ab adsorption to humic materials. We have investigated the effects of pH and I on the adsorption of Cry1Ab, bovine serum albumin (BSA), and hen egg white lysozyme (HEWL) to Leonardite humic acid (LHA). Cry1Ab was chosen because of its expression in several commercially important Bt crops. BSA and HEWL served as reference proteins, as their adsorption to various surfaces has been well-studied. LHA was selected because it is one of the most apolar HA among the commercially available HS (i.e., LHA has a high aromaticity and low ratio of heteroatoms oxygen, nitrogen, and sulfur to carbon28) and therefore served as a model for apolar components of SOM, including apolar HA and humin. Protein adsorption to LHA was compared to adsorption to polystyrene and gold, as models for apolar sorbent surfaces, and to poly(acrylic acid) (PAA) films and SiO2, as models for polar, negatively charged surfaces. The results from this work show that both PCEA and the hydrophobic effect play important roles in the adsorption of Cry1Ab to LHA.

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ΔmQCM ‐ D = Δmadsorbate + Δm water = −C

Δfn n

(1)

where C (17.7 ng Hz−1 cm−2) is the mass sensitivity constant, Δmadsorbate (ng cm−2) and Δmwater (ng cm−2) are the masses of “dry” adsorbed sorbate and of adlayer-associated water that couples to the oscillation, respectively, and n is the overtone number. Most systems showed overlapping Δf n/n for n = 3−11 and small dissipation values (Δf > 10 Hz per ΔD = 10−6), indicating that eq 1 was applicable. All data presented are for n = 5 (25 MHz). Adsorption experiments were completed with LHA and PAA films (details on the film preparation are provided below) and gold-coated sensors (QSX 301, Q-Sense) for all three

EXPERIMENTAL SECTION

Materials. Proteins. Purified and lyophilized Cry1Ab protein (M = 66.6 kDa) was obtained from M. Pusztai-Carey (Case Western Reserve University, Cleveland, OH), and high purity BSA (M = 66.4 kDa) and HEWL (M = 14.3 kDa) (>99%, 9924

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proteins and with polystyrene-coated sensors (QSX 305) for Cry1Ab. OWLS Measurements. These measurements were run on an OWLS 110 instrument (Microvacuum Ltd., Budapest, Hungary), equipped with a laminar slit shear flow cell, holding a silica-coated waveguide12 (OW2400, Microvacuum). A diffractive grating on the surface of the waveguide incouples He−Ar laser light at two well-defined incident angles for the transverse electric and magnetic polarization modes.30 Adsorption to the waveguide surface alters the interfacial refractive index and, therefore, the incoupling angles of the laser light, which are monitored. Assuming an optically uniform adsorbed layer, the mass of adsorbed protein, ΔmOWLS (ng cm−2) is given as31,32 nadlayer − nsolution ΔmOWLS = ladlayer (dn/dC)adsorbate (2)

kads diff kads

(3)

where α varies between 0 (= no adsorption) and 1 (= diffusion−2 limited adsorption rate), and kdiff min−1) is the ads (ng cm diffusion-limited initial adsorption rates for Cry1Ab, BSA, and HEWL, previously determined in QCM-D and OWLS experiments at the same volumetric flow rates and protein concentrations.11 Following adsorption, rinsing was initiated, and the reversibility of Cry1Ab adsorption to LHA was analyzed by fitting the OWLS-measured desorption data with a model that assumes a reversibly adsorbed fraction, f rev, and an irreversibly adsorbed fraction, (1 − f rev), of Cry1Ab molecules on the LHA surface Cry rinse Cry final ΔmOWLS (trinse) = ΔmOWLS [frev exp( −kdestrinse)

+ (1 − frev )] rinse ΔmCry OWLS

RESULTS AND DISCUSSION

Figure 1a,b shows the results of duplicate QCM-D and OWLS experiments at pH 7 and I = 50 mM, including the sequential adsorption of PLL to SiO2, LHA to PLL, and Cry1Ab to LHA. Figure 1 serves as an illustrative example for the following discussion on LHA film formation and Cry1Ab adsorption to LHA films. Preparation and Properties of LHA Films. Running solutions containing polycationic PLL over the SiO2 sensors/ waveguides resulted in adsorption of thin PLL films, as evidenced by the small increases in the adsorbed masses sensed by both QCM-D and OWLS (Figure 1 and Supporting Information, Tables S2 and S3). PLL adsorption was virtually irreversible, as indicated by only small decreases in ΔmQCM‑D and ΔmOWLS upon rinsing (R) with adsorbate-free solutions. Subsequent adsorption of LHA to PLL was highly reproducible in both initial rates and final LHA final final masses, ΔmLHA (Supporting InformaQCM‑D and ΔmOWLS tion, Tables S2, S3). Similar to PLL adsorption to SiO2, LHA adsorption to PLL was irreversible, consistent with strong LHA− PLL electrostatic attraction. Irreversible adsorption suggests that LHA final LHA final final adsorbed LHA masses, ΔmQCM‑D and ΔmOWLS , corresponded to the maximum (jamming) concentrations of LHA on the PLL surfaces, which was supported by comparable final ΔmLHA QCM‑D when adsorbing LHA from solutions with LHA concentrations from CLHA= 2.5 to 50 mg mL−1 (Supporting Information, Figure S2). Irreversible adsorption further suggests that the entire PLL surface was coated by LHA, which is supported by the following discussion. At all tested pH and I, LHA adsorption resulted in about 4 LHA final LHA final times larger ΔmQCM‑D than ΔmOWLS (Figure 1 and Supporting Information, Tables S2 and S3), demonstrating that LHA films contained a large amount of coadsorbed water. The gravimetric water contents of the LHA films, calculated as 1 final LHA final − (ΔmLHS OWLS /ΔmQCM‑D ), varied between 83% at pH 5 and 75% at pH 8. The estimated thicknesses of LHA films decreased from 5.4 nm at pH 5 to 3.1−3.3 nm at pH 7−8 (Supporting Information, Table S4). The water contents and thicknesses corresponded well to previously published values for dissolved and adsorbed HAs determined by other techniques.37−41 Previous work also showed that HA films adsorbed to positively charged polyelectrolytes had subnanometer averaged roughness,42 which is about a factor of 5−6 smaller than the molecular dimensions of Cry1Ab. It is therefore reasonable to expect that the LHA film surfaces were relatively smooth compared to the size of the adsorbing Cry1Ab. Due to their high water contents, the LHA films had densities close to that of bulk water (ρfilm= 1.07−1.13 g cm−3; Supporting Information, Table S4). Similarly, the polarizabilities of the LHA films and bulk water were likely comparable. As a consequence, Cry1Ab molecules likely experienced comparable vdW interaction forces with water molecules in the bulk solution as with the LHA films, resulting in only small contributions from vdW forces to Cry1Ab adsorption to LHA. Weak Cry1Ab−LHA vdW interactions are consistent with the low Hamaker constants of organic polymers and proteins,43−45 relative to some inorganic materials. We previously reported only weak vdW interactions of Cry1Ab also with the SiO2 surfaces of the QCM-D sensors and OWLS waveguides.11,12 Note that these weak Cry1Ab-SiO2 vdW interactions were attenuated in the present study by the PLL− LHA films on the SiO2 surfaces.46 The expected negative surface charge of the LHA films on the sensors and waveguides could not be directly measured in the

where ladlayer (cm) and nadlayer are the thickness and the refractive index of the adlayer, respectively, (dn/dC)adsorbate is the refractive index increment of the respective adsorbate [0.182 cm3 g−1 for the proteins,33 0.29 cm3 g−1 for LHA, as measured by refractometry (Supporting Information, Figure S1), and 0.139 cm3 g−1 for PAA and PLL34], and nsolution (=1.3336, measured, constant over all measured pH and I) is the refractive index of the solutions. In contrast to QCM-D, OWLS senses only the dry adlayer adsorbed mass. OWLS experiments were conducted for Cry1Ab adsorption to LHA and PAA films. LHA and PAA Films. Films were prepared for each protein adsorption experiment as detailed in earlier work.20 Briefly, PLL solution (CPLL = 100 μg mL−1) was run over SiO2 sensors/ waveguides, resulting in the formation of positively charged PLL adlayers (IEPPLL = 9.0−9.8),35 and then negatively charged LHA or PAA (CLHA = 50 μg mL−1; CPAA = 100 μg mL−1) was run over the PLL surface to form films, followed by rinsing (Figure 1a). Protein-containing solutions (Cprotein = 10 μg mL−1, unless otherwise stated) were then run through the flow cells. In most systems, initial fast protein adsorption rates, kads (ng cm−2 min−1), slowed over time, and adsorption plateaued at final final final adsorbed masses, Δmprotein and Δmprotein (ng cm−2). Initial QCM‑D OWLS adsorption rates, determined by linear fitting, were compared on the basis of adsorption efficiencies, α,36 according to

α=

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(4)

−2

where (trinse) (ng cm ) is the adsorbed Cry1Ab mass at time trinse (min) of rinsing, and kdes (min−1) is the first-order desorption rate constant for Cry1Ab molecules in the reversibly adsorbed fraction. 9925

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Figure 2. (a) Effect of solution pH on the adsorption efficiency, α, for Cry1Ab to Leonardite humic acid (LHA) films at ionic strength I = 50 mM, as measured by quartz crystal microbalance with dissipation monitoring (QCM-D) and optical waveguide lightmode spectroscopy (OWLS). The dashed line at α = 1 corresponds to diffusion-limited initial adsorption rates in the absence of adsorption energy barriers. (b) Effect of solution pH on the final Cry final final and ΔmCry “wet” and “dry” Cry1Ab adsorbed masses, ΔmQCM‑D OWLS , as measured by QCM-D and OWLS, respectively. The horizontal gray lines final Cry final correspond to the estimated wet and dry adsorbed masses of Cry1Ab at the jamming limit on the LHA surface. f Cry = ΔmCry OWLS /ΔmQCM‑D corresponds to the mass contribution of Cry1Ab to the total sensed wet mass of the Cry1Ab adlayer, assuming a constant water content of the underlying PLL−LHA layer during Cry1Ab adsorption.

QCM-D and OWLS flow cells. Therefore, micrometer-sized SiO2 particles were coated with PLL and then LHA at the same solution concentrations as used in the QCM-D and OWLS experiments and subsequently analyzed for zeta-potentials, ζ, as detailed in the Supporting Information. As expected, ζ values shifted from negative for bare SiO2, to positive after PLL adsorption, and back to negative after LHA adsorption (Supporting Information, Figure S3). The ζ values for LHAcoated particles became more negative from pH 5 to 7, consistent with increasing negative charge with increasing pH due to the successive deprotonation of carboxylic and phenolic moieties in LHA.47 In summary, the coating procedure resulted in the reproducible formation of stable, relatively smooth, and negatively charged LHA films that were subsequently used to systematically study the interaction of Cry1Ab with LHA. pH Dependence of Cry1Ab Adsorption to LHA (I = 50 mM). At all tested pH values, high initial adsorption rates of Cry1Ab to LHA decreased over time, and adsorption plateaued final Cry final at reproducible ΔmCry QCM‑D and ΔmOWLS (Figure 1 for pH 7). Figure 2a shows the effect of pH on the adsorption efficiency, α, of Cry1Ab to LHA. In OWLS measurements, α values ranged between 0.4 and 0.6 and showed no clear pH trend. Values determined by QCM-D increased from α ≈ 0.5 at pH 5 to α ≈ 1 at pH 8. This increase with pH was possibly due to a slight increase in the water mass that coadsorbed with Cry1Ab to the LHA surface. More importantly, both techniques consistently showed large α values >0.4 over the entire tested pH range (Supporting Information, Tables S5, S6), meaning that the initial Cry1Ab adsorption rates to LHA were close to diffusion-limited (α = 1). Cry1Ab molecules diffusing to the negatively charged LHA surface therefore experienced only minor, if any, energy barriers to adsorption, even at pH >6, at which both the surfaces of Cry1Ab and LHA were net negatively charged. QCM-D and OWLS also consistently showed decreasing Cry1Ab adsorption to LHA from pH 5 to pH 7 and 8 and, as final Cry final expected, larger ΔmCry QCM‑D than ΔmOWLS at all tested pH (Figure 2b and Supporting Information, Tables S5, S6). At pH 5, Cry1Ab final −2 Cry final adsorption plateaued at ΔmCry QCM‑D ≈ 825 ng cm and ΔmOWLS −2 ≈ 380 ng cm . These adsorbed masses corresponded well to the estimated “wet” and “dry” adsorbed masses for random

sequential adsorption of Cry1Ab to the jamming limit of the LHA film surfaces11 (gray lines in Figure 2b). Cry1Ab adsorption to the surface jamming limit at pH 5 and to a surface coverage below the jamming limit at pH 7 and 8 is supported by the decrease in the mass contribution of Cry1Ab to the total QCM-D final Cry final sensed mass, f Cry = ΔmCry OWLS /ΔmQCM‑D , from 0.46 at pH 5 to 0.18 at pH 7 and 8 (Figure 2b). Previous studies reported similar mass contributions of globular proteins to the total QCM-Dsensed masses of 40−50% at jamming coverage and 6, increasing adsorption of net negatively charged Cry1Ab (IEPCry1Ab ∼ 6; Supporting Information, Table S1) to likecharged LHA with decreasing I precludes Cry1Ab−LHA electrostatics repulsion, which would have resulted in an Idependence opposite to that observed. Instead, increasing adsorption with decreasing I is consistent with Cry1Ab−LHA electrostatic attraction, which likely resulted from Cry1Ab being oriented with its net positively charged domains II and III (IEPs of 9.6 and 9.4, respectively; Supporting Information, Table S1) toward the LHA film surface. We previously reported such patch9926

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Article final BSA showed decreasing α and ΔmBSA QCM‑D with increasing pH and with decreasing I, consistent with electrostatic repulsion of BSA from like-charged LHA. In contrast, lysozyme, which carried a uniform, positive surface charge at all tested pH (IEPlysozyme = 10.5; Supporting Information, Table S1), showed diffusionfinal ≈ 350 ng cm−2 limited adsorption rates (α = 1) and Δmlysozyme QCM‑D (Supporting Information, Table S5) that corresponded well to the estimated adsorbed mass at the surface jamming limit.11 These findings are consistent with strong lysozyme−LHA electrostatic attraction at both I = 10 and 50 mM. The pH and I dependencies of BSA and HEWL adsorption to LHA support PCEA of Cry1Ab to LHA. Cry1Ab Desorption from LHA. Following adsorption at all tested pH and I = 50 mM, rinsing with Cry1Ab-free solutions while constant pH and I were maintained resulted in partial Cry1Ab desorption from LHA (shown for pH 7 in Figure 1). The extent of desorption was quantified by fitting eq 4 to the decrease in ΔmOWLS during rinsing (Supporting Information, Figure S5). Figure 4a shows that the reversibly adsorbed fraction of Cry1Ab molecules, f rev, increased from pH 5 to 7 and decreased slightly from pH 7 to 8. Consistently, the irreversibly adsorbed mass of Cry1Ab at the end of the rinsing step decreased from pH 5 to 7 and increased to pH 8, which paralleled the pH dependence of final ΔmCry OWLS during the adsorption steps. Cry1Ab molecules in the reversibly adsorbed fraction were in equilibrium with solution phase molecules; therefore, they experienced weaker interactions with LHA than the molecules that did not desorb from LHA during extensive rinsing. Unfavorably oriented Cry1Ab molecules would have changed orientation to yield more favorable interactions with LHA or would have desorbed to be replaced by favorably oriented molecules during the adsorption step. As a result, the LHA surface at the end of the adsorption step would have been covered with favorably oriented and irreversibly adsorbed molecules, which contradicts experimental observations. Therefore, the distribution in the interaction energies of Cry1Ab molecules with LHA likely did not result from different orientations and/or conformations of adsorbed Cry1Ab molecules. Alternatively, it is more likely that the distribution of interaction energies of adsorbed Cry1Ab molecules resulted from the chemical nonuniformity of the LHA film surfaces. To further assess chemical nonuniformity as potential cause, we determined the effect of changes in solution pH and I during rinsing on Cry1Ab desorption from LHA. Following adsorption at pH 6 and I = 50 mM, increasing the pH of the rinsing solution to pH 7, 8, and 9 resulted in increasing rates and extents of Cry1Ab desorption from LHA (Figure 4b). Figure 4c shows that, following adsorption at pH 7 and I = 10 mM, desorption of Cry1Ab from LHA increased with increasing I of the rinsing buffer from 50 to 100 mM. The incremental increase in desorption with increasing pH and I implies that the adsorbed Cry1Ab molecules experienced a range of interaction energies with LHA, which likely originated from chemical surface nonuniformity of the LHA films. The increase in Cry1Ab desorption with increasing pH is consistent with the weakening of PCEA of Cry1Ab to LHA due to decreasing net positive surface charges of domains II and III in Cry1Ab. Desorption increased with increasing I at constant pH because of the increase in charge screening. Yet, incomplete Cry1Ab desorption even at pH 9 (I = 50 mM; Figure 4b), at which domains II and III carried only weak positive net charges (Supporting Information, Table S7), and at I = 100 mM (pH 7, Figure 4c), which led to strong

Figure 3. Effect of solution pH and ionic strength, I, on the adsorption efficiency, α, and the final adsorbed wet mass, ΔmQCM‑Dfinal, of (a) Cry1Ab, (b) bovine serum albumin (BSA), and (c) hen egg white lysozyme (HEWL) to Leonardite humic acid (LHA).

controlled electrostatic attraction (PCEA) for Cry1Ab adsorption to negatively charged SiO2 adsorbents.11−13 PCEA is consistent with the decrease in Cry1Ab adsorption to LHA at I = 50 mM with increasing pH, which results in successive deprotonation of positively charged amino acids in domains II and III (Supporting Information, Table S7) and, hence, attenuation of electrostatic attraction. Note that Cry1Ab adsorption at I = 10 mM in excess of the jamming limit reflects Cry1Ab−Cry1Ab PCEA on the sorbent surface at low I, as described previously.12,13 Figure 3b,c shows the adsorption of the reference proteins BSA and HEWL to LHA under the same solution conditions. BSA has a size very similar to that of Cry1Ab but a more uniform negative surface charge distribution (IEPBSA = 4.7−5.0; Supporting Information, Table S1). Therefore, as expected, 9927

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desorption from LHA. The following discussion will provide evidence that this additional force resulted from the hydrophobic effect. On the basis of the high water contents of LHA films, vdW interactions likely did not account for the nonelectrostatic contribution to Cry1Ab adsorption. The scenario that a fraction of adsorbed Cry1Ab molecules underwent extensive conformational changes also seems unlikely. First, increasing pH and I enhanced Cry1Ab desorption from LHA, which would not be expected for Cry1Ab molecules that extensively and irreversibly unfolded on the LHA surface. Second, Cry1Ab was shown to have a relatively high conformational stability in adsorbed states, including on apolar surfaces,11 on which conformationally unstable proteins have a high propensity to unfold. Third, adsorption of BSA, which has a low conformational stability on final various surfaces,49 resulted in comparable ΔmBSA QCM‑D when varying the supply rate of BSA to the LHA surface (Supporting Information, Figure S2). This finding suggests that the LHA surface did not facilitate time-dependent extensive unfolding of adsorbed BSA; if unfolding had occurred, the area of LHA occupied by each BSA molecule would have increased over time and would have resulted in decreasing final adsorbed masses with decreasing supply rates.49,50 In contrast to BSA, the decrease in Cry1Ab adsorption with decreasing Cry1Ab supply rate to the LHA surface reflected concentration-dependent adsorption, as expected on the basis of partial adsorption reversibility. Finally, previous work provided evidence for retained insecticidal activity of Cry proteins adsorbed to humic substances, as assessed in diet incorporation bioassays with susceptible test insects.25 Consistently, we demonstrate retained insecticidal activity of Cry1Ab adsorbed to a model humic acid in the companion paper.27 Retained insecticidal activity is inconsistent with extensive conformational changes of adsorbed Cry1Ab, as this process would have resulted in irreversible loss of the bioactive conformation and hence reduced insecticidal activity. Contributions from the Hydrophobic Effect to Cry1Ab Adsorption to LHA. On the basis of the amphiphilic character of HA, a nonuniform distribution of polar (e.g., alcohol and keto groups), ionogenic (e.g., phenolic and carboxylic groups with pH-dependent negative charges), and apolar moieties (e.g., aromatic and aliphatic groups) on the surfaces of the LHA films is expected. The chemical nonuniformity of the LHA surface may have affected the contributions of the hydrophobic effect and PCEA to Cry1Ab adsorption. Contributions from the hydrophobic effect are expected because of the apolar character of LHA, as evident from its relatively high aromaticity28 (58% of C) and low molar ratio of oxygen, nitrogen, and sulfur to carbon [(O + N + S)/C = 0.39], as compared to other HS. To assess the role of the hydrophobic effect in Cry1Ab−LHA interactions, Cry1Ab adsorption to LHA was compared to its adsorption to apolar polystyrene and gold and to polar, negatively charged PAA and SiO2, all at I = 50 mM. Cry1Ab adsorption to apolar polystyrene and gold showed diffusion-limited initial adsorption rates (α ≈ 1) and was final −2 extensive with ΔmCry at all tested pH (Figure QCM‑D > 600 ng cm 5a,b). The high affinity of Cry1Ab to these apolar model surfaces was likely due to the hydrophobic effect. In addition, the high density and polarizability of gold, as reflected in its high Hamaker coefficient,44,45 likely resulted in additional, strong vdW interactions. Conversely, increasing pH from 5 to 8 resulted in final pronounced decreases in α and ΔmCry QCM‑D values for Cry1Ab adsorption to PAA and SiO2 (Figure 5c,d), consistent with the attenuation of PCEA due to decreasing positive charges on

Figure 4. (a) Reversibility of Cry1Ab adsorption to LHA at ionic strength I = 50 mM as a function of solution pH, as measured by OWLS. The filled bars correspond to the final adsorbed masses during adsorption and are replotted from Figure 2b. The open bars represent the fitted final adsorbed masses after extensive rinsing with Cry1Ab-free solutions, as detailed in the Supporting Information. f rev corresponds to the reversibly adsorbed fraction defined as the ratio of the final adsorbed masses after desorption and adsorption. (b) Effect of rinsing using Cry1Ab-free solutions (indicated by R) and increasing solution pH during rinsing (indicated by ΔpH) on the desorption of Cry1Ab from LHA adsorbed to and initially desorbed from LHA at pH 6, as measured by QCM-D. All experiments were carried out at I = 50 mM. (c) Effect of ionic strength I on the desorption of Cry1Ab preadsorbed to LHA at pH 7 and I = 10 mM, as measured by QCM-D. Rinsing solutions were Cry1Ab-free and had a constant pH 7 but varied in I [I = 10 mM (black trace), 50 mM (blue trace), and 100 mM (red trace)]. The latter two systems were finally rinsed with solutions readjusted to I = 10 mM, as indicated by the gray vertical lines (ΔI). The final decrease in I from 50 Cry , demonstrating and 100 to 10 mM had only small effects on ΔmQCM‑D that the observed decreases in wet adsorbed masses during rinsing were due to desorption of Cry1Ab and not due to mass changes of the underlying PLL−LHA adlayers.

attenuation of PCEA by charge screening, suggests that an additional, nonelectrostatic force prevented complete Cry1Ab 9928

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Cry final Figure 5. Effect of solution pH on the adsorption efficiency, α, and on the final wet adsorbed mass, ΔmQCM‑D , of Cry1Ab to (a) polystyrene, (b) gold, (c) 11 poly acrylic acid, (d) silicon dioxide (SiO2) (data from earlier work ), and (e) LHA (replotted from Figure 2), as measured by quartz crystal microbalance with dissipation monitoring (QCM-D). (f) Proposed contributions from patch-controlled electrostatic attraction and the hydrophobic effect to Cry1Ab adsorption to LHA.

The pH dependencies of Cry1Ab adsorption to LHA (Figure 5e) and to apolar polystyrene and gold were comparable, whereas adsorption to the polar, negatively charged PAA and SiO2 showed a strong decrease in adsorption with pH. Adsorption to gold and polystyrene at pH >5 and I = 50 mM was only partially reversible, similar to Cry1Ab adsorption to LHA, whereas adsorption to PAA and SiO2 under the same solution conditions was highly reversible. The finding of similar adsorption characteristics of Cry1Ab to the apolar, uncharged

domains II and III of Cry1Ab and the absence of significant contributions from the hydrophobic effect. Less extensive Cry1Ab adsorption to PAA than to SiO2 at pH >5 may have resulted from weaker vdW interactions with PAA and from (electro)steric repulsion of Cry1Ab by the well-hydrated, charged PAA polymer strands that extended from the PAA surface into solution. On the contrary, extensive Cry1Ab adsorption to LHA indicated that steric repulsion effects on the LHA surface were small, if not absent. 9929

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rather than to the polar, negatively charged model surfaces strongly supports that the hydrophobic effect contributed to Cry1Ab adsorption to LHA. The proposed adsorption mechanism of Cry1Ab to LHA is summarized in Figure 5f. The contribution of PCEA increased with decreasing pH and I, resulting in enhanced and less reversible adsorption. At any given pH and I, contributions to Cry1Ab adsorption from the hydrophobic effect were larger for more apolar than polar adsorption sites on the LHA surface. Note that also BSA adsorption to LHA showed characteristics intermediate to BSA adsorption to the polar, charged PAA and SiO2 and to the apolar gold (Supporting Information, Table S5 and Figure S6), suggesting that the hydrophobic effect affects adsorption of other, net negatively charged proteins to HS. In contrast to Cry1Ab and BSA, HEWL was net positively charged and experienced only attractive interactions with LHA, PAA, SiO2, and gold, resulting in diffusion-limited initial adsorption rates and extensive adsorption to these surfaces (Supporting Information, Figure S7).

IMPLICATIONS On the basis of the finding of extensive adsorption to LHA between pH 5 and 8, Cry1Ab is expected to strongly adsorb to the surfaces of relatively apolar organic matter in agricultural soils, including apolar humic acids and humin. This work provides strong evidence that SOM surface polarity is a key factor determining the kinetics, extent, and reversibility of adsorption of Cry1Ab, and likely of other (Cry) proteins. The relative importance of the hydrophobic effect to Cry1Ab adsorption to SOM is further evaluated in the companion paper,27 in which we studied the adsorption of Cry1Ab to a set of humic substances with different polarity and charge characteristics. Energetic contributions to protein adsorption to SOM from the hydrophobic effect are not accounted for in traditional adsorption models that only describe electrostatic and dispersion interactions, such as the Derjaguiun−Landau−Verwey−Overbeek (DLVO) theory. Results from such models, therefore, should be carefully reinterpreted when applied to protein adsorption to apolar organic and possibly also to apolar mineral surfaces. ASSOCIATED CONTENT

S Supporting Information *

Additional information on protein physicochemical properties, LHA film formation and properties, and tables and figures with results from protein adsorption experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 0041-(0)44 6328314; fax: 0041-(0)44 6331122; email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Swiss National Science Foundation, National Research Program 59 (Project 405940-115662) for funding and Marianne Pusztai-Carey (Case Western Reserve University) for the Cry1Ab protein. We thank Joel A. Pedersen for helpful comments and suggestions and Janos Vörös for access to OWLS instrumentation. 9930

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