Adsorption of transgenic insecticidal Cry1Ab protein to SiO2. 1. Forces driving adsorption

Genetically modified Bt crops express insecticidal Cry proteins (Bt toxins) that may enter agricultural soils. A mechanistic understanding of Cry protein adsorption to soils is critical for risk assessment, as this process governs Cry protein fate and bioavailability. We used quartz crystal microbalance and optical waveguide lightmode spectroscopy to elucidate the driving forces of the adsorption of monomeric Cry1Ab to negatively charged quartz (SiO(2)) and positively charged poly-L-lysine (PLL) at pH 5-8 and constant ionic strength of 50 mM (NaCl). Bovine serum albumin and hen egg white lysozyme were used as reference proteins because of their known adsorption behavior. Electrostatics governed Cry1Ab adsorption; as pH increased above the isoelectric point of Cry1Ab, the initial rate and the extent of adsorption decreased on SiO(2) and increased on PLL. Reversible adsorption to SiO(2) suggested weak Cry1Ab-SiO(2) electrostatic interactions and no irreversible conformational changes of Cry1Ab at the surface. High conformational stability of Cry1Ab was further supported by supply rate-independent extent of adsorption of Cry1Ab to apolar gold. Some evidence is presented that the nonuniform surface charge distribution of Cry1Ab resulted in patch-controlled electrostatic attraction with sorbents that carried the same net charge as Cry1Ab. A more detailed discussion of this mechanism is given in a companion paper.     

Adsorption of transgenic insecticidal Cry1Ab protein to silica particles. Effects on transport and bioactivity

Bt crops are genetically modified to be resistant against insect pests by expressing insecticidal Cry proteins. The processes governing the fate and bioavailability of the expressed transgenic Cry proteins in soils are poorly understood. We studied adsorption of Cry1Ab to negatively charged silica (SiO(2)) particles, a major soil constituent and a model for negatively charged mineral surfaces, at pH 5 to 10 and ionic strengths I = 10 mM to 250 mM, both in solution depletion and saturated column transport experiments. Cry1Ab-SiO(2) interactions were dominated by patch-controlled electrostatic attraction (PCEA), as evident from increasing Cry1Ab attraction to SiO(2) with decreasing I at pH at which both Cry1Ab and SiO(2) were net negatively charged. Experimental and modeling evidence is provided that the surface heterogeneity of SiO(2) particles modulated PCEA, leading to a fraction of adsorption sites with slow Cry1Ab desorption kinetics. Desorption rates from these sites increased upon increasing the solution pH. In toxicity bioassays, we demonstrated that Cry1Ab retained insecticidal activity when adsorbed to SiO(2), suggesting high protein conformational stability during adsorption-desorption cycles. Models predicting Cry1A protein adsorption in soils therefore need to account for combined effects of the nonuniform protein surface charge distribution and of sorbent surface heterogeneity.     

Adsorption of Insecticidal Cry1Ab Protein to Humic Substances. 2. Influence of Humic and Fulvic Acid Charge and Polarity Characteristics

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.     

Adsorption of Insecticidal Cry1Ab Protein to Humic Substances. 1. Experimental Approach and Mechanistic Aspects

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 diffusion-limited 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.     

Validation of an enzyme immunoassay for monitoring Cry1Ab toxin in soils planted with Bt-maize (MON810) in a long-term field trial on four South German sites

For monitoring the environmental impacts of a genetically modified crop well-established methods are a key requirement for the collection of appropriate data. With regard to the fate of Cry1Ab protein from Bt-maize MON810 released by root exudates, plant residues and pollen deposition into soil, a highly specific and sensitive sandwich enzyme immunoassay for detection and quantification of Cry1Ab in soil matrix was developed. The assay was validated according to the criteria of EU-Decision 2002/657/EC for a range of soils originating from four selected experimental field sites in South Germany, where Bt-maize MON810 has been cultivated. On three sites Bt-maize was grown permanently for eight years whereas one site was cropped with Bt-maize for one year only. The validation of the method showed that the assay fulfils the validation criteria as prescribed in the guidelines of the EU-Decision with minor restrictions. When the assay was applied to field soil samples collected from the selected four experimental sites six months after harvest, no Cry1Ab protein was detectable. By ELISA-determination and quantification of Cry1Ab protein in soils collected from these sites at further time points more profound knowledge about the long-term behaviour of Cry1Ab protein in soil will be obtained.     

Effect of pH on the properties of soy protein–pectin complexes

The interactions of a commercial soy protein isolate (SPI) and a 2:1 SPI:high methoxy pectin (PEC) complex were evaluated over a range of pH values (3-7). The SPI formed very large (> 50 ??m) and largely insoluble aggregates (< 10%) close to its isoelectric point (IEP, pH 4 and 5) and smaller, more soluble (> 80%) particles at higher and lower pH values. The addition of PEC increased the solubility of SPI close to its IEP (pH 4 and 5) and prevented the formation of very large aggregates. However, PEC reduced the solubility of SPI at higher and lower pH values presumably via a depletion mechanism. The ??-potential of diluted SPI dispersions decreased from positive to negative with increasing pH, passing through zero at pH 4.6, the isoelectric point (IEP) of the protein. At pH < 6, the addition of PEC reduced the charge of the protein suggesting the formation of a complex while at pH 6 or 7 there was no evidence of complex formation. The increased SPI solubility in the IEP in the presence of PEC is probably due to the formation of charged complex which do not aggregate while the decreased solubility of protein in the presence at high and low PEC is probably due to the formation of insoluble complexes and a depletion interaction respectively. Thermal treatment (30 min, 90 °C) enhanced the solubility of the SPI:PEC complexes close to the IEP (pH 4 and 5), but reduces it at low pH (pH 3). The SPI:PEC complexes could be manufactured in the form of a beverage at pilot scale where their solubility was enhanced by homogenization.     

Clay particles destabilize engineered nanoparticles in aqueous environments

Given the ubiquity of natural clay minerals, the most likely interaction of nanoparticles released into an aquatic environment will be with suspended clay minerals. Thus, the transport of engineered nanoparticles in the subsurface and the water column will most likely be altered by their interaction with these minerals. We studied the interactions of two of the most produced nanoparticles, Ag and TiO(2), and montmorillonite to determine how heteroaggregation can alter the stability of nanoparticle/clay mineral mixtures. Since at low pH montmorillonite has a negatively charged basal plane and positively charged edges, its interaction with these nanoparticles at different pH lead to unusual behaviors. There are six different interactions for each clay-nanoparticle pair. At pH values below the IEP of montmorillonite edge site, montmorillonite reduced the stability of both negatively charged Ag and positively charged TiO(2) nanoparticles. Surprisingly this enhanced coagulation only occurs within an intermediate ionic strength range. The spillover of the montmorillonite basal plane electric double layer to the montmorillonite edge may screen the electrostatic attraction between Ag and the montmorillonite edge at low ionic strength, whereas a repulsion between TiO(2) and montmorillonite face sites may restabilize the mixture.     

Adsorption characteristics of oligopeptides composed of acidic and basic amino acids on titanium surface

The adsorption characteristics of octapeptides, containing different numbers of aspartic acid, lysine, and alanine residues (i.e., D(4)K(0)A(4), D(4)K(1)A(3), D(4)K(3)A(1), D(4)K(4)A(0), and D(0)K(4)A(4)) on the surface of titanium (Ti) particles were investigated in the pH range of 3.0-8.8 at 30 degrees C. The adsorption isotherms for octapeptides having four plural aspartic acid residues with or without lysine residues showed two distinct adsorption modes, i.e., irreversible and reversible modes, at pHs 3.0-6.5; at pH 7.0 or higher, the adsorption mode was reversible. Increasing the number of lysine residues at a fixed number of aspartic acid residues (i.e., 4) decreased the amount of peptides adsorbed in both modes. D(4)K(4)A(0) adsorbed irreversibly at pHs 3.0-6.5, due to the fact that negatively charged carboxyl groups directly interact with a positively charged Ti surface, whereas positively charged amino groups of lysine residues are directed in an opposite direction toward the solution side, as predicted by molecular mechanics/dynamics calculations.     

Effect of pH and Ionic Strength on the Physicochemical Properties of Coconut Milk Emulsions

ABSTRACT:  Coconut milk (16% to 17% fat, 1.8% to 2% protein) was extracted from coconut ( Cocos nucifera L.) endosperm and diluted in buffer to produce natural oil-in-water emulsions (10 wt% oil). The effect of pH (3 to 7) and NaCl (0 to 200 mM) on the properties and stability, namely, mean particle size, ζ-potential, viscosity, microstructure, and creaming stability, of the natural coconut milk emulsions was investigated. At pH values close to the isoelectric point (IEP) of the coconut proteins (pH 3.5 to 4) and in the absence of NaCl, coconut milk flocculated, but did not coalesce. Flocculation corresponded to low surface charges and was accompanied by an increase in emulsion viscosity. Adding up to 200 mM NaCl to those flocculated emulsions did not change the apparent degree of flocculation. Coconut milk emulsion at pH 6 was negatively charged and not flocculated. Upon addition of salt, the ζ-potential decreased from –16 to –6 mV (at 200 mM NaCl) but this was not sufficient to induce flocculation in coconut milk emulsions. At low pH (< IEP), the positively charged droplets of coconut milk emulsions only flocculated when the NaCl concentration exceeded 50 mM, as the ζ-potential approached zero.     

Polyelectrolyte Multilayers from Cationic and Anionic Starch: Influence of Charge Density and Salt Concentration on the Properties of the Adsorbed Layers

The purpose of the present work was for identify limits for the formation of stable polyelectrolyte multilayers (PEMs) from cationic and anionic starches (with degrees of substitution of 0.04–0.09) on SiO₂ surfaces, taking account of the effect of the charge density of the starches and the salt concentration in the surrounding water phase. The experiments were performed at a pH of 6.3 using stagnation point adsorption reflectometry (SPAR) and quartz crystal microbalance with dissipation (QCM‐D). From these experiments it was concluded that it is possible to form PEMs by the adsorption of oppositely charged starches on SiO₂ surfaces; it was also found that adsorption of the first layer is controlled both by electrostatic, non‐ionic interactions and by pure steric restrictions, i.e. geometrical restrictions, at the surface. The results also indicate that the charge density of the starch must exceed a certain value to allow multilayer formation and that this critical charge density increases with increasing salt concentration. The combination of charge densities of the cationic/anionic starches was also found to influence the adsorption behaviour, and the formed polyelectrolyte multilayers had a high water content of 69–92%.     

Distinct effects of humic acid on transport and retention of TiO2 rutile nanoparticles in saturated sand columns

The distinct effects of humic acid (HA, 0-10 mg L(-1)) on the transport of titanium dioxide (rutile) nanoparticles (nTiO(2)) through saturated sand columns were observed under conditions of environmental relevance (ionic strength 3-200 mM NaCl, pH 5.7 and 9.0). Specifically, the transport of nTiO(2) was dramatically enhanced in the presence of HA at pH 5.7, even at a low HA concentration of 1 mg L(-1). The mobility of nTiO(2) was further increased with greater concentrations of HA. In contrast, this enhancement of the nTiO(2) transportability due to the presence of HA was limited at pH 9.0 because of the negligible adsorption of HA onto nTiO(2), regardless of the concentrations of HA examined in this study. The distinct effects can be explained by the adsorption behaviors of HA to nTiO(2) and sand surfaces and the resulting interactions between nTiO(2) and sand surfaces under different conditions, which resulted in a large variation of the nTiO(2) transport and deposition behaviors at various conditions. In addition, theoretical interaction energy calculations and additional elution experiments indicate that the secondary energy minimum played an important role in controlling the nTiO(2) transport and deposition in porous media observed in this study. Moreover, the interaction energy calculations suggest that at pH 5.7, HA affected nTiO(2) transport by increasing the negative surface charge of nTiO(2) at low HA adsorption densities; whereas, combinations of increased electrostatic and steric interactions due to the presence of HA were the main mechanisms of enhanced transportability of nTiO(2) at high HA adsorption densities. Overall, results from this study suggest that natural organic matter and solution pH are likely key factors that govern the stability and mobility of nTiO(2) in the natural aquatic environment.     

表面等离子共振传感检测Cry2A蛋白

目的：建立利用表面等离子共振（surface plasmon resonance,SPR）传感器检测转基因植物中苏云金芽孢杆菌Cry2A蛋白的方法。方法：利用SPR检测技术,根据生物分子间的相互作用,在金片表面修饰Cry2A蛋白的特异性单克隆抗体,对不同质量浓度梯度的Cry2A蛋白进行检测研究。结果：该方法可有效地检测到Cry2A蛋白,检测灵敏度可达10 ng/mL,与同为抗虫基因编码的Cry1Ac和Cry2Ab蛋白未出现交叉反应。结论：利用SPR方法检测Cry2A蛋白操作简单,省时且灵敏度高、特异性强,可用于对Cry2A蛋白的定性检测。     

Calcium-(organo)aluminum-proton competition for adsorption to tomato root cell walls: experimental data and exchange model calculations

Aluminum interacts with negatively charged surfaces in plant roots, causing inhibition of growth and nutrient uptake in plants growing on acid soils. Pectins in the root cell wall form the major cation adsorption surface, with Ca2+ as the main adsorbing cation. Adsorption of Al3+ and Ca2+ to isolated cell wall material of tomato (Lycopersicon esculentum L.) roots was examined at pH 3.00-4.25 and in the presence of the aluminum chelators citrate and malate. Al3+ displaced Ca2+ from its pectic binding sites in the cell wall to a large extent but apparently also bound to non-Ca binding groups, displacing protons. Aluminum adsorption depended on the pH of the solution, with little Al adsorbing to the cell wall material at very low pH (<3.50). Under very acid conditions Al3+ replacing Ca2+ at pectic cross-links is therefore not expected to play a role in Al toxicity. Equimolar concentrations of citrate decreased Al competition for Ca binding sites almost completely, whereas malate only had an intermediate effect. The competition of (organo) Al3+, Ca2+, and H+ for cell wall binding sites was described adequately using the Gaines-Thomas exchange model.     

Effects of Cry1F and Cry34Ab1/35Ab1 on storage pests

Two crystalline protoxins from Bacillus thuringiensis (Bt), Cry1Fa1 and Cry34Ab1/Cry35Ab1 (Cry1F, Cry34/35Ab1), were evaluated for efficacy against lepidopteran and coleopteran storage pests. Cry1F was tested against the lepidopterans Sitotroga cerealella (Angoumois grain moth) and colonies of Plodia interpunctella (Indian mealmoth) that are susceptible or resistant to Bt Cry1Ab and Cry1Ac toxins, Bt subspecies entomocidus, and the commercial formulation Dipel^®. Cry1F was also tested against the coleopterans Cryptolestes pusillus (flat grain beetle) and Tribolium castaneum (red flour beetle). Cry34/35Ab1 was tested against S. cerealella, C. pusillus, and T. castaneum, and against additional coleopteran storage pests, including Tenebrio molitor (yellow mealworm), Trogoderma variabile (warehouse beetle), Oryzaephilus surinamensis (sawtoothed grain beetle), Rhyzopertha dominica (lesser grain borer), and Sitophilus oryzae (rice weevil). Strains of Bt-susceptible or -resistant P. interpunctella generally were more sensitive to Cry1A protoxin or toxin than either Cry1F protoxin or Dipel. Despite difficulties with the bioassay of S. cerealella larvae, the data suggest that Cry1F and Cry34/35Ab1 caused increased larval mortality, and a developmental delay was observed and no pupae emerged with 0.9% Cry1F. Neither Cry1F nor the corn rootworm-active toxin Cry34/35Ab1 significantly affected the biological parameters of the coleopteran species evaluated.     

Organic nature of colloidal actinides transported in surface water environments

Elevated levels of (239,240)Pu and 241Am have been present in surficial soils of the Rocky Flats Environmental Technology Site (RFETS), CO, since the 1960s, when soils were locally contaminated in the 1960s by leaking drums stored on the 903 Pad. Further dispersion of contaminated soil particles was by wind and water. From 1998 until 2001, we examined actinide ((239,240)Pu and 241Am) concentrations and phase speciation in the surface environment at RFETS through field studies and laboratory experiments. Measurements of total (239,240)Pu and 241Am concentrations in storm runoff and pond discharge samples, collected during spring and summer times in 1998-2000, demonstrate that most of the (239,240)Pu and 241Am transported from contaminated soils to streams occurred in the particulate (> or = 0.45 microm; 40-90%) and colloidal (approximately 2 nm or 3 kDa to 0.45 microm; 10-60%) phases. Controlled laboratory investigations of soil resuspension, which simulated storm and erosion events, confirmed that most of the Pu in the 0.45 microm filter-passing phase was in the colloidal phase (> or = 80%) and that remobilization of colloid-bound Pu during soil erosion events can be greatly enhanced by humic and fulvic acids present in these soils. Most importantly, isoelectric focusing experiments of radiolabeled colloidal matter extracted from RFETS soils revealed that colloidal Pu is in the four-valent state and is mostly associated with a negatively charged organic macromolecule with a pH(IEP) of 3.1 and a molecular weight of 10-15 kDa, rather than with the more abundant inorganic (iron oxide and clay) colloids. This finding has important ramifications for possible remediation, erosion controls, and land-management strategies.     

Adsorption mechanism of arsenic on nanocrystalline titanium dioxide

Arsenate [As(V)] and arsenite [As(III)] interactions at the solid-water interface of nanocrystalline TiO2 were investigated using electrophoretic mobility (EM) measurements, Fourier transform infrared (FTIR) spectroscopy, extended X-ray absorption fine structure (EXAFS) spectroscopy, and surface complexation modeling. The adsorption of As(V) and As(III) decreased the point of zero charge of TiO2 from 5.8 to 5.2, suggesting the formation of negatively charged inner-sphere surface complexes for both arsenic species. The EXAFS analyses indicate that both As(V) and As(III) form bidentate binuclear surface complexes as evidenced by an average Ti-As(V) bond distance of 3.30 A and Ti-As(III) bond distance of 3.35 A. The FTIR bands caused by vibrations of the adsorbed arsenic species remained at the same energy levels at different pH values. Consequently, the surface complexes on TiO2 maintained the same nonprotonated speciation at pH values from 5 to 10, and the dominant surface species were (TiO)2AsO2- and (TiO)2AsO- for As(V) and As(III), respectively. The surface configurations constrained with the spectroscopic results were formulated in the diffuse layer model to describe the adsorption behavior of As in the pH range between 4 and 12. The study suggests that TiO2 is an effective adsorbent for As removal due to its high surface area and the presence of high affinity surface hydroxyl groups.     

Analysis of malts by capillary electrophoresis

A range of malts, as well as their high‐ and low‐molecular‐mass fractions, has been examined by capillary electrophoresis in phosphate buffer, pH 2.5, and in carbonate buffer, pH 9.5, and the results have been compared with those for roasted barley and for caramels. The malts fall into two categories: (i) the lightly roasted malts, where the high‐molecular‐mass coloured fraction is negatively charged at pH 9.5 and positively charged at pH 2.5; and (ii) the highly roasted malts (and the roasted barley), where the high‐molecular‐mass fraction migrates close to the electro‐osmotic flow at both pH 9.5 and 2.5, implying that it carries little or no charge. The former category shows migration patterns similar to Class III caramels, whereas the latter migrates differently from Class I, III and IV caramels as well as from the former. Capillary electrophoresis therefore has considerable potential for differentiating between malts and between malts and caramels and roasted barley. © 2002 Society of Chemical Industry     

A real-time immuno-PCR assay for the detection of transgenic Cry1Ab protein

The adaptation and production of transgenic crops harboring Cry1Ab protein is increasing every year globally due to their potent insecticidal activity. The Cry1Ab protein produced by Bacillus thuringiensis confers resistance against several lepidopteran insect pests. The release of transgenic crops/produce in the market worldwide has increased the need of regulatory affairs to monitor and verify the presence of transgenic protein in crops/produce. In this regard, the real-time immuno-PCR (IPCR) assay was developed for the detection and quantification of Cry1Ab protein. The IPCR assay showed high sensitivity with minimum detection limit of 100 pg/mL (0.1 ppb) and found to be 10 times more sensitive than sandwich ELISA. Under the optimized assay conditions, Cry1Ab protein can be determined in the concentration ranged from 100 pg/mL to 100 ng/mL. As results suggest, this assay could be a powerful tool for the detection of even trace amounts of Cry1Ab protein in transgenic crops.     

Investigation of nanoparticle transport inside coarse-grained geological media using magnetic resonance imaging

Quantifying nanoparticle (NP) transport inside saturated porous geological media is imperative for understanding their fate in a range of natural and engineered water systems. While most studies focus upon finer grained systems representative of soils and aquifers, very few examine coarse-grained systems representative of riverbeds and gravel based sustainable urban drainage systems. In this study, we investigated the potential of magnetic resonance imaging (MRI) to image transport behaviors of nanoparticles (NPs) through a saturated coarse-grained system. MRI successfully imaged the transport of superparamagnetic NPs, inside a porous column composed of quartz gravel using T(2)-weighted images. A calibration protocol was then used to convert T(2)-weighted images into spatially resolved quantitative concentration maps of NPs at different time intervals. Averaged concentration profiles of NPs clearly illustrates that transport of a positively charged amine-functionalized NP within the column was slower compared to that of a negatively charged carboxyl-functionalized NP, due to electrostatic attraction between positively charged NP and negatively charged quartz grains. Concentration profiles of NPs were then compared with those of a convection-dispersion model to estimate coefficients of dispersivity and retardation. For the amine functionalized NPs (which exhibited inhibited transport), a better model fit was obtained when permanent attachment (deposition) was incorporated into the model as opposed to nonpermanent attachment (retardation). This technology can be used to further explore transport processes of NPs inside coarse-grained porous media, either by using the wide range of commercially available (super)paramagnetically tagged NPs or by using custom-made tagged NPs.