Kinetics and thermodynamics of interaction between sulfonamide antibiotics and humic acids: Surface plasmon resonance and isothermal titration microcalorimetry analysis

Journal of Hazardous Materials 302 (2016) 262–266

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Kinetics and thermodynamics of interaction between sulfonamide antibiotics and humic acids: Surface plasmon resonance and isothermal titration microcalorimetry analysis Juan Xu, Han-Qing Yu, Guo-Ping Sheng ∗ CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei 230026, China

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• HA would significantly affect the migration and transformation of SMZ. • Kinetics and thermodynamics of HA–SMZ interactions were studied using SPR and ITC. • The interaction is enhanced by increasing ionic strength and decreasing temperature. • Hydrogen bond and electrostatic interaction play important roles in the process.

a r t i c l e

i n f o

Article history: Received 17 June 2015 Received in revised form 10 September 2015 Accepted 27 September 2015 Keywords: Humic substances Isothermal titration microcalorimetry (ITC) Sulfonamide Surface plasmon resonance (SPR)

a b s t r a c t The presence of sulfonamide antibiotics in the environments has been recognized as a crucial issue. Their migration and transformation in the environment is determined by natural organic matters that widely exist in natural water and soil. In this study, the kinetics and thermodynamics of interactions between humic acids (HA) and sulfamethazine (SMZ) were investigated by employing surface plasmon resonance (SPR) combined with isothermal titration microcalorimetry (ITC) technologies. Results show that SMZ could be effectively bound with HA. The binding strength could be enhanced by increasing ionic strength and decreasing temperature. High pH was not favorable for the interaction. Hydrogen bond and electrostatic interaction may play important roles in driving the binding process, with auxiliary contribution from hydrophobic interaction. The results implied that HA existed in the environment may have a significant influence on the migration and transformation of organic pollutants through the binding process. © 2015 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Fax: +86 551 63601592. E-mail address: [email protected] (G.-P. Sheng). http://dx.doi.org/10.1016/j.jhazmat.2015.09.058 0304-3894/© 2015 Elsevier B.V. All rights reserved.

Antibiotics residues in the environment are considered to be emerging pollutants due to their acute and chronic toxic effects on public health [1–3]. Among the major classes of antibiotics, sul-

J. Xu et al. / Journal of Hazardous Materials 302 (2016) 262–266

fonamide antibiotics are produced in large quantities and heavily used in human therapy and livestock production [4]. They have been widely detected in the effluents from municipal wastewater treatment plants, natural waters and soils [5–8]. Immigration of these sulfonamides into environments would induce spread of antibiotic resistance in response to increased selective pressure, potentially leading to proliferation of resistant pathogens [5]. Furthermore, it was reported that up to 90% of sulfonamides were excreted within 1–2 days into a wastewater treatment system or in the natural environment, and some metabolites of sulfonamides could convert back to parent compounds [9]. As a result, the present of these residual sulfonamides in the environment may arouse possible environmental risks. Humic acids (HA) are main component of natural organic matters presented in the natural environment. They are high molecular weight and heterogeneous organic materials with various functional groups [10], and can affect the transport, persistence and bioavailability of pollutants in the environment [11,12]. Thus, understanding the interaction between sulfonamides and HA is essential for assessing the potential of sulfonamides to leach into the natural environment. However, little information is available about the kinetics and thermodynamics for such interactions. Furthermore, due to the solubility of HA in aqueous solutions, it is difficult to study their binding characteristics for pollutants. Several methods have been developed to analyze the binding characteristics of HA, such as solubility enhancement [13,14], reverse phase separation [15,16] and dialysis [17]; however, these approaches are generally time-consuming and insensitive. Novel sensitive methodologies are still needed to effectively characterize the interaction between HA and pollutants. In this study, surface plasmon resonance (SPR) combined with isothermal titration calorimetry (ITC) was used to investigate the interaction between HA and sulfamethazine (SMZ), a typical sulfonamide widely found in the environment [18]. SPR is a widely used sensor technology to measure molecular interaction affinities between soluble analytes and ligands immobilized on the metal sensor surface [19–21]. It is a sensitive and quantitative biophysical approach that can measure binding affinity and kinetics simultaneously [22–24]. ITC, as a principal microcalorimetric technique, can be used to directly obtain thermodynamic information about biochemical binding processes at a constant temperature [25,26]. With the integration of these simple and effective techniques, the kinetics and thermodynamics of the interaction between HA and SMZ could be determined readily and accurately, allowing for a better understanding of the migration and transformation of organic pollutants in the environment.

263

CM5 chip was activated using an amine coupling reagent mixture containing 0.4 M EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodi-imide-HCl] and 0.1 M NHS (N-hydroxysuccinimide) at a flow rate of 5 ␮L/min for 12 min. SMZ solution with a concentration of 0.5 mg/mL in PBS buffer (50 mM, pH 7.0) was injected into channel 1 on the CM5 chip at a flow rate of 5 ␮L/min for 20 min, while PBS buffer without SMZ was injected into channel 2 as reference. The remaining N-hydroxysuccinimide was blocked by a 2 min pulse of ethanolamine (pH 8.5, 10 ␮L/min). After immobilization, the stability of the SMZ immobilized surface was confirmed by three washes with regeneration solution (50 mM NaOH contained 0.05% SDS followed by 50 mM NaOH). HA with various concentrations (0.13–2.88 mg C/L) in PBS buffer passed over the SMZ-immobilized SPR chip surface (10 ␮L/min, 30 ␮L injection) for 3 min to allow association. Then PBS buffer passed over the surface for 7 min for dissociation. Afterwards, the chip surface was regenerated. The signal, in resonance units, was monitored with respect to time for real-time monitoring. Each analyte solution was injected into both channel 1 and channel 2. The specific response of the SMZ surface was obtained by subtracting the channel 2 response from the channel 1 response. All SPR sensorgrams were processed using BiaEvaluation 4.1 software. Sensorgrams were firstly zeroed on the y-axis and then x-aligned at the initial injection. Kinetic parameters, such as the dissociation rate constant (kd ) and the association rate constant (ka ), were determined by fitting the sensing curves at dissociation stage and association stage based on 1:1 molecular binding model. As these kinetic parameters were independent of the HA concentrations used, the average values of kinetic parameters were calculated from five curves with gradient HA concentrations. Thus, the dissociation rate constant (kd ) could be derived from the equation: Rd = R0 e−kd (t1 −t1,0 ) + Roffset

(1)

where Rd is the SPR response at time t1 at dissociation stage; R0 is the SPR response at time t1,0 at dissociation stage; and Roffset is the residual response at infinite time. The association rate constant (ka ) could be derived from the equation: Ra =

ka CRmax [1 − e−(ka C+kd )(t2 −t2,0 ) ] + RI ka C + kd

(2)

2. Material and methods

where Ra is the response at time t2 at association stage; Rmax is the maximum response related to analyte binding capacity; C is the molar concentration of HA; t2,0 is the fitting start time during the association stage; and RI is the bulk shift. The value of the equilibrium affinity constant (KA ) for the binding reaction could be calculated from the quotient ka /kd .

2.1. Chemicals

2.3. ITC analysis

HA were purchased from RCNC Corp., China and were purified prior to use according to the method proposed by Stevenson [27]. SMZ was purchased from Sigma–Aldrich Corp. In this work, HA were dissolved in phosphate buffers (PBS) to the desired concentration, and the actual concentration was measured by total organic carbon (TOC) Analyzer (Vario TOC, Germany) and expressed as TOC concentration.

The thermodynamics of the interaction between HA and SMZ was investigated using a VP-ITC calorimeter (MicroCal, Northampton, MA). HA and SMZ solutions were prepared at concentrations of 595.2 mg-C/L (measured as TOC) and 500 mg/L, respectively, in PBS buffer (50 mM, pH 7.0). In order to achieve a better resolution for the binding test using the ITC technique, high concentrations of SMZ and HA solutions were used due to the low heat release during binding process. All Solutions were previously degassed for 15 min under vacuum before titration. Experiments were carried out with a working volume of 1468.5 ␮L at 25 ◦ C under a stir rate of 306 rpm. Titrations of SMZ into the buffer and HA solution were completed in 13 ␮L aliquots injected over 26 s with 180 s between injections to ensure complete equilibration. Analysis of the data was performed using Origin 7.0 as described in previous work [28].

2.2. SPR analysis The interaction between HA and SMZ was measured using an SPR device with dual-channel detection (Biacore 3000 system, GE, USA). Before analysis, SMZ was firstly immobilized on the commercial CM5 (carboxymethylated dextran) sensor chip. The

J. Xu et al. / Journal of Hazardous Materials 302 (2016) 262–266

0.18 mg/L 0.36 mg/L 0.72 mg/L

Response Units (RU)

400

1.44 mg/L 2.88 mg/L

(a)

25

Response Units (RU)

264

300 200 100

1.12 mg/L 2.24 mg/L

(b)

450 300 150

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0.14 mg/L 0.28 mg/L 0.56 mg/L

600

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(a)

0.14 mg/L 0.28 mg/L 0.56 mg/L

1.12 mg/L 2.24 mg/L

(b)

0.18 mg/L 0.36 mg/L 0.72 mg/L

1.44 mg/L 2.88 mg/L

(c)

15 10 5

600 450 300 150

0

0

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0.13 mg/L 0.26 mg/L 0.52 mg/L

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(c)

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0.17 mg/L 0.34 mg/L 0.68 mg/L

0

0

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20

750 600 450 300 150 0

0

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800

Time (s) Fig. 1. SPR kinetic analysis of binding of SMZ to humic acids at (a) pH 6; (b) pH 7; and (c) pH 8. Other experimental conditions: PBS concentration 50 mM, temperature 25 ◦ C.

0

200

400

600

800

Time (s) Fig. 2. SPR kinetic analysis of binding of SMZ to humic acids at PBS concentration of (a) 10 mM; (b) 50 mM; and (c) 100 mM. Other experimental conditions: pH 7, temperature 25 ◦ C.

3. Results and discussion SPR was used to investigate the binding kinetics between SMZ and HA. The typical association and dissociation SPR curves for HA interacting with SMZ are shown in Figs. 1–3. The reflected signal remained constant when the PBS buffer flowed through the SMZ immobilized chip during the first 130 s. When the HA solution flowed through the chip, the HA would bind with the SMZ immobilized on the SPR chip and increase the amount of molecules layered on the sensor chip, which resulted in an increase in the reflected signal during the association period from 130 to 310 s. Further flow of the PBS buffer caused part of the HA to dissociate from the chip, which resulted in a decreased SPR response during the dissociation period from 310 to 730 s. The association and dissociation curves for interactions between SMZ and HA under different conditions are shown in Figs. 1–3. The increasing and decreasing tendency of the signal clearly indicated that the binding of SMZ to HA could be influenced by characteristics of the solution environment, such as pH, ionic strength and temperature. The kinetic parameters ka , kd , and affinity constant KA are listed in Table 1. 3.1. Effects of solution conditions on the interaction between SMZ and HA To explore the effects of pH on the interactions between SMZ and HA, PBS buffers of pH 6.0, 7.0 and 8.0 were used for the SPR experiments (Fig. 1). Results show that both association and disso-

ciation processes were influenced by the solution pH (Table 1). The association rates at various pHs were similar; however the dissociation rate at pH 8 was the highest (Table 1). Thus, the affinity constant KA would be low at pH 8 due to the high dissociation rate, suggesting that the interaction could be depressed at high pH conditions. SMZ contains two ionizable functional groups: the aniline amine (pKa = 2.65 ± 0.20) and the amide (pKa = 7.40 ± 0.20) moieties [29]. At alkaline pH, the anionic form of SMZ is prevalent, but at neutral or acidic pH, the non-ionized form will be dominant. Percentages of anionic sulfamethazine at pH 6, 7, and 8 are 3.8%, 28.5% and 79.9%, respectively [29]. Also, as pH increases, more acidic functional groups in HA will dissociate and ionize, thereby giving it more negative charges [30]. The significant electrostatic repulsions between the negatively charged SMZ and HA might lead to the decrease in KA at alkaline pH. The binding between HA and SMZ under various ionic strength were also studied (Fig. 2). The ionic strength was controlled by preparing HA and SMZ solution with PBS buffer (pH 7) of 10, 50 and 100 mM, respectively. When the PBS concentration increased from 10 to 100 mM, the dissociation rates decreased significantly. As a result, the binding affinity between HA and SMZ increased dramatically. This might be due to the decrease in electrostatic repulsive force between HA and SMZ under high ionic strength condition. According to the DLVO theory, the electrostatic repulsion between negative HA and SMZ could be greatly reduced by compressing the electric double layer at high ionic strength [31]. This may be the

J. Xu et al. / Journal of Hazardous Materials 302 (2016) 262–266

0.14 mg/L 0.28 mg/L 0.56 mg/L

8.7

(a)

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(a)

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450 8.5

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ΔQ (μcal)

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90

1.12 mg/L 2.24 mg/L

(c)

-8 0

20

40

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80

SMZ (mg/L)

60

Fig. 4. (a) Thermogram of SMZ binding to HA; and (b) non-linear regression of the heat vs SMZ dosage.

30

3.2. Mechanism of the interaction between SMZ and HA 0 0

200

400

600

800

Time (s) Fig. 3. SPR kinetic analysis of binding of SMZ to humic acids at temperature of (a) 25 ◦ C; (b) 30 ◦ C; and (c) 35 ◦ C. Other experimental conditions: pH 7, PBS concentration 50 mM.

reason that the affinity between SMZ and HA increased with the increase of ionic strength. Temperature was another important parameter affecting the interaction between HA and SMZ (Fig. 3). With increasing environmental temperature, the association rate between HA and SMZ decreased, while the dissociation rate increased. The affinity constant KA reduced from 2.31 × 106 to 9.10 × 105 L/mol as temperature increased from 25 to 35 ◦ C, suggesting that the binding between SMZ and HA is strengthened at lower temperature (Table 1). These results implied that high temperature was not favorable for interactions between SMZ and HA, which was further supported by the following ITC experiment.

ITC results could reveal the driving force for the binding of SMZ to HA, and the thermodynamic parameters of the interaction between SMZ and HA were calculated by non-linear regression of the heat vs SMZ dosage. Fig. 4a is a typical thermogram of SMZ binding to HA. The released heat was detected after each titration of SMZ into HA. Binding capacity N, binding constant K, and binding enthalpy H between HA and SMZ were calculated to be 3.20 × 10−5 mmol/mg C-HA, 4.61 × 103 L/mol and −19.29 kJ/mol, respectively. The Gibbs’ energy change during the binding process was calculated to be −19.94 kJ/mol from the equationG = −RT lnK. This indicated that the binding reaction was thermodynamically favorable, and a stable HA–SMZ complex could be formed. The negative value of the enthalpy change suggested that the binding reaction was exothermic; therefore, high temperatures would not favor the binding reaction. This finding agreed well with the temperature effects found in the SPR analysis. The entropy change of the reaction was calculated from the equationS = (H − G)/T. Entropy change was calculated to be 2.20 J/mol/K, suggesting that the disorder of system increased after HA binding with SMZ. A positive S value is frequently taken as evidence for

Table 1 Influences of environmental conditions on dissociation rate (kd ), association rate (ka ) and affinity constant (KA ) of the interaction between SMZ and HA. Impact conditions

Kinetic parameters

pH

PBS conc. (mM)

T (◦ C)

kd (×10−4 L/s)

ka (×102 L/mol/s)

KA (×106 L/mol)

6 7 8 7 7 7 7

50 50 50 10 100 50 50

25 25 25 25 25 30 35

1.15 1.42 3.34 32.54 0. 70 3.41 3.39

2.17 3.83 3.72 3.66 1.85 3.64 3.10

1.99 2.31 0.99 0.10 3.04 1.07 0.91

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hydrophobic interactions, and a negative H value is frequently taken as evidence for hydrogen bond and electrostatic interaction [32]. Since |H| > |TS|, this suggests that the binding reaction is driven mainly by the entropy change of the reaction. While this is primarily attributed to hydrogen bond and electrostatic interactions, it is possible that hydrophobic interaction also contribute to the binding process. 3.3. Significance of this study SPR and ITC methods are simple, sensitive, and reliable. They are applicable for investigating interactions between HA and various micropollutants. Kinetic parameters for the interaction such as association constant ka and dissociation constant kd under different experimental conditions can be obtained quickly. Simultaneously, the important thermodynamic parameters enthalpy H, entropy S and Gibbs’ energy change G can be calculated from a onetime titration during ITC experiment. All these information are critical for understanding the transformation of micropollutants in the environments, which are very limited in previous study due to the methodology scarcity. Especially, the sensor chip used in SPR analysis can be modified with specific group to capture the target pollutant from a complex samples, such as a mixture of antibiotics, endocrine disrupting chemicals, etc. Kinetic parameters for the binding of the specific contaminant to humic substances can be obtained readily. This extends the methodology for studying the migration and transformation of micropollutants in the environments. 4. Conclusions SPR combined with ITC was used to effectively and efficiently investigate the interactions between organic contaminants and HA. Results show that organic pollutants such as SMZ could bind to the HA existed broadly in the environment spontaneously. The negative value of G revealed that the binding process was favorable, and a stable SMZ–HA complex could be formed. This interaction could significantly influence the migration and transformation of SMZ, depending on aqueous environmental conditions. With increasing temperature or decreasing ionic strength, SMZ might desorb from the SMZ–HA complex, generating the risk of rereleasing these organic pollutants into the environment. This study may provide useful information for better understanding of the fate of organic pollutants in the natural environment. Acknowledgements The authors wish to thank the Natural Science Foundation of China (51322802 and 21377123), the Program for Changjiang Scholars and Innovative Research Team in University, and the Fundamental Research Funds for the Central Universities for the partial support of this study. References [1] S.D. Costanzo, J. Murby, J. Bates, Ecosystem response to antibiotics entering the aquatic environment, Mar. Pollut. Bull. 51 (2005) 218–223. [2] W.H. Xu, G. Zhang, X.D. Li, S.C. Zou, P. Li, Z.H. Hu, J. Li, Occurrence and elimination of antibiotics at four sewage treatment plants in the Pearl River Delta (PRD), South China, Water Res. 41 (2007) 4526–4534. [3] S.F. Yang, C.F. Lin, A.Y.C. Lin, P.K.A. Hong, Sorption and biodegradation of sulfonamide antibiotics by activated sludge: experimental assessment using batch data obtained under aerobic conditions, Water Res. 45 (2011) 3389–3397. [4] L.L. Ji, W. Chen, S.R. Zheng, Z.Y. Xu, D.Q. Zhu, Adsorption of sulfonamide antibiotics to multiwalled carbon nanotubes, Langmuir 25 (2009) 11608–11613.

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