Hydrophobic Interactions between Spin-Label 5-SASL and Humic

Environ. Sci. Technol. 2001, 35, 761-765

Hydrophobic Interactions between Spin-Label 5-SASL and Humic Acid As Revealed by ESR Spectroscopy JULIETA A. FERREIRA,† OTACIRO R. NASCIMENTO,‡ AND L A D I S L A U M A R T I N - N E T O * ,§ Embrapa Instrumentac¸ a˜o Agropecua´ria, R. XV de Novembro 1452, C.P. 741, 13560-970, Sa˜o Carlos (SP), Brazil, IQSC-USP, R. Dr. Carlos Botelho 1465, CEP 13560-970, Sa˜o Carlos (SP), Brazil, and IFSC-USP, R. Dr. Carlos Botelho 1465, CEP 13560-970, Sa˜o Carlos (SP), Brazil

The spin-label probe 5-SASL (stearic acid spin-label with nitroxide free radical in position 5 of hydrocarbon chain), detectable by electron spin resonance (ESR), was tested to evaluate pH and reaction time dependencies of hydrophobic interactions with humic acid (HA). Strong changes were observed in 5-SASL ESR spectra in the presence of HA suspensions below pH 5, with disappearance of the three isotropic narrow hyperfine lines of the nitroxide group (typical of free spin-label) and formation of “immobilized” 5-SASL spectra. These changes were interpreted as due to 5-SASL bonding with hydrophobic groups of HA, by van der Waals forces and/or hydrogen bonds, in very hydrophobic sites (probably water-protected) existent in HA below pH 5. However, such sites are absent above pH 5, as demonstrated by a specific experiment to check 5-SASL spectra reversibility. On the other hand, the HA suspension was more efficient in dissolving 5-SASL than water above pH 5. This fact also suggests the existence of “surface” hydrophobic sites, where the spin-label binds to HA while maintaining the nitroxide group in contact with water, as evidenced by the typical free spin-label spectrum and hyperfine interaction splitting (a0) 1.574 mT). Also experiments checking 5-SASL reversibility bonding with HA were consistent with the supramolecular association model to HA.

Introduction Pollutant binding to soil organic matter (SOM) is usually accompanied by drastic changes in pollutant mobility, leaching, and bioavailability, including degradation, which affects subsequent absorption by plants and fauna. There are many binding mechanisms, mainly physical, physicalchemical, and chemical between organic pollutants and SOM. However, the nature and extent of these binding mechanisms are not yet fully understood (1). Soil sorption of most hydrophobic organic compounds, e.g., nonpolar pesticides, is directly related to SOM presence (2, 3). Humic substances (HS) are the major SOM components, containing carboxylic, phenolic, amine, quinone, and other functional groups (4-6), and specific structural configurations (conformation) (7-13). * Corresponding author telephone: 55-16-2742477; fax: 55-162725958; e-mail: [email protected]. † IQSC-USP. ‡ IFSC-USP. § Embrapa Instrumentac ¸ a˜o Agropecua´ria. 10.1021/es0010251 CCC: $20.00 Published on Web 01/20/2001

 2001 American Chemical Society

Reactivity of HS with pesticides is modulated by their functional groups as well as by structural arrangements (1421). Significant interactions are the covalent and ionic bond formations in addition to other charge-transfer mechanisms, such as electrons or protons (16, 17, 22, 23). Reactions of specific functional groups [e.g., carboxylic groups in hydrogen bonding or proton transfer (16, 22, 23), quinone groups in electron-transfer reactions (24) or covalent bonding (25)] generally are well-defined and experimentally detectable (16, 17, 22-24). Another very important reaction mechanism between several pesticides and SOM is hydrophobic bonding as proposed in the literature (6, 19, 26, 27, 29). However, hydrophobic bonding experimental evidence or detection is hard to obtain because generally binding energy is low, as occurs in the van der Waals interaction (20, 27). Furthermore, the hydrophobic interactions of pesticides, for example, also depend on HS structural conformation (19, 28, 29). With respect to HS structure, until recently the dominant model was that of macromolecular polymers (4, 26, 30). In this model, conformational changes take place as in other biological macromolecules such as proteins, polysaccharides, nucleic acids, and lignin (31, 32). Schnitzer and Khan (26) and, more recently, Schulten and Schnitzer (7) proposed the existence of voids in this structure, which would be excellent sorption sites for nonpolar pesticides. However, Wershaw (33) was first to postulate an alternative description for macromolecular HS structure. He proposed (8) that HS in solution form mixed aggregates of amphiphilic molecules of plant degradation products and lignin-carbohydrate complexes. In his view, humic aggregates are held together by weak bonding mechanisms such as H-bonding and hydrophobic interactions. By considering the well-established micelle concept (34), he suggested that humic aggregates are like micelles in which liquid-like interiors are composed of hydrophobic portions while highly charged components are positioned at the external surfaces. Engebretson and Wandruska (19) using the fluorescent probe pyrene have also shown experimental evidence in favor of the micelle model. Piccolo et al. (20, 27) and Kenworthy and Hayes (35) were the first to present direct evidence showing that the macromolecular structure of HS may not be entirely polymeric. The large molecular sizes often observed may be considered to arise from associations of smaller molecules held together by weak and easily disreputable force (36). The relevance and consequences of having a definitive model for HS are obvious. However, regardless of what models are proposed, a consensus exists on the existence of hydrophobic regions that would be excellent sorption sites for nonpolar pesticides and other xenobiotics. Recently Hu et al. (37) associated poly(methylene) chains, detected through 13C nuclear magnetic resonance (NMR), with hydrophobic domains in HA. Hydrophobic bonding occurrence is associated with conformational HS changes dependent on parameters such as pH, ionic strength, temperature, concentration of HS, and others (19, 21, 28, 29). Greater knowledge about these hydrophobic sites, including their dependence on different parameters, could help in understanding the fate of pesticides and other xenobiotics in soil and aquatic environments and eventually increase information on HS structure. An available alternative for identifying hydrophobic regions in HS is specific markers, such as hydrophobic spinlabel probes detectable by ESR spectroscopy (38). Spin-label molecules are stable free radicals usable as reporter groups or probes. The most commonly used spin-labels contain the VOL. 35, NO. 4, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. (a) Chemical structure of spin-label stearic acid 5-SASL (N-oxyl-4′,4′-dimethyloxazolidine derivative of 5-ketostearic acid). (b) ESR spectrum of 10 µM 5-SASL in aqueous solution after 5 d of shaking, pH 4.2. The value of hyperfine interaction parameter (a0) is 1.574 mT. relatively inert nitroxide group, which is stable up to about 80 °C and over the pH range of 3-10 (38-40). The ESR spectra of spin-label molecules are sensitive to the molecular neighborhood and can provide useful information about the presence and characteristics of HS hydrophobic sites. Using a spin-label method, Chien et al. (41) hypothesized that humic molecules form “micelle-like” aggregate with hydrophobic interiors into which nonpolar organic molecules can penetrate. Working with the paramagnetic TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) probe, they concluded that instead of penetrating the HA micelle, TEMPO molecules were sorbed on humic micelle surfaces, thereby maintaining continual contact with water. In this paper, the spin-label probe 5-SASL (N-oxyl-4′,4′-dimethyloxazolidine derivative of 5-ketostearic acid), detectable by ESR, was used to evaluate hydrophobic interactions with HA at different pH values and for various reaction times.

Materials and Methods Soil Humic Acid Extraction. HA used in this investigation was extracted from the 0-5-cm-depth layer of a Brazilian Oxisol of a nonagricultural site in the State of Sa˜o Paulo, using the procedure recommended by the International Humic Substances Society (IHSS) (42). The extracted HA was dialyzed against water; silver nitrate was then used to test for removal of excess chloride ions. The resulting sample was stored as a homogenized freeze-dried powder. The elemental analysis (C, H, N, S) of this sample was done using Carlo Erba equipment. Spin-Label Samples. The spin-label utilized was a nitroxide derivative of stearic acid 5-SASL (Figure 1a) obtained from Aldrich. The 5-SASL aqueous solution was prepared starting from an alcoholic solution of the spin-label that was evaporated (under flowing N2) inside a tube. After water was added from minutes to several days under constant shaking, dissolved 5-SASL adhered to tube sides. When the label nitroxide group is tumbling rapidly and isotropically, as in aqueous solution, the ESR spectrum consists of three narrow lines (Figure 1b). The motion is fast enough on an ESR time scale to average out spectral anisotropy. The hyperfine interaction occurrence depends 762

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in part on the unpaired electron density at the nitrogen atom. The free electron relevant in ESR localized in a 2p π-orbital of the nitrogen-oxygen bond interacts with the nitrogen nuclear magnetic moment (I ) 1), giving rise to the characteristic three-line hyperfine structure of the spin-label spectrum due to the three nuclear spin projections (mI ) +1, 0, and -1) as shown in Figure 1b (38, 39). A high degree of mobility prevails when spin-label molecules freely tumble in nonviscous solutions. Increasing spin-label immobilization leads to a differential line broadening in the spectrum. It is the anisotropy of this hyperfine splitting with respect to the magnetic field direction that confers both structural and motional sensitivity on the spin-label method. The anisotropy of the spin-label hyperfine splitting is a consequence of the particular charge distribution symmetry within the molecule. Isolated paramagnetic molecules, as in the present case, have their charge distribution distorted by the polarity of their environment. The isotropic splitting constant (a0) provides a relative polarity indicator, which is calibrated by measuring its value for the label tumbling isotropically in solvents of different polarities. Clearly, a0 is greater in highly polar solvents, such as water (a0 ) 1.574 mT), and decreases with solvent polarity decline to low values in hydrophobic solvents such as ethanol (a0 ) 1.497 mT) and n-decane (a0 ) 1.39 mT). Different values of a0 occur in this case because the hyperfine interaction depends in part on the unpaired electron density at the nitrogen atom. Consider the electronic structure of nitroxide radical as represented by two forms:

The higher the polarity of environment, the more structure b is favored because of hydrogen-bonding possibilities. This structure increases electron density at the nitrogen atom, resulting in larger a0 values. On the other hand, when hydrogen bonding is less probable, as in nonpolar solvent mediums, the free electron remains close to the O atom (I ) 0) (structure a), decreasing interaction with nuclear spin of the N atom. Since a0 is a polarity indicative of the environment in which the spin-label is situated, this parameter could be used to probe hydrophobic environments in HA molecules. HA/5-SASL Samples. HA was dissolved in water and then shaken for at least 3 d while protected from light. The HA suspension concentration was 3 g L-1, and the pH was adjusted to between 3.0 and 6.5 by NaOH or HCl. Some additional experiments to check reversibility and velocity of HA/5-SASL reactions were conducted at pH 8. The 5-SASL was prepared from an alcohol solution evaporated using N2 gas inside a tube (as described before in the Spin-Label Samples section); HA suspension was then added and shaken to react with 5-SASL molecules. In these experiments, after 5-SASL in HA suspension was added, the pH was adjusted to 3.3, 4.1, 5.5, and 6.5 and then measured daily until the completion of the experiments. To study the spin-label behavior, aliquots of the HA/ 5-SASL sample were collected for periods ranging from minutes to several days. Thus, under different pH values and for various reaction times between 5-SASL and HA molecules, an ESR spectrum was obtained. Electron Spin Resonance (ESR) Spectra. The ESR spectra of the HA and HA/5-SASL samples were obtained with a Bruker EMX ESR spectrometer operating at X-band frequency (9 GHz) at room temperature. Organic free radicals of HA freeze-dried sample were quantified using an approximation where spin density is proportional to intensity × line width2 (I × ∆H 2) (43) based

on a secondary standard, a ruby crystal calibrated with strong pitch, according to Singer’s method (44, 45). The secondary standard produces more accurate spin quantification than conventional calibration with a strong pitch reference because it detects very small variations of Q-factor in the microwave cavity during experiments. Experimental conditions were carefully checked using low microwave power (around 1 mW), to avoid saturation of the semiquinone signal, and adequate modulation amplitude (0.05 mT), to prevent signal deformation by increasing signal line width (41). For ESR detection of 5-SASL signal in aqueous 5-SASL (control samples) and HA/5-SASL suspension samples, microwave power was set at 5 mW. Scan rate and time constant were adjusted so that spectra distortion was avoided. Modulation amplitude was 0.05 mT. All spectra were obtained with receiver gain equal to 2 × 104. Aqueous samples were placed in 50-µL microcapillaries inside quartz ESR tubes; the sample amount used for each spectrum was around 20 µL. Spectra of HA suspension were also obtained for comparison with HA/5-SASL suspension samples (data not shown).

Results and Discussion Elemental analyses (C, H, N, S) of HA freeze-dried sample were performed resulting in carbon (48.1%), hydrogen (5.5%), and nitrogen (4.0%) contents. Sulfur was undetected in this sample, and oxygen content (42.4%) was calculated by difference: O% ) 100 - (C + H + N + S) %. Ash content was 6%, all values typical of those for HA shown in the literature (4, 46). The semiquinone free-radical content of the HA freezedried sample was quantified using the approximation intensity × line width2 (I × ∆H 2) based on a secondary standard (16, 44) with a value of 2.65 × 1017 spins/g of HA, typical of that from Brazilian Oxisols (16, 48) within the range reported in the literature (24, 49). Figure 2 presents ESR spectra of 5-SASL (10 µM) in aqueous solution at pH 3.3 in both the presence (Figure 2a) and the absence (Figure 2b) of HA (3 g L-1). Figure 3 displays spectra of 5-SASL (10 µM) in HA (3 g L-1) suspension at pH 4.1 (Figure 3a) and 6.5 (Figure 3b). At pH 5.5, the spectra of 5-SASL in the presence of HA (spectra not shown) were similar to those obtained at pH 6.5 (Figure 3b). There are significant changes in spectra of spin-label 5-SASL in the presence of HA at pH values below 5 (Figures 2a and 3a), mainly when reaction time between spin-label and HA increases. In the beginning of the 5-SASL and HA reaction, spectra show three-line hyperfine structure, typical of free spin-label spectra (as described in Material and Methods, Spin-Label Samples section, and Figure 1b). On the other hand, by increasing reaction time, the 5-SASL spectra become broad, indicating interaction with a new environment while the initially present sharp three-line spectrum decreases until its practically total disappearance. The situation existing at low pH is strongly modified when the pH of a HA/5-SASL suspension is increased to values above 5, where only typical three narrow lines of the “free” spin-label is observed in all spectra (Figure 3b). Independent of reaction time, there is no differential spectrum broadening, typical of immobilized spectra. Besides, a strong increase is apparent in the intensity of 5-SASL spectral lines in the presence of HA as reaction time passes (Figure 3b). Figure 4a shows the hyperfine line intensity (mI ) +1, as seen in Materials and Methods, Spin-Label Samples section) at pH 3.3 and pH 4.1 for HA/5-SASL and at pH 4.1 for 5-SASL in water. Clearly the intensity decrease of this hyperfine line at pH 3.3 and pH 4.1 is associated with formation of the immobilized spectrum of HA/5-SASL (Figures 2a and 3a) while 5-SASL in water increases with time of contact, under constant shaking. The hyperfine line intensity (mI ) +1) of

FIGURE 2. ESR spectra of 10 µM 5-SASL aqueous solutions at pH 3.3 (a) in HA (3 g L-1) presence and (b) absence. All spectra were obtained with receiver gain ) 2 × 104. 5-SASL in water reaches similar values at both pH values below 5 (Figure 4a) and above 5 (Figure 4b). However, these intensity values of hyperfine lines observed in water are lower than those observed in 5-SASL in HA suspension at pH above 5 (Figure 4b). The choice of hyperfine line with mI ) +1 to follow the intensity of 5-SASL was arbitrary, and other hyperfine lines could be used alternatively (mI ) -1 or 0, Figure 1b). A possible explanation for our experiments is that, in HA at low pH values, the carboxylic groups in particular are protonated reducing charge repulsion between functional groups and permitting occurrence of hydrogen bonds (4) with formation of more expanded HA conformation. This expanded structure, existing at pH below 5, probably can create hydrophobic environments in the HA interior that would be excellent sorption sites for nonpolar molecules, such as 5-SASL. In these hydrophobic sites, interactions between 5-SASL and hydrophobic groups of HA must occur, for example, with formation of van der Waals and/or hydrogen bonds, and probably also due to the absence of water in these sites, tumbling time of 5-SASL decreases considerably, resulting in immobilized spectrum (Figures 2a and 3a). The proposal of changes in HA molecular environment at low pH, including the existence of protected hydrophobic sites, is consistent with other ESR data obtained from power saturation curves and line width changes of HS semiquinone signals (16, 17, 29). However at pH values above 5, carboxylic groups are deprotonated (4) and strong charge repulsion between charged carboxylate groups can occur, disrupting the HA expanded structure existing at low pH. In this same pH range (>5), a significant amount or all water-protected hydrophobic HA sites probably disappear. So water molecules can again access places where the spin-label was “protected”, resulting VOL. 35, NO. 4, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. ESR spectra of 10 µM 5-SASL solutions in the presence of 3 g L-1 HA at (a) pH 4.1 and (b) pH 6.5. in increased tumbling rates, thus regenerating the typical three narrow lines of the “free” spin-label spectrum. In agreement with this hypothesis, the value of a0 in HA/ 5-SASL suspension samples at pH above 5 is 1.574 mT (extracted from spectra of Figure 3b), typical of 5-SASL in water (38, 39). This explanation is also totally consistent with spectra observed in Figure 5 where a HA/5-SASL suspension sample that reacted for 16 d at pH 4.1 (only the immobilized spectrum of 5-SASL was observed, Figure 5a) was treated with NaOH, reaching a stable pH 8, with the free spin-label spectrum again appearing with typical three narrow lines (Figure 5, panels b and c for 30 min and 1 d, respectively, after adding NaOH at pH 8 to the HA/5-SASL sample). The hyperfine interaction value (a0) for this HA/5-SASL at pH 8 is 1.570 mT, indicating interaction with water (38, 39). From these data and considering the higher capacity of HA to dissolve 5-SASL as compared to water (Figure 4b), we propose that “surface” hydrophobic sites in HA, such as poly(methylene) groups detected by Hu et al. (37), have a chemical affinity to the spin-label compound. This result is also in agreement with work of Chien et al. (41) in experiments on interaction between spin-label TEMPO and HA at pH 11.8. Summarizing our experiments, we propose existence of at least two kinds of hydrophobic sites at HA molecules. First, “interior” (probably water-protected) hydrophobic sites at pH below 5 have a very strong affinity with nonpolar chemical compounds, possibly including nonpolar pesticides. These interior hydrophobic sites, however, would be destroyed at higher pH due to conformational HA changes. Second, “surface” hydrophobic sites, at pH above 5, are where hydrophobic moieties such as the poly(methylene) groups are exposed to water but keep their capacity to bind nonpolar chemical compounds. These less protected (not “closed”) hydrophobic sites certainly have lower affinity than those 764

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FIGURE 4. Line-intensity variation of isotropic hyperfine signal of nitroxide radical (the intensity of the low-field line, mI ) +1) from 5-SASL spectra at (a) pH 3.3 and pH 4.1 for HA/5-SASL suspensions and at pH 4.1 for aqueous control sample; (b) pH 6.5 and pH 5.5 for HA/5-SASL suspensions and at pH 6.4 and 5.2 in HA absence (aqueous control sample).

FIGURE 5. (a) ESR spectrum of 10 µM 5-SASL after 16 d of contact with 3 g L-1 HA, initial pH 4.1; same sample as in panel a treated with NaOH, pH 8, after 30 min (b) and 24 h (c). interior sites existing at low pH, explaining the increase of HA sorption capacity at low pH of several nonpolar com-

pounds (14, 15, 29, 50, 51). However, these HA surface hydrophobic moieties could be very important at the pH above 5 that is normally observed in soils and natural water solutions. These components potentially constitute sorption sites for nonpolar compounds, thereby explaining the HA capacity to dissolve and/or associate nonpolar molecules, such as 5-SASL. On the basis of the HA/5-SASL spectra at pH above 5, our data can fit the model of a polymeric macromolecule, possessing voids, as suggested by Schulten and Schnitzer (7) with formation of water-protected hydrophobic sites only at low pH and subjected to breaking at higher pH. In accordance with this interpretation is the relatively slow spin-label “immobilization” in HA at low pHs (Figures 2a and 3a) that apparently indicates that these sites are accessed by slow diffusion. To check this hypothesis, we prepared HA/5-SASL suspension at pH 8 for 1 d, which resulted in a typical threehyperfine line display of free 5-SASL. When the pH of this sample was then decreased to 4.1, we observed the rapid formation of a 5-SASL immobilized spectrum (not shown). This result indicates that the water-protected sites were easily accessible and were promptly occupied by 5-SASL when the pH was lowered. Moreover, if HA was a polymeric macromolecule, it would be expected that at least part of the interior hydrophobic sites detected at low pH should be also found at higher pH. However, this was not the case (Figures 2b, 3b, and 5c), thereby raising questions about the polymeric macromolecular model with respect to our HA/5-SASL data. Another HA model currently proposed is a supramolecular association of small molecules (21, 36). In this view, the creation of water-protected hydrophobic sites at low pH would be consistently explained by H-bonding association of mainly carboxylic groups from different small molecules generating an expanded HA structure with hydrophobic interior voids. With the pH increase, the H-bonds are broken due to deprotonation of carboxylic groups and smaller conformational arrangement occur, stabilized by van der Waals forces (36), with a consequent reduction of internal voids and inner hydrophobic sites. In view to this, our HA/ 5-SASL experiments are apparently more consistent with the model of HA association of small molecules (21, 36). New experiments are in progress using other spin-label probes with different chemical characteristics with the potential to provide additional information on both hydrophobic sites characteristics and HA structure.

Acknowledgments This research was supported by PADCT/CNPq (Project 62.0324/98-8), Embrapa (Project 12.1998.810), and FAPESP (fellowship to J.A.F., 98/03472-9) (Brazilian agencies). Gratitude is expressed to four anonymous referees for very helpful reviews.

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Received for review February 21, 2000. Revised manuscript received November 27, 2000. Accepted November 27, 2000. ES0010251 VOL. 35, NO. 4, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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