Adsorption of Insecticidal Cry1Ab Protein to Humic Substances. 2

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Adsorption of Insecticidal Cry1Ab Protein to Humic Substances. 2. Influence of Humic and Fulvic Acid Charge and Polarity Characteristics Jeanne E. Tomaszewski,† Michael Madliger,† Joel A. Pedersen,‡,§ René P. Schwarzenbach,† and Michael Sander*,† †

Swiss Federal Institute of Technology, ETH Zurich, Institute of Biogeochemistry and Pollutant Dynamics (IBP), Zurich, Switzerland Environmental Chemistry and Technology Program and §Department of Soil Science, University of WisconsinMadison, Madison, Wisconsin 53706, United States

‡

S Supporting Information *

ABSTRACT: Assessing the fate and potential risks of transgenic Cry proteins in soils requires understanding of Cry protein adsorption to soil particles. The companion paper provided evidence that patch-controlled electrostatic attraction (PCEA) and the hydrophobic effect contributed to Cry1Ab protein adsorption to an apolar humic acid (HA). Here, we further assess the relative importance of these contributions by comparing Cry1Ab adsorption to seven humic substances varying in polarity and charge, at different solution pH and ionic strength, I. Cry1Ab adsorption to relatively apolar HAs at I = 50 mM exhibited rapid initial rates, was extensive, and was only partially reversible at pH 5−8, whereas adsorption to more polar fulvic acids was weak and reversible or absent at pH >6. The decrease in adsorption with increasing HS polarity at all tested pH strongly supports a large contribution from the hydrophobic effect to adsorption, particularly at I = 50 mM when PCEA was effectively screened. Using insect bioassays, we further show that Cry1Ab adsorbed to a selected HA retained full insecticidal activity. Our results highlight the need to consider adsorption to soil organic matter in models that assess the fate of Cry proteins in soils.

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INTRODUCTION Adsorption to soil particle surfaces is a key process that affects the fate and activity of proteins, such as insecticidal toxins, prions, and enzymes, in soils. Adsorption of proteins may slow their transport,1−3 alter their activity,4−6 and protect them from proteolysis and other degradation reactions, 7,8 thereby increasing their stability in soils. Adsorption of infectious protein aggregates (e.g., prions, viruses) may impact disease transmission.9,10 Adsorption may also affect the fate and activity of protein toxins, including insecticidal Cry proteins expressed by genetically modified Bt crops (e.g., maize, soybeans, cotton) that carry Bacillus thuringiensis transgenes. While adsorption of these and other proteins to mineral surfaces is reasonably well studied, e.g. refs 5 and 11−13, adsorption to soil organic matter (SOM) has received substantially less attention.14−17 Humic substances (HS), the base-soluble components of SOM, include humic acids (HAs; acid insoluble) and fulvic acids (FAs; acid soluble). HS are amphiphilic, containing both apolar domains, dominated by aliphatic and aromatic hydrocarbons, and polar domains, containing oxygen-, nitrogen-, and sulfur-bearing moieties.18 HS polarity increases with the molar ratio of (O + N + S) to C.19,20 A significant fraction of the polar moieties in HS are carboxylic and phenolic groups that confer pH-dependent negative charges to HS. FAs are generally more polar [exhibit © 2012 American Chemical Society

higher (O+N+S)-to-C ratios] and more charged than HAs extracted from the same source material. As we previously demonstrated,21 using Cry1Ab as a model protein and Leonardite humic acid (LHA) as a model HS, the amphiphilicity of LHA resulted in pH- and I-dependent contributions to adsorption from both electrostatic interactions and the hydrophobic effect. The hydrophobic effect arises when apolar surfaces, such as apolar domains on HS and proteins, are in contact with water.22 The solvation of such surfaces is energetically unfavorable, as it disrupts the hydrogen-bonding network of water. The hydrophobic effect gives rise to numerous association processes in water, including protein folding,22,23 stabilization of HS associations,18,24 and protein adsorption to apolar surfaces.25 Koopal and co-workers14,15 recently provided evidence that both electrostatics and the hydrophobic effect contributed to the complexation of positively charged hen egg white lysozyme with Aldrich humic acid. Such contributions were also discussed in studies on protein encapsulation by humic substances.16,26,27 A Received: Revised: Accepted: Published: 9932

June 5, 2012 August 1, 2012 August 6, 2012 August 6, 2012 dx.doi.org/10.1021/es302248u | Environ. Sci. Technol. 2012, 46, 9932−9940

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Table 1. Selected Physicochemical Properties of Tested Humic and Fulvic Acids calcd bulk charge densitya [meq (gHS)−1] pH 5 | 6 | 7 | 8 | | | |

3.90 3.92 4.25 3.79

| | | |

4.45 4.46 4.79 4.39

| | | |

Suwannee River (SRHA) Elliott soil (ESHA) Pahokee peat (PPHA) Leonardite (LHA)

3.14 3.15 3.46 2.97

4.84 4.80 5.11 4.80

Elliott soil (ESFA) Pahokee peat (PPFA)

4.92 | 5.73 | 6.28 | 6.62 4.87 | 5.84 | 6.49 | 6.88

Aldrich (AHA)

2.34 | 3.17 | 3.69 | 4.06

apparent zeta-potential (ζ) of HS filmsb [mV] pH 5 | 7 IHSS Humic Acids ± 0.8 | −34.6 ± 0.8 ± 0.7 | −35.6 ± 0.7 ± 0.9 | −36.1 ± 1.5 ± 0.7 | −39.2 ± 1.8 IHSS Fulvic Acids −19.6 ± 0.9 | −37.1 ± 1.0 −20.6 ± 1.0 | −33.6 ± 1.5 Industrial Humic Acid −27.7 ± 1.0 | −38.7 ± 1.0 −24.6 −26.4 −26.1 −21.6

a

(O + N + S)/C [(molO + molN + molS)(molC)−1]

aromatic | aliphatic carbonc [kgC kg−1] | | | |

0.62c 0.50c 0.56c 0.39c

0.16 0.29 0.26 0.37

0.73c 0.68c

0.15e | 0.11e 0.17e | 0.10e

0.61d

−

0.15 0.09 0.11 0.09

30,31 b

Charge densities calculated from carbon contents and acid titration data reported in the literature. Duplicate samples with three readings each; average ± standard deviation; measured at I = 50 mM. cData from http://ihss.gatech.edu/ihss2/ (accessed May 21, 2012). dDetermined by elemental analysis (Microelement Analysis, Laboratory of Organic Chemistry, ETH Zurich); ratio not ash corrected; approximate ash content of Aldrich HA: 12.32 wt % (determined as 100% − ∑ %X (w/w); where X = C, H, N, O, S determined for two vacuum-dried samples). eAromatic and aliphatic carbon contents of ESFA (1S102F) and PPFA (1S103F) were taken as representative of those used here, 2S102F and 2S103F.

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EXPERIMENTAL SECTION Humic Substances and Cry1Ab Protein. Elliott soil HA and FA, Pahokee peat HA and FA, and Suwannee River HA were purchased from the International Humic Substances Society (IHSS, St Paul, MN). Aldrich HA (Na-salt form) was obtained from Aldrich. All HS were used as received. Selected physicochemical characteristics of the HS are provided in Table 1. More details on the HS are provided in the Supporting Information (Tables S1 and S2). Poly-L-lysine hydrobromide (PLL; 70−150 kDa), used for assembling adlayers, was purchased from Fluka. Purified and lyophilized Cry1Ab protein was obtained from M. PusztaiCarey (Case Western Reserve University, Cleveland, OH). Key physicochemical properties of Cry1Ab are given in the Supporting Information (Table S3). Solutions of HS (0.05 mg of HS mL−1), PLL (0.1 mg of PLL mL−1), and Cry1Ab (0.01 mg of Cry1Ab mL−1) were prepared as detailed in the companion paper.21 Adsorption Experiments. Adsorption was studied by two complementary in situ adsorption techniques, QCM-D and OWLS, as detailed in the companion paper.21 In brief, adsorption was studied at 25 °C from solutions delivered at a volumetric flow rate of 20 and 50 μL min−1 over the QCM-D sensors and OWLS waveguides, respectively. OWLS quantifies the absolute (“dry”) adsorbed masses, whereas QCM-D senses the dry mass and the mass of adlayer associated water. The adsorption experiments were mostly conducted at I = 50 mM to exclude Cry1Ab−Cry1Ab interactions in adsorbed states that occur at lower I = 10 mM that may result in more than monolayer adsorption.28,29 Following equilibration of SiO2-coated QCM-D sensors (QSX303, Q-Sense) or OWLS waveguides (custom coated21), each experiment consisted of three consecutive adsorption-rinsing steps, at constant pH and I: (i) PLLcontaining solutions were flowed over the sensors/waveguides, resulting in PLL adsorption, following by rinsing; (ii) HS-containing solutions were flowed over the sensors or waveguides, resulting in HS adsorption to the PLL-films, followed by rinsing; and (iii) Cry1Ab-containing solutions were flowed over the HScoated sensors or waveguides, followed by rinsing. The initial Cry1Ab adsorption rate, kads (ng cm−2 min−1); the final Cry1Ab mass at which adsorption plateaued during the final Cry final −2 adsorption step, ΔmCry QCM‑D and ΔmOWLS (ng cm ); and the

systematic assessment of the effects of HS charge and polarity on protein adsorption, however, has not yet been reported. The overall goal of the work reported in this and the companion paper21 was to determine the mechanism by which Cry1Ab adsorbs to HS. In earlier work, we showed that the highly nonuniform surface distribution of charged amino acid residues on Cry1Ab resulted in patch-controlled electrostatic attraction (PCEA) of net negatively charged Cry1Ab to SiO2 via positively charged domains II and III at pH values exceeding the global IEP (i.e., pH >6).3,28,29 At no tested pH nor I did van der Waals (vdW) interactions or the hydrophobic effect contribute significantly to overall adsorption of the protein to SiO2. Finally, using an insect bioassay, we also demonstrated that Cry1Ab retained full insecticidal activity when adsorbed to SiO2. In the companion paper,21 we showed that PCEA was also responsible for increasing Cry1Ab adsorption to Leonardite humic acid (LHA) with decreasing I at pH >6, similar to Cry1Ab adsorption to SiO2. However, compared to SiO2, Cry1Ab adsorption to LHA was extensive and much less reversible at pH ≥6 and I = 50 mM. The higher Cry1Ab affinity for the more apolar LHA than for the polar SiO2 was ascribed to the hydrophobic effect. This interpretation was supported by the results from adsorption of Cry1Ab and two reference proteins to model polar and apolar surfaces. However, LHA is a relatively apolar HA, leaving open the question to what extent the hydrophobic effect is also important for the adsorption of Cry1Ab and of other proteins to more polar SOM components. In the present work, we investigated the effects of HS polarity and charge characteristics on Cry1Ab adsorption. To this end, we studied the pH- and I-dependence of Cry1Ab adsorption to six chemically diverse HAs and FAs (see Table 1 for an overview) by quartz crystal microbalance with dissipation monitoring (QCM-D) and optical waveguide lightmode spectroscopy (OWLS). Furthermore, we assessed the insecticidal activity of Cry1Ab adsorbed to a selected HA with intermediate charge and polarity characteristics by diet incorporation bioassays with larvae of the susceptible insect Ostrinia nubilalis. Finally, using the results of this and our previous work, we provide a more general picture of the protein and adsorbent characteristics that govern the adsorption of Cry proteins to soil organic matter. 9933

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adsorption efficiency (α) (i.e., the ratio of kads to the maximum possible, diffusion-limited initial adsorption rate in the absence of an energy barrier to adsorption) were calculated as detailed in the companion paper.21 The reversibly adsorbed fraction (f rev) was defined as the fraction of the final wet adsorbed final Cry1Ab mass attained during the adsorption step, ΔmCry QCM‑D , that desorbed during rinsing with Cry1Ab-free solution of the same pH and I: frev = 1 −

QCM-D sensors and OWLS waveguides resulted in sequential adsorption of PLL to SiO2 and HS to PLL, consistent with our findings for LHA.21 HS adsorption to PLL was initially fast and then slowed and plateaued at reproducible final “wet” and “dry” final HS final masses, ΔmHS QCM‑D and ΔmOWLS , respectively. HS adsorption was largely irreversible, as rinsing with HS-free solution resulted in only small decreases in the adsorbed masses. Adsorption irreversibility implies that HS adsorption plateaued when the jamming limit of HS on the PLL surface was attained,16,21 likely reflecting strong electrostatic attraction between positively charged PLL and negatively charged HS. Much larger final wet than dry adsorbed masses were measured for ESHA and ESFA (the two HS for which OWLS measurements were made) at pH 5 and 7 (Supporting Information, Tables S4, S5), as previously reported for LHA.21 The gravimetric final HS final water contents, 1 − (ΔmHS OWLS /ΔmQCM‑D), of the ESHA and ESFA films ranged between 78 and 90% (Supporting Information, Table S6). Using a partial specific volume of 0.55 cm3 g−1 for HS, an average of published values for HS, we estimated densities between 1.05 and 1.14 g cm−3 for the ESHA and ESFA films (details in the Supporting Information and Table S6). For all other HS, we assumed an average HS film density of 1.09 g cm−3 to estimate HS film thicknesses from the final measured ΔmHS QCM‑D. Film thicknesses ranged from 1.2 nm for PPFA at pH 8 to 6.5 nm for ESHA at pH 5 (Supporting Information, Table S7). These dimensions agree well with the sizes of dissolved and adsorbed HS estimated by other techniques.33−35 A comparison of ESHA and ESFA and of PPHA and PPFA shows that HAs had higher final adsorbed masses and film thicknesses than did FAs extracted from the same source materials. Despite high water contents, all HS films were relatively rigid. This rigidity was evident from QCM-D measurements that showed similar or overlapping frequency shifts for the different overtones and small dissipation values (data not shown). As discussed in the companion paper, the high gravimetric water contents of the HS films in combination with relatively small Hamaker coefficients of organic macromolecule (including proteins and polymers)36−38 strongly suggest that Cry1Ab experienced only weak vdW interactions with the HS films. We previously showed that vdW interactions did not significantly contribute to Cry1Ab adsorption to amorphous silica, which has a Hamaker coefficient comparable to those of organic polymers.37,39,40 Cry1Ab Adsorption to HS Films. Comparison of QCM-D and OWLS. Figure 1 and the Supporting Information (Tables S8 and S9) show highly reproducible Cry1Ab adsorption to ESHA and ESFA (pH 5 and 7) and to LHA (pH 5−8)21 in both QCM-D and OWLS experiments. Both techniques showed an initial phase of fast Cry1Ab adsorption to HS, followed by a second phase of slower adsorption to final adsorbed masses. QCM-D and OWLS experiments both showed that the affinity of Cry1Ab decreased from LHA to ESHA and ESFA. The slight difference in Cry1Ab adsorption at pH 7 to ESFA between QCM-D and OWLS experiments (no adsorption vs final adsorbed mass of 20 ng cm−2) may have resulted from the different flow regimes and Cry1Ab mass transfer conditions to the HS in the QCM-D and OWLS flow cells. Cry1Ab affinity to ESFA measured by OWLS was, however, very weak, as adsorption plateaued at only ∼5% of the maximum (jamming) concentration of Cry1Ab on a planar HS surface21 and was fully reversible upon rinsing.

Cry final rinse ΔmQCM ‐D Cry final ΔmQCM ‐D

(1)

final rinse where ΔmCry (ng cm−2) corresponds to the adsorbed QCM‑D Cry1Ab mass remaining at the end of the rinsing step. The use of eq 1 slightly underestimates Cry1Ab desorption because the contribution of water to the wet mass, as sensed by QCM-D, increases as the adsorbed protein mass decreases.32 Nonetheless, application of eq 1 to all tested Cry1Ab−HS systems allows comparison of the relative differences in Cry1Ab adsorption reversibility to the different HS. Apparent Zeta Potential Measurements. Silica particles (0.82 μm median hydrodynamic diameter) were sequentially coated with PLL and HS at constant pH and I. The apparent zeta potentials (ζ) of the coated particles were estimated from electrophoretic mobility measurements on a Zetasizer ZS instrument (Malvern Instruments, UK). Details are provided in the Supporting Information. Bioassays. The insecticidal activity of Cry1Ab adsorbed to Pahokee peat HA (PPHA) films was determined by diet incorporation bioassays using neonatal larvae of the pest insect O. nubilalis (European cornborer; INRA, Surgeres, France), as previously detailed for silica particles.3 PPHA was coated on SiO2 particles at pH 6 using the procedure described in the Supporting Information. Different masses of dissolved Cry1Ab were added to suspensions of PPHA-coated particles. Cry1Ab concentrations in solution following adsorption were below the quantification limit (0.1 ng mL−1)28 of the enzyme-linked immunosorbent assay used, demonstrating that most, if not all, added Cry1Ab adsorbed to the particles. Both particle-adsorbed Cry1Ab and dissolved Cry1Ab (controls) were mixed into the insect diet with final concentrations ranging from 1 to 65 ng Cry1Ab gdiet−1. For each concentration, 20 larvae plus diet were placed into individual wells on bioassay trays and allowed to grow for 7 days (25 °C; 70% relative humidity; 16 h/8 h light/ dark cycle). Growth inhibition (GI) of the larvae was determined as the toxicity end point:

GI =

or free) wt0= 7d − wtCry1Ab(PPHA = 7d

wt0= 7d − wt = 0d

(2)

where wt=0d (mg) are the initial masses of the larvae, and w0t=7d (PPHA or free) (mg) and wCry1Ab (mg) are the masses of the larvae t=7d after feeding 7 days on Cry1Ab-free and Cry1Ab-containing diets (HA-adsorbed or directly added), respectively. Regression analysis of dose−response curves yielded the median effect concentration, EC50 (ng gdiet−1).3 Cry1Ab-free controls with and without HA-coated SiO2 particles showed that addition of particles to the diet did not impact larval growth (data not shown).

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RESULTS AND DISCUSSION Formation and Properties of HS Films. Flowing PLLand, subsequently, HS-containing solutions over the SiO2-coated 9934

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Figure 1. Duplicate Cry1Ab adsorption experiments to Elliot soil humic acid (HA) and fulvic acid (FA) and to Leonardite HA (data from companion paper21) monitored by (a) QCM-D and (b) OWLS. Cry1Ab protein at a concentration cCry1Ab = 10 μg mL−1 was introduced at time t = 0 min. R: Initiation of rinsing with Cry1Ab-free solutions. Constant pH 7 and ionic strength I = 50 mM were maintained throughout the adsorption and rinsing steps.

The decrease was largest for SRHA and smallest for LHA. Even though Cry1Ab adsorption decreased with increasing pH, extensive adsorption was still measured at pH 7 and 8, at which both Cry1Ab and HAs carried net negative surface charges. In contrast to the HAs, Cry1Ab adsorption to ESFA and PPFA at pH 7 and 8 was weak or not detectable. The pronounced final decrease in ΔmCry QCM‑D for adsorption to the FAs with increasing pH paralleled the decreases in α values (Figure 2). final The ΔmCry QCM‑D values indicated Cry1Ab affinity to the HS decreased in the order Leonardite HA > Aldrich HA > Elliot soil HA ≥ Pahokee peat HA ≈ Suwanee River HA ≫ Pahokee peat FA > Elliot soil FA. Cry1Ab−HS interactions were therefore strongly affected by HS surface properties and more favorable for HAs than FAs, including HA−FA pairs extracted from the same source material (Pahokee peat and Elliot soil). final As shown in Figure 2, the pH dependencies of α and ΔmCry QCM‑D for Cry1Ab adsorption to HA paralleled that to apolar, uncharged polystyrene, whereas Cry1Ab adsorption to the FAs was more similar to adsorption to polar, negatively charged poly(acrylic acid). Reversibility of Cry1Ab Adsorption to HS. Following adsorption, rinsing with Cry1Ab-free solutions at the same pH and I resulted in partial to complete Cry1Ab desorption from the HS surfaces (Figure 1 and Supporting Information, Figure S1). Figure 3a and Table S10 (Supporting Information) show that the reversibly adsorbed fraction of Cry1Ab, f rev, increased final with decreasing ΔmCry QCM‑D . The extensive adsorption at pH 5 was only slightly reversible for both HAs (f rev = 0.12−0.16) and FAs (f rev = 0.18−0.29). The less extensive adsorption at pH 7 and 8 was more reversible with f rev ≈ 0.36−0.55 for HAs and even >0.9 for FAs. The adsorption and desorption data therefore consistently showed higher Cry1Ab affinities for HAs than for the FAs and, for all HS, decreasing affinities as pH increased. The increase in reversibility of Cry1Ab adsorption to HS with increasing pH implies that a fraction of the HS-adsorbed Cry1Ab molecules were in exchange with solution-phase Cry1Ab molecules. This equilibrium likely explains plateauing Cry1Ab adsorption at subjamming limit surface coverages at pH ≥6. Furthermore, partial adsorption reversibility implies a

Since we employed identical solution conditions to examine Cry1Ab adsorption to the three HS, the differences in observed rates and extents of adsorption, and hence in Cry1Ab−HS interaction energies, are attributable to differences in the properties of the HS films. QCM-D and OWLS measured similar adsorption behaviors for Cry1Ab to HS in this work, for Cry1Ab, lysozyme, and albumin to LHA21 and to SiO2,28,29 and for several additional proteins and peptides to various surfaces.41−43 Therefore, we used QCM-D as the primary technique to investigate the effects of HS polarity and charge characteristics on Cry1Ab adsorption. Initial Rates and Final Extents of Cry1Ab Adsorption to HS. We studied Cry1Ab adsorption to films of seven HS that spanned a wide range of polarity and charge characteristics (Table 1 and Supporting Information, Tables S1 and S2) to better assess the effects these HS properties exert on adsorption. Representative traces of Cry1Ab adsorption to all seven HS at pH 5 to 8 and I = 50 mM are presented in Figure S1 (Supporting Information). The corresponding initial rates, expressed as adsorption efficiencies, α, and final adsorbed final masses during the adsorption step, ΔmCry QCM‑D , are summarized in Figure 2 and compiled in Table S8 (Supporting Information). While α values for adsorption to the HAs varied little with pH, α values decreased with increasing pH for the FAs. Initial Cry1Ab adsorption rates to the HAs were close to diffusionlimited (α ≈ 1) over the entire pH range tested. In contrast, initial adsorption rates to the FAs were close to diffusionlimited only at pH 5 and 6, but much lower at pH 7 and 8. These results suggest Cry1Ab interactions with HAs were attractive over the entire pH range studied, whereas Cry1Ab interactions with FAs at pH 7 and 8 were much weaker. At pH 5, Cry1Ab adsorption to all HAs was extensive with final −2 for SRHA to ΔmCry QCM‑D values ranging from 720 ng cm −2 860 ng cm for LHA, corresponding well to the wet mass of an adlayer of Cry1Ab at the jamming limit (i.e., maximum surface coverage) of 650−750 ng cm−2, estimated for random sequential adsorption to a planar surface.28,29 At pH ≥6, final Cry1Ab adsorption to all HAs plateaued at smaller ΔmCry QCM‑D (below the jamming limit), which decreased as pH increased. 9935

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Cry final Figure 2. pH dependencies of Cry1Ab adsorption efficiencies α (symbols) and final adsorbed masses during the adsorption step, ΔmQCM‑D (vertical bars), for (a) Aldrich humic acid (HA), (b) Elliot soil HA, (c) Elliot soil fulvic acid (FA), (d) Suwanee River HA, (e) Pahokee peat HA, (f) Pahokee peat FA, (g) Leonardite HA, (h) polystyrene (apolar, uncharged), and (i) poly(acrylic acid) (negatively charged, polar), as measured by quartz crystal microbalance with dissipation monitoring (QCM-D). All experiments were conducted at I = 50 mM and at a Cry1Ab concentration of 10 μg mL−1. *Panels g−i contain data from the companion paper.21 Error bars represent ranges (for duplicate measurements) and standard deviations.

pH. Instead, the observed I and pH dependencies were fully consistent with PCEA, which is weakened by increasing I, due to charge screening, and by increasing pH, due to the decrease in the net positive charges of Cry1Ab domains II and III. The calculated net charges of domains II and III decreased substantially from pH 5 to 7 (viz., from +11 to +4 and from +7 to +4, respectively), but only slightly from pH 7 to 8 (viz., from +4 to +3 for both domains) (Supporting Information, Table S11). The increases in the net negative charges of the HAs from pH 7 to 8 were much smaller than from pH 5 to 7 (Supporting Information, Table S1). PCEA therefore provides a plausible explanation for the final decreases in ΔmCry QCM‑D for the HS from pH 5 to 7, and the small Cry final changes, by comparison, in ΔmQCM‑D from pH 7 to 8. To assess the relative importance of PCEA to total Cry1Ab− final HS interactions at I = 50 mM, we plotted the ΔmCry QCM‑D values for all pH and HS combinations versus the pH-dependent HS bulk charges (Figure 3b) calculated from reported acid−base titration data of the tested HS (Table 1).30 If PCEA had dominated Cry1Ab−HS interactions at a given pH (for pH >6), increasing the negative charges of the HS would have resulted in comparable or final increased Cry1Ab adsorption. Instead, ΔmCry QCM‑D values decreased with increasing charges of the HS (e.g., from the HAs to the FAs;

distribution of Cry1Ab−HS interaction energies across the HS film surfaces. We previously hypothesized that this distribution resulted from nonuniformity in HS surface polarity, with larger contributions from the hydrophobic effect to Cry1Ab adsorption to more apolar than polar adsorption sites.21 Here we provide further evidence in support of this hypothesis by relating Cry1Ab adsorption to the charge and polarity characteristics of the tested HS. Cry1Ab−HS Electrostatic Interactions and the Hydrophobic Effect. The decrease in Cry1Ab affinity to HS with increasing pH is consistent with the contribution of electrostatics to overall Cry1Ab−HS interactions. To further assess the electrostatic interactions, we repeated selected Cry1Ab adsorption experiments at a lower I of 10 mM. These experiments were conducted at pH 7, at which Cry1Ab carried a net negative charge. We found significantly higher Cry1Ab adsorption at I = 10 mM than at 50 mM (all at pH 7) to HAs and FAs from both Pahokee peat and Elliot soil (Supporting Information, Figure S2). Increasing affinity with decreasing I rules out electrostatic repulsion at pH >6 between net negatively charged Cry1Ab and the like-charged HS as the cause of decreasing Cry1Ab−HS adsorption with increasing 9936

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Cry final Figure 3. Final adsorbed masses, ΔmQCM‑D , of Cry1Ab to humic substances during the adsorption step as a function of solution pH plotted versus (a) the reversibly adsorbed fraction of Cry1b molecules, f rev, (b) the titrated charge of bulk HS calculated from titration data,30 (c) the apparent zeta potential, ζ, of humic substance-coated SiO2 particles at pH 5 and 7 and ionic strength I = 50 mM, and (d) the polarity of HS expressed as the molar ratio of oxygen, nitrogen, and sulfur to carbon. Error bars represent ranges (for all duplicate measurements) and standard deviations.

(in contrast to I = 10 mM, as shown in the Supporting Information, Figure S2), due to charge screening. The differences in Cry1Ab affinity to the different HS, therefore, must have resulted from a nonelectrostatic interaction force. As detailed in the following discussion, the observed differences are consistent with adsorptive contributions from the hydrophobic effect. To assess whether the differences in Cry1Ab adsorption to HS can be explained by varying contributions from the hydrophobic effect, we related the extents of Cry1Ab adsorption to the polarity characteristics of the tested HS. The macroscopic polarity of solid surfaces is often described by the contact angles of sessile drops of water or other liquids on the adsorbent.25 However, we consider this characterization method inappropriate for the thin, layered HS films for a number of reasons, as detailed in the Supporting Information. Attempts to characterize the polarity of the HA films in this manner resulted in contact angles that ranged from 26° to 36° that did not correlate with dmQCM-DCryfinal (Table S12). We instead described the polarities of the tested HS by their molar ratio of O, N, and S to C. Aldrich HA was excluded from

Figure 3b). Furthermore, at a given pH, and hence Cry1Ab charge final state, ΔmCry QCM‑D differed substantially for HS with comparable bulk charges (e.g., SRHA, ESHA, and LHA at pH 8). The surface charges of the HS films rather than the titrated HS bulk charges may have been more pertinent to Cry1Ab−HS electrostatic interactions. Therefore, we assessed the surface charge properties of the tested HS films by measuring the ζ of HS-coated SiO2 particles at pH 5 and 7 and I = 50 mM. We used the same PLL and HS solution concentrations to coat the SiO2 particles and the QCM-D sensors, so that the ζ of the HScoated particles scaled with the surfaces charges of the respective HS films on the QCM-D sensors. Figure 3c shows that the HS films had more negative ζ at pH 7 than 5. At pH 7, the ζ of the HS covered a relatively narrow range from −34 mV for PPFA to −39 mV for LHA (Supporting Information, Table S12), while the amount of adsorbed Cry1Ab varied final −2 for substantially among the HS from ΔmCry QCM‑D = 0 ng cm −2 ESFA to 570 ng cm for LHA. The lack of clear trends of Cry1Ab affinity to HS with both increasing negative titrated bulk charges and ζ suggests that PCEA was weak at I = 50 mM 9937

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this analysis due to its high ash and iron contents.44 Figure 3d final shows that ΔmCry QCM‑D decreased with increasing (O + N + S)/C and, hence, polarity of the HS at all tested pH. For example, LHA and ESFA, the least and most polar HS, showed the strongest and weakest Cry1Ab adsorption at each pH value. For final HS with intermediate polarity characteristics, ΔmCry QCM‑D tended to decrease with increasing HS polarity. The clear dependence final of ΔmCry QCM‑D on HS polarity strongly supports a significant contribution of the hydrophobic effect to Cry1Ab adsorption to HS. HS polarity seemed to have affected Cry1Ab adsorption even under solution conditions that resulted in relatively strong Cry1Ab−HS electrostatic attraction, as suggested by the final decrease in ΔmCry QCM‑D with increasing HS polarity at pH 5. The systematic analysis of adsorption to different HS spanning a wide range in charge and polarity characteristics is consistent with both the hydrophobic effect and PCEA contributing to Cry1Ab−HS interactions. Cry1Ab adsorption decreased with increasing HS polarity, due to the reduced contribution to adsorption from the hydrophobic effect. The effect of HS polarity on adsorption was modulated by PCEA between Cry1Ab and HS. PCEA, and hence adsorption, decreased with increasing I and pH, due respectively to charge screening in solution and to decreasing positive net charges of domains II and III. As HS polarity increases, the concentration of H-bond accepting and donating moieties in HS increase; thus, the decrease in Cry1Ab adsorption with increasing HS polarity indicates that H-bond interactions, while undoubtedly present between Cry1Ab and the HS surfaces, had a minor contribution relative to the hydrophobic effect and, at low I, PCEA. Lastly, vdW interactions were likely small between Cry1Ab and the water-rich HS films, as previously discussed. Insecticidal Activity of Cry1Ab Adsorbed to HA. We assessed the activity of Cry1Ab adsorbed to HS by adsorbing the protein to Pahokee peat HA films coated to silica particles, followed by their incorporation into the diet of neonate larvae of O. nubilalis. PPHA was used because of its intermediate polarity characteristics and Cry1Ab affinity among the tested HS. O. nubilalis was chosen because it has an alkaline and high ionic strength midgut (pH 10, I > 200 mM),45,46 in which Cry1Ab is expected to desorb from the PPHA, and because as the target organisms for Cry1Ab, it carries specific receptors for this protein in its midgut. Increasing desorption of Cry1Ab from PPHA with increasing I was independently confirmed in QCM-D experiments (Supporting Information, Figure S3). Figure 4 shows that PPHA-adsorbed and nonadsorbed Cry1Ab (control) resulted in comparable EC50 values [EC50PPHA = 10.2 ng gdiet−1, 95% confidence interval (CI95%) 8.4−12.4 ng gdiet−1; and EC50control = 12.7 ng gdiet−1, CI95% 10.2− 15.5 ng gdiet−1]. PPHA-adsorbed Cry1Ab therefore was fully active, in agreement with earlier reports that Cry retained insecticidal activity when adsorbed to SiO2 and other soil constituents.8,17,47,48 Retention of activity implies that Cry1Ab desorbed from PPHA in the organism and that the desorbed Cry1Ab was in an active (near native) conformation. Cry1Ab therefore did not undergo extensive and irreversible conformational change upon adsorption to and desorption from PPHA and very likely also other HS.

Figure 4. Growth inhibition (GI) of O. nubilalis larvae fed on artificial diet containing Cry1Ab, either adsorbed to Pahokee peat humic acidcoated SiO2 particles (red symbols) or not adsorbed (directly added to the diet) (open symbols). Error bars represent standard deviations in GI of 20 replicates. EC50 values (Cry1Ab concentration resulting in 50% GI) were determined by regression analysis (solid lines for fits and dashed lines for 95% confidence intervals).

charge characteristics. Adsorption is expected to decrease with increasing SOM polarity, due to attenuation of the hydrophobic effect, and with increasing solution pH and I, due to weakening of Cry1Ab−SOM PCEA. These trends lead to the expectation that the apolar humin fraction of SOM, provided it is accessible, is a major sorbent for this protein in soils. Furthermore, at circumneutral pH and elevated I, at which PCEA is weak, stronger Cry1Ab adsorption is expected to SOM than to more polar mineral surfaces, including SiO2, as studied in our earlier work.3,28,29 The results from this work suggest that changes in soil pH and I, for instance associated with rain events, irrigation, or the application of manure, affect the fate and availability of Cry1Ab, and likely also other chemically similar Cry1A proteins, in soils. Increases in solution pH and I may facilitate Cry1Ab desorption from SOM, possibly resulting in temporarily and locally high Cry1Ab concentrations in the pore water. Desorption of Cry1Ab and other Cry1A proteins from SOM (as well as from other soil constituents) may result in enhanced microbial degradation and hence dissipation of these proteins in soils.49,50 The results from this work suggest that Cry1Ab will likely also associate with dissolved organic matter, potentially leading to colloid-facilitated transport of Cry1Ab through the soil profile and in overland flow during surface runoff-producing events. Colloid-facilitated transport may contribute to the occurrence and stability of Cry1Ab in the streams of agricultural landscapes planted with Bt crops.51−53 The finding that Cry1Ab retains full insecticidal activity over short-term sorption−desorption cycles to HAs highlights the need to include SOM-adsorbed Cry proteins in the assessment of the environmental fate and potential risks of Cry proteins. The systematic approach to assess the forces driving Cry1Ab adsorption to HS presented in this and the companion paper21 lays the groundwork for further mechanistic studies on the adsorption of other proteins, biomacromolecules, and engineered particles to SOM. Such studies will assist in prediction of the environmental fate of these molecules and particles of

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ENVIRONMENTAL IMPLICATIONS The results presented in this and the companion study21 show that SOM is an important sorbent for Cry1Ab in agricultural soils and that Cry1Ab adsorption to SOM under the same solution conditions may vary widely with SOM polarity and 9938

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emerging concern. We hypothesize that the hydrophobic effect also contributes to the association of proteins and viruses54−57 and carbonaceous nanoparticles58−60 to natural organic matter in both terrestrial and aquatic systems.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information on protein physicochemical properties, HS 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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AUTHOR INFORMATION

Corresponding Author

*Telephone: 0041-(0)44 6328314; fax: 0041-(0)44 6331122; e-mail: [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, Marianne Pusztai-Carey for the Cry1Ab protein, Tenzing C. Gyalpo for help on the bioassays, and Janos Vörös for access to the OWLS instrument. J.A.P. gratefully acknowledges sabbatical support from ETH Zürich.

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REFERENCES

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