Article pubs.acs.org/est
Environmental Conditions That Influence the Ability of Humic Acids to Induce Permeability in Model Biomembranes Loice M. Ojwang’ and Robert L. Cook* Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States S Supporting Information *
ABSTRACT: The interaction of humic acids (HAs) with 1-palmitoyl-2-oleoylSn-glycero-3-phosphocholine (POPC) large unilamellar vesicle (LUV) model biomembrane system was studied by fluorescence spectroscopy. HAs from aquatic and terrestrial (including coal) sources were studied. The effects of HA concentration and temperature over environmentally relevant ranges of 0 to 20 mg C/L and 10 to 30 °C, respectively, were investigated. The dosage studies revealed that the aquatic Suwannee River humic acid (SRHA) causes an increased biomembrane perturbation (percent leakage of the fluorescent dye, Sulforhodamine B) over the entire studied concentration range. The two terrestrial HAs, namely Leonardite humic acid (LAHA) and Florida peat humic acid (FPHA), at concentrations above 5 mg C/L, show a decrease or a plateau effect attributable to the competition within the HA mixture and/or the formation of “partial aggregates”. The temperature studies revealed that biomembrane perturbation increases with decreasing temperature for all three HAs. Kinetic studies showed that the membrane perturbation process is complex with both fast and slow absorption (sorption into the bilayer) components and that the slow component could be fitted by first order kinetics. A mechanism based on “lattice errors” within the POPC LUVs is put forward to explain the fast and slow components. A rationale behind the concentration and temperature findings is provided, and the environmental implications are discussed.
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nanoparticles, in other cases.18−26 4) It has also been shown that HSs can act as an electron shuttle between a microorganism and a pollutant or a mineral phase as well as it has been hypothesized that HSs can act as sunscreen.3,27,28 In all of the above cases, the interaction between HSs and a biological membrane (biomembrane) is of great importance and interest, both of which have collectively led to a number of studies utilizing a range of analytical techniques, such as electrophoretic mobility measurements, adsorption isotherms, transmission electron microscopy (TEM), and nuclear magnetic resonance (NMR).3−7 The findings from these studies prompted interest in the mechanism by which HSs interact with biomembranes. Work by Maurice and co-workers, which focused on the fulvic acid fraction (FA) of HSs, showed preferential sorption of the more hydrophobic−FA fractions to biomembranes at acidic pH.5,6 In parallel studies, Campbell and co-workers showed that HSs sorbed more efficiently to biomembranes at acidic pH conditions and that, once sorbed, the HSs increased the permeability of phytoplankton and model biomembranes, with humic acid inducing the largest effect.3,4 A membrane-focused study by Elayan et al. utilized wide line 31P NMR and humic acids from three different sources to show that, at acidic pH, HSs perturbed the structure of model biomembranes.7
INTRODUCTION Humic substances (HSs) are the major components of natural organic matter (NOM). They are prevalent throughout the environment, including terrestrial, aquatic, and coal areas, where they play diverse and significant environmental roles. They are formed as a result of the microbial breakdown of plant matter that contains proteins, cellulose, lignin, lipids, cutins, and cutans. This leads to the formation of a complex, heterogeneous, and polydisperse mixtures with diverse functional groups, ranging from hydrophobic to hydrophilic entities, such as aliphatic, aromatic, aldehyde, methoxyl, ketone, thiol phenolic, quinoline, carboxylic, and hydroxylic moieties.1,2 This endows HSs with unique properties, such as the potential to interact with organic substances, metals, nutrients, and biological surfaces.1−7 The interaction of HSs with biomembranes is of interest to a large number of areas of study for a variety of reasons, some of which are listed below. 1) The utilization of HSs as a food source is believed to involve the secretion of enzymes to help breakdown the HSs into smaller entities, which are then consumed by microorganisms as food.8,9 2) The ability of HSs to modify biological functions has been shown for both plants and animals via promoting plant root growth, inducing antiandrogenic effect, influencing sodium metabolism, altering cell potential, and influencing carbon fixation.10−15 3) HSs have been shown to affect the bioavailability and toxicity of metal ions, organic molecules, and nanoparticles, by inhibiting the bioavailability of toxic compounds in some cases,16,17 while increasing the bioavailability of toxic compounds, including © 2013 American Chemical Society
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February 4, 2013 June 13, 2013 June 27, 2013 June 27, 2013 dx.doi.org/10.1021/es4004922 | Environ. Sci. Technol. 2013, 47, 8280−8287
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lipid film. The film was then dissolved in 10 mL of 50 mM SRB in 0.01 M phosphate buffer, vortexed using a Vortex Genie series (G560) for 30 min, and then underwent four freeze/thaw cycles (placed in dry ice and acetone until the mixture was completely frozen and then heated to 40 °C). The result was a pink heterogeneous mixture of large multilamellar vesicles. The solution was then extruded three times through a 100 nm-pore Whatman Nuclepore polycarbonate track-etched membrane at ambient temperatures using a Lipex Lipid Extruder (North Lipids, Vancouver, BC, Canada), generating large unilamellar vesicles (LUVs) with a diameter of approximately 100 nm (Figures 1 SI and 2 SI in Supporting Information), which will be referred to as model biomembranes from here on in. The nonencapsulated dye was separated from the encapsulated dye by gel filtration using a column packed with Sephadex G-50 resin (column diameter: 1 cm; length: 15 cm; applied sample volume: 2 mL) and 10 mM phosphate buffer as an elution buffer for a total of five times. HA (FPHA, LAHA, and SRHA) stock solutions were prepared by dissolving 8.6 mg of each HA in 100 mL of 0.01 M phosphate buffer. The appropriate amount of a HA stock solution was then added to the appropriate volume of the LUV solution to carry out the fluorescence leakage experiments. Fluorescence Leakage Measurements. All fluorescence measurements were carried out using a Horiba Jobin Yvon Fluorolog 3 spectrofluorimeter with a FL1073 detector, Spectra Acq computer, and a model LFI3751 temperature control, blank subtracted, and inner filter corrected. SRB-loaded LUVs were exposed to FPHA, LAHA, and SRHA to investigate the perturbation effect of HAs on the model biomembranes. This was accomplished by measuring 5.00 mL of SRB-loaded LUVs and adding 5.00 mL of 0.01 M phosphate buffer solution for the blank and 5.00 mL of HAs in phosphate buffer for the test sample. The excitation wavelength used was 565 nm, and the emission was monitored from 570 to 700 nm with 1.0 nm increments and an integration time of 1 s. The excitation and emission slits were both set at 1.5 nm. For the kinetics measurements, emission was monitored at 588 nm with an integration time of 1 min. The signal generated was compared to the one obtained after the sample was spiked with 100 μL of 1% detergent TX-100. Dynamic light scattering (DLS) and cryogenic (cryo-) TEM were used to verify the sizes of the prepared LUVs and the completely lysed LUVs as induced by the TX-100, see the Supporting Information. The solution pH was confirmed at all stages of the analysis. The one notable exception was during the kinetic runs, in which the pH of the two mixing solutions was confirmed just before the mixing step and at the end of the run. All measurements were done in triplicate, and the results were plotted, as indicated in the Results section. Leakage of the SRB dye was expressed as a percentage relative to the total amount of dye released by the addition of 100 μL of 1% TX-100, which represented 100% leakage and was calculated according to the equation
All of these studies have led to important insights; however, they have left a number of open questions, such as the following: (i) what is the effect of temperature and (ii) what are the kinetics of these interactions. Also, the aforementioned studies have either focused on aquatic or terrestrial HSs but not on both in order to determine whether a universal behavior for HSs can be found. In this study, the nature and kinetics of HS interactions with POPC LUV model biomembranes, as well as the effects of various environmental parameters, such as temperature and HS concentration were investigated using three standard humic acids (HAs) from terrestrial (including low grade coal) and aquatic sources.
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EXPERIMENTAL SECTION Materials. 1-Palmitoyl-2-oleoyl-Sn-glycero-3-phosphocholine (POPC) was purchased from Avanti Polar Lipids (Alabaster, AL); Florida (Pahokee) Peat Humic Acid (FPHA; a terrestrial HA sourced from a typical agricultural peat soil HA), Leonardite Humic Acid (LAHA; a terrestrial HA sourced from a low grade coal), and Suwanee River Humic Acid (SRHA; an aquatic HA sourced from a black water source) were purchased from the International Humic Substances Society (IHSS, St. Paul, MN). More details on each of these HAs, including chemical composition, are available on the IHSS Web site.29 Methanol and chloroform were purchased from Fisher Scientific Company (Somerville, NJ). Nitrogen gas was supplied by the chemistry department (upon being sourced from Capital Welders Supply Co., Baton Rouge, LA). Sulforhodamine-B and Triton X-100 (TX-100) were obtained from Sigma Aldrich (Milwaukee, WI), while Sephadex G-50 GE was obtained from Healthcare Biosciences (Piscataway, NJ). High quality (18MΩ) deionized water was made in our laboratory using an apparatus manufactured by US Filter. Experimental Design. A major issue with studying the interaction of HSs with biomembranes from a mechanistic point of view is the complexity of both HSs and biomembranes. In order to simplify the biomembranes, a large unilamellar vesicle (LUV) biomembrane model system was chosen. POPC was chosen as the model phospholipid for these LUVs for the following reasons: 1) it is ubiquitous in eukaryote cell membranes;30,31 2) its bilayer phase exists within an environmentally and easily accessible temperature range (Tm; is approximately −2 °C),32 and 3) it is well studied. Humic acids were chosen to represent HSs as 1) they are a major portion of HSs, 2) they have been shown to induce the largest membrane perturbations,4 and 3) well characterized standards are commercially available. A pH of 4.8 was chosen as it has been shown that the ability of HSs to induce membrane perturbation increases with decreasing pH,4,7 and this pH is of environmental relevance to both water bodies and pore waters.33,34 As the goal of this study was to gain deeper insight into the mechanism by which HSs induce membrane permeability, the focus was on HS samples and conditions that had been previously shown to induce the largest membrane perturbation, while still being within environmentally relevant constraints. Sample Preparation. The sulforhodamine-B (SRB) vesicles were prepared in accordance with Ladokhin et al. and Graslund et al.35,36 In short, 100 mg of POPC was dissolved in 132 μL of methanol and 264 μL of chloroform, thoroughly mixed for 30 min after which the solvents were evaporated under nitrogen gas for 35−45 min and the remnants were dried overnight under vacuum, resulting in the formation of a thin
Percent release = 100 × (IH − IB)/(IT − IB)
where IH is the fluorescence intensity after the addition of the HA solution, IB is the background fluorescence of SRB-loaded vesicles before the addition of either the HA or TX-100 solutions, and IT is the total fluorescence intensity after the complete rupture of the vesicles caused by the addition of TX100.4 8281
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RESULTS AND DISCUSSION From the previous reports in the literature it has become apparent that the interactions of humic materials with biomembranes is most prevalent at acidic pHs; however, much of this work has focused on aquatic humics. As discussed in the Introduction, the interaction of both aquatic and terrestrial humic substances with biomembranes is important in terms of their biological functions and pollutant toxicity. This led to a rough guiding experiment, in which the potential of FPHA to perturb model biomembranes at a range of FPHA concentrations at two different temperatures (15 and 25 °C) and two pH conditions (an acidic pH of 4.8 and a slightly basic pH of 7.6) was examined via the fluorescent dye leakage of the model biomembrane system (see Figure 3SI in the Supporting Information). This guiding experiment showed that all three factors were important. By far, pH plays the most important role, with little leakage occurring at the slightly basic pH, as expected based on previous studies; consequently, we will focus only on the acidic pH from here on in.4,7 At the acidic pH conditions it was found that both the FPHA concentration and the temperature strongly influenced the amount of model biomembrane perturbation (the higher the percentage leakage observed, the larger the perturbation of the model biomembrane). This led to more detailed studies of these two factors, as discussed below. Dose Response Study. Dose response studies have also been carried out at 25 °C using three different types of HAs at pH 4.8, see Figure 1. These data can be analyzed within at least
by LAHA plateaus. This plateau can be explained by a saturation of available adsorption and/or absorption sites (see the detailed discussion below). Within the second concentration range (5−20 mg C/L), a number of interesting observations can be made. First of all, and possibly counterintuitively, the leakage induced by LAHA decreases with increasing HA concentration. It is important to mention here that, due to the complexity of the HAs studied here and that of natural organic matter as a whole, any explanation of such behavior is speculative in nature. With this in mind, one plausible, though unlikely, explanation is that, at concentrations close to or higher than 5 mg C/L, LAHA is starting to form aggregates; however, a number of diffusion-ordered NMR studies have shown that the same or similar humic substances to those used in this study maintain molecular weights of 5000 Da or less at concentrations 2 or 3 magnitudes higher than those used in this study.38−40 The same studies also showed that, as the concentration of the HS solution decreased, so did the apparent molecular weight. Extrapolating these findings to the concentrations used in this work, it becomes more than reasonable to conclude that there is little aggregation of any of the three HA used, at the concentrations studied, here. There is the possibility of small associations or ‘partial aggregates’ of membrane-perturbing entities within LAHA at concentrations above 5 mg C/L, especially if one considers HSs to be supramolecular assemblies.38,41 Accordingly, if the most hydrophobic components associated with each other, they would become less likely to perturb the biomembrane system (see the detailed discussion below) and, hence, induce less perturbation per unit C. Another explanation for the turnover in the LAHA dose curve is a possible competition between membrane-perturbing and membrane-protecting entities within LAHA, if there is a higher concentration of membraneperturbing entities. At lower LAHA concentrations there may not be enough membrane-protecting entities to compete with the membrane-perturbing entities for the adsorption sites, and hence, as the concentration of the membrane-perturbing entities increases with increasing LAHA concentration, an increase in dye leakage is observed. However, as the LAHA concentration is further increased, there will be a large enough population of the membrane-protecting entities able to adsorb to enough sites on the model biomembrane so that a lower number of total sites are available for the membrane-perturbing entities to occupy than at lower LAHA concentration, and hence, less dye leakage. For FPHA it can be seen that leakage increases until about 7.5 mg C/L and causes more perturbation than LAHA at just below 7.5 mg C/L for the conditions used in this work. After about 7.5 mg C/L, as discussed above, the leakage plateaus out with increasing FPHA concentration, again with the possible explanation being saturation of available adsorption and/or absorption sites. For SRHA there is an increasing dye leakage with increasing concentration for the entire concentration range studied here, consistent with the work of Campbell and co-workers.4 Effects of Temperature on Leakage. The interaction of HAs with POPC LUVs was investigated at five temperatures ranging between 10 and 30 °C (in 5 °C increments), as shown in Figure 2, at a constant HA concentration of 5 mg C/L and at pH 4.8. From these data it can be seen that the ability of all three HAs to induce leakage decreases with increasing temperature, across the entire temperature range studied, and it does so in a nearly linear manner (with R2 ranging between 0.99, 0.96, and 0.82 for LAHA, FPHA, and SRHA,
Figure 1. Dose response study of SRHA, FPHA, and LAHA, HAs at pH 4.8 and room 25 °C with a contact time of 15 min between the humic acid and the POPC LUVs.
two HA concentration ranges: 0−5 and 5−20 mg C/L. Within the first range, and for all three HAs studied, one observes an increase in dye leakage with increasing concentration. The ability of HAs to induce leakage and, hence, cause model biomembrane perturbation, follows the order LAHA > FPHA > SRHA. This trend directly correlates with the composition of these three HAs, and in particular with the concentration of aromatic moieties, as determined via 13C NMR analysis (58, 47, and 31% for LAHA, FPHA, and SRHA, respectively).29 An inverse dependence is observed with respect to the polarity ((O+N)/C) (0.51, 073, and 0.83, as obtained via elemental analysis for LAHA, FPHA, and SRHA, respectively).29,37 Even at a HA concentration as low as 5 mg C/L, the leakage induced 8282
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phases could be coexisting in the vesicles’ membranes, resulting in packing defects in phase boundaries between these coexisting phases. Such packing defects occur as a result of either the fluctuations in the lateral compressibility of the bilayers at Tm or due to the acyl chain packing mismatch at the interfacial region between gel and fluid domains.42−45 This, therefore, could be the reason for the high leakage observed as the temperature decreases from 30 to 10 °C. Leakage Time Course Data and Kinetics. The time course data of leakage versus concentration are shown in Figure 3. From these data it is clear that LAHA induces the fastest perturbation, while SRHA induces the slowest perturbation. Focusing only on the LAHA data, it can be seen that at the highest concentrations (7.5 and 10 mg C/L) full perturbation is established in less than 10 min; however, at the lowest concentration (0.625 mg C/L) the perturbation is still ongoing even after 240 min (4 h). It is also interesting to note that, as with the LAHA data from the dosage study, the LAHA time course leakage experiments reveal a sudden transition between a steep increase in leakage with time versus a shallow increase in leakage with time after 75 min or less for LAHA concentrations of 2.5 mg C/L and higher. The onset time of this sudden transition is concentration dependent, with the most rapid onset corresponding to the highest LAHA concentration. Time course data for SRHA, on the other hand, all show a trending toward, but never quite reaching, a plateau. The FPHA data are in between the LAHA and SRHA data, with an apparent trend toward a plateau between 7.5 and 10 mg C/L, with a crossover point around 75 min. The findings reported for the leakage experiments are consistent with the dosage data and show that the competition or the formation of
Figure 2. Effects of temperature on leakage of SRB dye as induced by 5 mg C/L of SRHA, FPHA, and LAHA HAs at pH 4.8 and a 60 min contact time (to allow for full thermal equilibration) between the humic acid and the POPC LUVs.
respectively). LAHA exhibits the largest (steepest) slope, while SRHA exhibits the smallest (shallowest) slope for the plots of leakage as a function of increasing temperature. These findings are consistent with the above discussion for the three different HAs and their ability to induce leakage and can be related to the chemical makeup of these HAs. These results agree with the literature finding that maximum leakage occurs at the Tm (approximately −2 °C)32 and decreases at temperatures above and below the Tm when the bilayers are either all-gel or all-fluid.42−45 At 10 °C, the temperature nearest to the Tm of POPC of the five temperatures investigated, the gel and the liquid crystalline
Figure 3. Kinetics of leakage of SRB dye as induced by a) SRHA b), FPHA, and c) LAHA at pH 4.8 and 25 °C, the 5 mg C/L line has no embedded dots while the 10 mg C/L has embedded dots. 8283
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‘partial aggregates’ phenomena discussed for the dosage data can also be used here to explain the differences among the different HAs in this study. In fact, if points are taken at 10, 20, 40, and 60 min and plotted as they were in Figure 1, the same trends are seen, but with greater leakage at longer times for SRHA and FPHA. For LAHA, on the other hand, at about 6 mg C/L there is a convergence of the leakage data and a decrease in the amount of leakage with increasing HA concentration. An initial inspection of the time course data presented in Figure 3 would lead one to expect a two-component kinetic fitting, with a fast and slow component. Such a fitting was achieved; however, the kinetic analysis yielded a rather complex picture, especially in regards to the fast component. More details of the fitting and the associated discussion are presented in the Supporting Information. The slow component in the mechanism can be fitted by first order kinetics for all HAs and is highly concentration dependent, with reductions in the rate occurring with increasing HS concentration, parallel to the plateauing or decreasing with concentration effects seen in the dosage studies for LAHA. These parallel findings imply that the competition or the formation of ‘partial aggregates’ phenomena lead to a decreased potential for membrane-perturbing HA entities to associate with each other and induce the slow component in the membrane perturbation mechanism (see the discussion below). Stated differently, the moieties within HAs that are outcompeted for the adsorption sites or form ‘partial aggregates’ − in all likelihood the least polar moieties − are the very moieties responsible for the slow biomembrane perturbation in this study. The fast component could not be readily fitted to either first or second order kinetics. A very weak second order fit could be obtained if 1) only the first 5 to 10 min of the time course data were considered and 2) the slow component discussed above was not in play, implying that the slow component requires a perturbation that takes longer than the first 5 to 10 min. The fact that the fast component cannot be fitted to the first or second order kinetics can be explained by more than one distinct process taking place. This conclusion is consistent with complex heterogeneous nature of HAs. A Conceptual Absorption Model for HA to POPC LUVs. The kinetic data and analysis allow for further insight into the mechanism by which HAs can interact with biomembranes. The use of vesicles in this study allows the highly complex interactions of HAs with biomembranes to be simplified, as only passive processes are being monitored. This means that the observed perturbation is not an HA-induced biochemical process, such as an interference with photosynthetic oxygen production, regulation of a variety of genes, impact on ion regulation and metabolism, and hyperpolarization of the transepithetial potential.11,13,14 Our previous wideline 31P NMR work has shown that HA can induce a structural perturbation within DPPC multilaminar vesicles, giving rise to the proposed adsorption/absorption mechanism.7 A full explanation and justification for the adsoprtion/absorption mechansims is provided in Elayan et al.7 In short, the adsoprtion step in this mechanism is a hydrogen bridge between the HAs and vesicle’s outer surface, explaining the pH dependence reported here and in a number of other studies.3,4,7 This adsorption step, which can be viewed as the association step, is followed by an absoprtion step, which can be viewed as the perturbation step, in which the hydrophobic entities within the HAs penetrate into the hydrophobic domains of the lipid
bilayer. For the leakage experiments presented here, the focus is on the absorption step as the membrane structure-perturbing event, as discussed in detail by Elayan et al.,7 leading to an alteration in the permeability of the bilayer and, hence, determining the amount of dye that can leak out. The kinetic data show that the absorption step is more complex than implied by the 31P NMR data, with at least two distinct processes taking place, namely 1) a fast absorption process and 2) a slow absorption process, as illustrated in Figure 4.
Figure 4. Proposed adsorption/absorption mechanism of HAbiomembrane interactions at acidic pH (It should be noted that the illustration does not imply that HA or any form of natural organic matter will uniformly coat a biomembrane; in fact TEM images in the literature clearly show the opposite.).3
A Tale of a Fast and a Slow Absorption (Perturbation) Process. The fast absorption process takes place on the minute (or faster, as the data in Figure 3 illustrate) time scale, while the slow absorption process can take place over the course of hours. The pH dependence of the perturbation has been explained by the formation of a hydrogen bridge between the HA and the biomembrane surface, and thus, prior to the absorption discussed below, an adsorption via a hydrogen bridge has to take place.2,7 In order to develop the concept of absorption, instead of looking at a biomembrane as a homogeneous surface, a better viewpoint of the surface would be as a bilayer with “lattice” defects.46−49 Due to the fact that these defect sites locally reduce the thickness of the phospholipids from a bilayer to a single layer, the hydrophobic domains of the HAs should be able to more directly interact with the hydrophobic tail groups of the phospholipids and lead to a rapid perturbation of the bilayer’s stucture, affecting its integrity. The temperature data presented here lend further credence to this view, as the changes in the bilayer integrity induced by the packing defects in phase boundaries between coexisting phases, especially close to the phase transition temperature, would result in more defects being present and, hence, larger leakages.42−45 Based on the above discussion, the conceptual illustration in Figure 4 is intended to to help guide further consideration. This loss of bilayer stuctural integrity would eventually lead to an increase in the permeability of the bilayer and, hence, the rapid leakage of the dye induced by all three HAs in the kinetic studies described above. On the other hand, the slower component is more difficult to explain; nevertheless, the aboveproposed mechansim is consistent with the hypothesis that at 8284
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SI illustrating the concentration, pH, and temperature study on the interaction of FPHA with POPC LUVs. Table 1 SI contains the relative amount of functionalities within HAs from different sources determined by 13C NMR, and Table 2 SI contains first order kinetic rate constants for the slow component of the leakage studies for the three HAs investigated. Finally, the structures of 1-palmitoyl-2-oleoyl-Sn-glycero-3-phosphocholine (POPC) and sulforhodamine B are provided in Figures 4 SI and 5 SI, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.
least two chemically distinct groups of entities within the mixture that a humic acid constitutes are involved with the fast and slow steps observed above. The mechanism of this slow bilayer perturbation can be viewed as akin to the mechanism just discussed; however, rather than a direct absoprtion step, the HA entity associated with the slower perturbation component diffuses over the bilayer surface until it can absorb to a “lattice” defect site. This leads to questions as to why there is a much higher probability of such a condition to take place for the HA entities involved with the fast component than with the slow component. As stated above, we propose that a hydrogen bridge between the HA and the bilayer, i.e. the adsorption step, must take place before the absorption step can take place. This requires that the HA entity, or entities, involved be able to support such a hydrogen bridge, strongly implying that they have a high density of polar groups (functionalized aromatic and O-alkyl moieties). On the other hand, HA entities with a low density of polar groups (protonated aromatic and alkyl moieties) will have a much lower probability of achieving the hydrogen bridge condition and, hence, will take longer to achieve the initial adsoprtion (association) step required prior to the absorption (perturbation) step. Environmental Implications. Simply stated, real biomembranes are highly complex systems with varying lipid composition, of lipids featuring different 1) headgroup sizes and charges, 2) hydrocarbon chain lengths and branching, and 3) positions and numbers of unsaturated bonds, not to mention the presence of 4) cholesterol and proteins. This complexity poses major problems for mechanistic studies, necessitating the use of highly simplified vesicle model systems. For this study, the complexity of HSs made the use of a model biomembrane system all the more logical. In the real world, however, this biomembrane complexity is, in all likelihood, at the core of what has allowed organisms to survive in environments with high HS concentrations. An example of such a protective strategy would be the incorporation of negatively charged phospholipids, even at acidic pHs, which has been shown to decrease the ability of HSs to induce permeability in model membrane systems by electrostatic repulsion.4 In the real environment this electrostatic repulsion may be countered by the presence of bridging cations, as shown by the work of Borrok et al. on the role of ternary bacteria-metal-HS complexes in the sorption of HSs to living surfaces.50 Another protective strategy would be the inclusion of cholesterol, which acts as a membrane stabilizer. With this mind, it has been shown or argued that HSs (NOM) directly perturb real membranes as well as model membrane systems, with fulvic acid inducing less perturbation than humic acid.4,7,14 The ability of HA to enhance membrane permeability at low environmental concentrations and on the minute time scale, with temperature playing a role as well, raises a large number of questions in regards to the role of humic acids, and NOM as a whole, in the environment, especially in regards to NOM’s relationship with living entities, from the viewpoint of pollutant toxicity, bioavailability, and bioremediation for metals, organic pollutants, and nanoparticles16−26 as well as NOM toxicity, both from the direct and indirect points of view.11−14
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Dr. Elayan for help in the initial LUV preparations. This material is based upon work supported by the National Science Foundation under the NSF CAREER Award CHE-05479. Any opinions, findings, and conclusion or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. Finally, the guiding insights of three anonymous reviewers and E. Michael Perdue are acknowledged for improving this manuscript. Elzbieta Cook is thanked for her editorial help.
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REFERENCES
(1) Buffle, J. Complex Reactions in Aquatic Systems: An Analytical Approach; Ellis Horwood: Chester, 1981. (2) Stevenson, F. J. Humus Chemistry; Genesis, Composition, Reactions, 2nd ed.; John Wiley & Sons: New York, 1994. (3) Campbell, P. G. C.; Twiss, M. R.; Wilkinson, K. J. Accumulation of natural organic matter on the surfaces of living cells: implications for the interaction of toxic solutes with aquatic biota. Can. J. Fish. Aquat. Sci. 1997, 54 (11), 2543−2554, DOI: 10.1139/cjfas-54-11-2543. (4) Vigneault, B.; Percot, A.; Lafleur, M.; Campbell, P. G. C. Permeability changes in model and phytoplankton membranes in the presence of aquatic humic substances. Environ. Sci. Technol. 2000, 34 (18), 3907−3913, DOI: 10.1021/es001087r. (5) Frost, P. C.; Maurice, P. A.; Fein, J. B. The effect of cadmium on fulvic acid adsorption to Bacillus subtilis. Chem. Geo. 2003, 200 (3−4), 217−224, DOI: 10.1016/S0009-2541(03)00192-X. (6) Maurice, P. A.; Manecki, M.; Fein, J. B.; Schaefer, J. Fractionation of an aquatic fulvic acid upon adsorption to the bacterium, Bacillus subtilis. Geomicrobiol. J. 2004, 21 (2), 69−78, DOI: 10.1080/ 01490450490266235. (7) Elayan, N. M.; Treleaven, W. D.; Cook, R. L. Monitoring the effect of three humic acids on a model membrane system using P-31 NMR. Environ. Sci. Technol. 2008, 42 (5), 1531−1536, DOI: 10.1021/ es7024142. (8) Reddy, K. R., DeLaune, R. D. Biochemistry of Wetlands: Science and Applications; CRC Press: Boca Raton, 2008. (9) Findlay, S. E. G.; Sinsabaugh, R. L. Aquatic Ecosystems: Interactivity of Dissolved; Academic Press: San Diego, CA, 2003. (10) Canellas, L. P.; Dobbss, L. B.; Oliveira, A. L.; Chagas, J. G.; Aguiar, N. O.; Rumjanek, V. M.; Novotny, E. H.; Olivares, F. L.; Spaccini, R.; Piccolo, A. Chemical properties of humic matter as related to induction of plant lateral roots. Eur. J. Soil Sci. 2012, 63 (3), 315−324, DOI: 10.1111/j.1365-2389.2012.01439.x. (11) Steinberg, C. E. W.; Meinelt, T.; Timofeyev, M. A.; Bittner, M.; Menzel, R. Humic substances (review series) Part 2: Interactions with organisms. Environ. Sci. Pollut. Res. 2008, 15 (2), 128−135, DOI: 10.1065/espr2007.07.434.
ASSOCIATED CONTENT
S Supporting Information *
Figures illustrating the sizes of the LUVs used in this study Figure 1 SI and Figure 2 SI are provided together with Figure 3 8285
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