Chemosphere 148 (2016) 241e247
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Evaluation of the toxicity of ionic liquids on trypsin: A mechanism study Yunchang Fan, Xing Dong, Lingling Yan*, Dandan Li, Shaofeng Hua, Chaobing Hu, Chengcheng Pan College of Physics and Chemistry, Henan Polytechnic University, Jiaozuo 454003, 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
The presence of ionic liquids (ILs) inhibited the trypsin activity. The IL-trypsin interaction was mainly driven by hydrogen bonding. Besides hydrogen bonding, hydrophobicity was also an important parameter. Relationship of IC50, hydrogen bonding ability and hydrophobicity was established.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2015 Received in revised form 7 January 2016 Accepted 8 January 2016 Available online xxx
The toxicity of ionic liquids (ILs) was evaluated by using trypsin as biomarker. Experimental results indicated that the trypsin activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. Primary analysis illustrated that hydrophobicity of ILs was one of the driven forces ruling the ILs-trypsin interaction. Thermodynamic parameters, Gibbs free energy change (DG), enthalpy change (DH) and entropy change (DS) were obtained by analyzing the fluorescence behavior of trypsin in the presence of ILs. Both negative DH and DS suggested hydrogen bonding was the major driven force underlying the IL-trypsin interaction. To assess the toxicity of ILs, it should be considered the combination of the hydrogen bonding ability and hydrophobicity of ILs. A regression based model was established to correlate the relationship of the inhibitory ability, hydrophobicity and hydrogen bonding ability of ILs. © 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Jim Lazorchak Keywords: Ionic liquids (ILs) Fluorescence spectroscopy Trypsin Hydrogen bonding Hydrophobicity
1. Introduction Over the past decade, ionic liquids (ILs) have attracted much attention because of their unique physicochemical properties such
* Corresponding author. E-mail address: [email protected] (L. Yan). http://dx.doi.org/10.1016/j.chemosphere.2016.01.033 0045-6535/© 2016 Elsevier Ltd. All rights reserved.
as extremely low vapor pressure, excellent thermal and chemical stability and good solubility for a wide range of organic and inorndez-Fern ganic compounds (Herna andez, 2015). Therefore, ILs have been regarded as a good alternative to conventional solvents and widely used in chemical engineering, biocatalysis and nanotechnology fields (Ajloo et al., 2013). As the environment grew in importance, people come to realize that the use of ILs on a large scale inevitably causes the release of these chemicals into
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environment. The high chemical stability of ILs may lead to their accumulation in the circumstance, thus causing damage to the ecosystem and affecting living organisms. Therefore, it is worth investigating the toxicity and ecotoxicity of ILs systematically to understand their impact on human life comprehensively. Up to now, many works have reported the biological effects of ILs on aquatic organisms like fishes (Dong et al., 2013; Wang et al., 2010; Li et al., 2012), algae (Ma et al., 2010; Das and Roy, 2014; Chen at al., 2014), and marine invertebrates (Shi et al., 2013; Kumar et al., 2011; Pretti et al., 2009; Costello et al., 2009). The obtained results indicate that ILs do exhibit some toxicity. Furthermore, there are many other testing models used to evaluate the toxicity of ILs, such as bacteria (Yan et al., 2015; Costa et al., 2015), crops (Liu et al., 2015, 2013, 2014), mice/rats (Dumitrescu et al., 2014; JodynisLiebert et al., 2010) and cell lines (Samori et al., 2010; Radosevic et al., 2013; Kaushik et al., 2012; Mikkola et al., 2015). Enzymatic inhibition assay was another popular method for evaluating the biological toxicity and environmental impact of the most commonly encountered ILs. In this context, enzymes like acetylcholinesterase (Stolte et al., 2012; Schaffran et al., 2009; €hn et al., 2011), carboxylesterase Torrecilla et al., 2009), lipase (Kla (Laicure et al., 2013; Costa et al., 2014), catalase (Pinto et al., 2011; Saadeh et al., 2009), cellulase (Wang et al., 2011; Adsul et al., 2009; Ilmberger et al., 2012; Bose et al., 2012), and adenosine deaminase (Ajloo et al., 2013) are usually used. It is demonstrated that the alkyl chain length on the IL cation and the types of IL anions are the key parameters determining the toxicity of ILs (Pinto et al., 2011). An IL with a longer alkyl chain is more toxic; anions species have little effect on the enzyme activity (Pham et al., 2010). Despite the progress made in this field, knowledge about the molecular interaction mechanisms between ILs and enzymes are still limited. Generally, the active sites of enzymes usually locate in their hydrophobic regions. Hydrogen bonding is also obligatory for maintaining the enzyme activity. The hydrophobic and hydrogen bonding interactions between ILs and enzymes may have negative influence on the enzyme stability (Fan et al., 2013a, 2012). These findings suggest that studies on the molecular mechanism of ILenzyme interaction are extremely important to better understand the toxicity of ILs. Trypsin is a common digestive protease excreted by the pancreas and takes part in the digestion of food proteins and other biological processes. If the pollutants enter gastrointestinal tract of humans or animals, they may interact with trypsin and affect the trypsin activity, causing pathological changes. Therefore, trypsin can be used as a biomarker to evaluate the toxicity of pollutants. Just recently, two papers have reported the use of trypsin as biomarker to evaluate the potential toxicity of dimethyl phthalate (Wang et al., 2015) and an azo dye (acid yellow) (Wang et al., 2012). The aims of this work were therefore to investigate the molecular mechanism of IL-trypsin interaction with the use of fluorescence technique. The thermodynamic parameters of IL-trypsin interaction were characterized and a model for predicting the toxicity of ILs on trypsin was established. 2. Experimental 2.1. Materials Trypsin from bovine pancreas (2500 units mg¡1) was purchased from Aladdin Reagent Co. (Shanghai, China). Benzoyl-DLarginine p-nitroanilide (BAPA) was purchased from SigmaeAldrich Co. (St. Louis, USA). 1-Butyl-3-methylimidazolium chloride (99%, [C4mim]Cl), 1-butyl-3-methylimidazolium bromide (99%, [C4mim] Br), 1-butyl-3-methylimidazolium trifluoromethanesulfonate (99%, [C4mim]TfMs), 1-butyl-3-methylimidazolium tetrafluoroborate
(99%, [C4mim]BF4), 1-butyl-3-methylimidazolium acetate (99%, [C4mim]Ac), 1-butyl-3-methylimidazolium nitrate (99%, [C4mim] NO3), 1-hexyl-3-methylimidazolium chloride (99%, [C6mim]Cl), 1hexyl-3-methylimidazolium bromide (99%, [C6mim]Br), 1-octyl-3methylimidazolium chloride (99%, [C8mim]Cl), 1-octyl-3methylimidazolium bromide (99%, [C8mim]Br), 1-decyl-3methylimidazolium chloride (99%, [C10mim]Cl), and 1-decyl-3methylimidazolium bromide (99%, [C10mim]Br) were obtained from Lanzhou Institute of Chemical Physics of the Chinese Academy of Sciences (Lanzhou, China). All the other chemicals are analytical grade unless stated otherwise. Ultrapure water (18.2 MU cm) produced by an Aquapro purification system (Aquapro International Co., Ltd., Dover, DE, USA) was used throughout the experiments. The stock solution of trypsin (0.5 g L¡1) was prepared by 1.0 mM HCl aqueous solution, TriseHCl buffer (50 mM, pH 8.2) containing 0.02 M CaCl2 was prepared by dissolving 6.05 g of tris(hydroxymethyl)aminomethane and 2.94 g of CaCl2$2H2O in 1.0 L of water; the substrate solution of BAPA was prepared as follows: dissolving 40 mg of BAPA in 1.0 mL of dimethyl sulfoxide and then diluting to 100 mL with TriseHCl buffer prewarmed to 37 C. Solutions of ILs (0.10 ¡ 2.5 M) were prepared by TriseHCl buffer and all the stock solutions were stored in the dark at 0e4 C. 2.2. Trypsin activity assays Trypsin activity, with BAPA as substrate, was measured via the method reported in the literature with minor modification (Ministry of Agriculture of the People's Republic of China, 2006). In brief: 0.1 mL of trypsin solution (0.5 g L1), 4.9 mL of TriseHCl buffer and 5 mL of BAPA solution (0.4 g L1) were added into a 10mL glass-stoppered tube. Before addition, all the solutions were incubated at 37 C for 20 min at least. After incubation at 37 C for 10 min, the absorbance of the resulting solution was measured by a spectrophotometer (model TU-1810, Purkinje General Instrument Co., Beijing, China) at 400 nm, wherein the trypsin activity was defined as the absorbance variation for a 10-min duration (DA0 min1). To study the effects of ILs, a specified amount of an IL was added into the above enzymatic reaction system, the trypsin activity in the presence of an IL was registered as DA min1; the relative activity of the enzyme with and without the presence of an IL was thus expressed as DA/DA0. The half maximal inhibitory concentration (IC50), indicating the concentration of each IL needed to inhibit trypsin activity by half was used to quantitatively evaluate the inhibition effectiveness of an IL on the trypsin activity. 2.3. Fluorescence measurements Fluorescence spectroscopic analysis was performed on a Cary Eclipse fluorescence spectrophotometer (Agilent, Santa Clara, CA, USA) equipped with 1.0 cm quartz cells and a thermostatic bath. Typically, 5.0 mL of trypsin solution (0.5 g L1), and a known concentration of an IL were added to a 10.0 mL standard flask and diluted with TriseHCl buffer solution to the volume. Fluorescence emission spectra of trypsin were measured in the range of 292e498 nm with excitation wavelength at 280 nm. The slit widths for both excitation and emission were 5 nm. 2.4. Measurements of the octanolwater partition coefficients (P) of ILs The P values of the twelve ILs, used in this work, were measured per the methods already reported (Fan et al., 2014). Typically, the solutions of ILs (0.1 M for each) were prepared with TriseHCl buffer saturated by octanol; 10.0 mL of the solution of a specific IL and
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10.0 mL of octanol saturated with TriseHCl buffer were mixed under stirring for 30 min at 298 K. After phase separation by centrifugation, the IL concentrations in the octanol and water phases were determined by two methods: (I) high performance liquid chromatography (HPLC). Both of the octanol and water phases were injected into an Agilent 1200 HPLC system (Agilent, Santa Clara, CA, USA) equipped with a variable wavelength detector (VWD) and an autosampler. An Amethyst C18-H column (4.6 mm 150 mm, 5 mm, Sepax Technologies Inc., Newark, DE, USA) was used to separate the ILs, and the column temperature was set at 25 C. The injection volume was 1.0 mL and detection wavelength was 220 nm. The mobile phase was the mixture of acetonitrile and 2.0 mM aqueous sodium 1-heptanesulfonate solution: for the analyses of [C6mim]Cl and [C6mim]Br, 40% (v/v) of acetonitrile was adopted; 50% of acetonitrile was used for determining [C8mim]Cl and [C8mim]Br; 70% of acetonitrile was adopted for analyzing [C10mim]Cl and [C10mim]Br, and 30% of acetonitrile was used to elute all the remaining ILs. The flow rate of mobile phase was set at 0.8 mL min1. (II) UVevis spectrophotometry. The octanol and water phases were properly diluted by anhydrous ethanol, and then the IL concentrations in the two phases were determined by a spectrophotometer at 220 nm. The P value was calculated by the equation:
P ¼ Coctanol =Cwater
(1)
where Coctanol and Cwater are the concentrations of a specific IL in the octanol and aqueous phases, respectively.
2.5. Measurements of KamleteTaft polarity parameters The KamleteTaft polarity parameters, a (hydrogen bond donating ability), b (hydrogen bond accepting ability) and p* (dipolarity/polarisability) of the ILs studied in this work were measured using the three dyes, Reichardt's dye (RD), N,N-diethyl4-nitroaniline (DENA) and 4-nitroaniline (NA). The concentrations of DENA and NA in the ILs were both 5.0 105 M and the concentrations of RD in ILs ranged from 2.2 104 to 1.7 102 M to control the absorbance not exceeding the signal range of the spectrophotometer. The KamleteTaft polarity parameters were calculated by the following equations (Hauru et al., 2012):
. vmax cm1 ¼ 104 lðnmÞ
(2)
ET ð30Þ ¼ 28592=lmax ðRDÞ
(3)
p* ¼ 0:314ð27:52 vmax ðDENAÞÞ
(4)
a ¼ 0:0649ET ð30Þ 2:03 0:72p*
(5)
b ¼ ð1:035vmax ðDENAÞ þ 2:64 vmax ðNAÞÞ=2:8
(6)
detailed information about the experimental temperature, maximum absorption wavelengths (lmax) of the three dyes, ET (30) and p* values is listed in Table S1 in the Supplementary Information (SI). All pH values were tested with a pHS-3B digital pH meter (Shanghai Leici Instrument Factory, Shanghai, China) equipped with a combined glass electrode. All the above experiments were conducted in triplicate and the data presented in this work are an average of the obtained values.
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3. Results and discussion 3.1. Effects of ILs on the trypsin activity To facilitate the evaluation of the effects of the IL chemical structures on the activity of trypsin, the ILs were divided into three groups: I) [C4mim]Cl, [C6mim]Cl, [C8mim]Cl and [C10mim]Cl; II) [C4mim]Br, [C6mim]Br, [C8mim]Br and [C10mim]Br; III) [C4mim]Br, [C4mim]Ac, [C4mim]Cl, [C4mim]BF4, [C4mim]TfMs and [C4mim] NO3. The ILs in groups I and II have the same anion but different cation; ILs in group III have the same cation but different anion. The experimental results are shown in Fig. 1 and the IC50 values for each IL are listed in Table 1. Distinctly, all the ILs indicate weak inhibitory effect on the trypsin activity, the inhibitory abilities of the ILs in groups I, II and III follow the order: [C10mim]Br > [C8mim] Br z [C6mim]Br > [C4mim]Br; [C10mim]Cl > [C8mim]Cl z [C6mim] Cl > [C4mim]Cl; and [C4mim]Br z [C4mim]NO3 ¼ [C4mim] Cl z [C4mim]BF4 z [C4mim]TfMs > [C4mim]Ac. Our previous work showed that the inhibitory abilities of ILs on the enzyme activity were closely related to their hydrophobicity (Fan et al., 2013a). Therefore, the hydrophobicity (logP) of the ILs was measured and the results are listed in Table 1. It should be noted that the logP values of some ILs used in this work have been reported (Jain and Kumar, 2016; Ropel et al., 2005; Ulbert et al., 2004; Kaar et al., 2003). As shown in Table S2 in the SI, the logP values of ILs determined in this work are close to those reported in literature. Furthermore, UVevis spectrophotometry is usually used to measure the logP values of ILs (Jain and Kumar, 2016; Ulbert et al., 2004; Kaar et al., 2003). To further validate the precision of HPLC method proposed in this work, the logP values of the ILs were also determined by the UVevis method. As can be seen from Table S2 in the SI, the logP values determined by HPLC method are in good agreement with those measured by the UVevis method, indicating HPLC is also an accurate method. Data shown in Table 1 indicate that the hydrophobicity of ILs increases with increasing the alkyl chain length in the IL cation. As mentioned above, for the ILs in groups I, their inhibitory ability to the trypsin activity also increases with increasing the alkyl chain length in the IL cation with the exception of [C8mim]Cl (its inhibitory ability is equal to [C6mim]Cl); similar results are also observed for the ILs in group II. These results suggest that hydrophobicity is one of the driven forces underlying the IL-trypsin interaction, but not the only one. As far as the ILs in group III are concerned, [C4mim]Ac has the lowest hydrophobicity but the weakest inhibitory ability. The ILs, [C4mim]Br, [C4mim]NO3, [C4mim]Cl, [C4mim]BF4 and [C4mim]TfMs have the same inhibitory ability but greatly different hydrophobicity, suggesting that hydrophobicity is a parameter but not the major one affecting the trypsin activity. Therefore, it is necessary to further explore the driven forces underlying the IL-trypsin interaction. 3.2. Interaction mechanism Fluorescence is a useful tool for sensing the molecular interaction mechanism (Fan et al., 2013a, 2012). The fluorescence behavior of trypsin in the presence of ILs was thus investigated in this work. As an example, the fluorescence emission spectra of trypsin in the presence of various concentrations of [C4mim]Cl are shown in Fig. 2, the fluorescence spectra of trypsin in the presence of other ILs are shown in the SI (Figs. S1eS11). Obviously, with the successive addition of ILs, the fluorescence of trypsin is effectively quenched, thus denoting that the interaction between ILs and trypsin occurs. Fluorescence quenching can occur by different mechanisms,
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Fig. 1. Effects of ILs on the trypsin activity.
Table 1 IC50 Values, logP values, hydrogen-bonding parameters and ab values of ILs. IL
[C4mim]Cl
IC50 (M) 0.29 logP 2.2 a 0.41 b 0.82 ab 0.34
[C6mim]Cl
[C8mim]Cl
[C10mim]Cl
[C4mim]Br [C6mim]Br [C8mim]Br [C10mim]Br [C4mim]TfMs
[C4mim]BF4 [C4mim]Ac [C4mim]NO3
0.23 1.2 0.41 0.86 0.35
0.23 0.057 0.41 0.83 0.34
0.15 1.1 0.38 0.88 0.34
0.28 2.0 0.41 0.85 0.35
0.31 1.8 0.63 0.37 0.23
0.22 0.91 0.46 0.75 0.35
0.20 0.25 0.44 0.79 0.35
0.07 1.4 0.46 0.76 0.35
0.30 0.96 0.63 0.48 0.30
0.39 2.2 0.47 1.1 0.52
0.29 2.0 0.48 0.66 0.32
static and dynamic quenching are two general types. They can be distinguished by their differing dependence on temperature. Higher temperature results in faster diffusion and leads to the dissociation of weakly bound complexes. Consequently, the quenching constant decreases as temperature increases for the static quenching, while the reverse effect will be observed for the dynamic quenching (Fan et al., 2013a, 2012). For exploring the fluorescence quenching mechanisms, the quenching mode was first assumed a dynamic process. The dynamic quenching data can be analyzed by the SterneVolmer equation (Fan et al., 2013a, 2012):
F0 =F ¼ 1 þ Ksv ½Q
Fig. 2. Fluorescence spectra of trypsin in the presence of different concentrations of [C4mim]Cl; from 1 to 6: 0, 0.01, 0.02, 0.035, 0.05, 0.065 M, respectively.
(7)
where F0 and F denote the fluorescence intensities of trypsin in the absence and presence of the quencher, respectively; [Q] is the concentration of quencher; Ksv is the SterneVolmer quenching constant, which can be obtained by plotting F0/F versus [Q] (shown in Table 2). The SterneVolmer plots of the quenching of trypsin by [C4mim]Cl at different temperatures are shown in Fig. 3 and the SterneVolmer plots for all the other ILs are shown in SI
Y. Fan et al. / Chemosphere 148 (2016) 241e247
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Table 2 The dynamic quenching constants of ILs. T (K)
[C4mim]Cl
[C4mim]Br
[C4mim]TfMs
[C4mim]BF4
[C4mim]Ac
[C4mim]NO3
[C6mim]Cl
[C6mim]Br
[C8mim]Cl
[C8mim]Br
[C10mim] Cl
[C10mim]Br
288 303 318
2.22 1.69 1.22
2.45 1.92 1.45
1.61 1.24 0.86
2.28 1.82 1.54
7.23 6.19 5.30
26.8 24.0 20.8
1.72 1.46 1.28
4.07 3.72 3.49
3.99 3.05 2.47
12.0 9.92 8.53
12.3 9.64 7.07
13.9 11.8 9.99
(Figs. S23eS33) and the results are listed in Table 3. It is obvious that the binding constant values are very low, denoting that the affinities between various ILs and trypsin are poor. The molecular interaction forces between biomacromolecules and small molecules are usually composed of hydrophobic interactions, van der Waals forces, hydrogen bonding and electrostatic forces. Thermodynamic parameters, enthalpy change (DH), free energy change (DG), and entropy change (DS), are the main evidence determining binding modes. The values of DG, DH, and DS can be obtained from the temperature dependence of the binding constants (Fan et al., 2013b):
DG ¼ RT ln K
(9)
where R, T and K are gas constant, absolute temperature and the binding constant, respectively. If DH and DS values are constant in the temperature range studied, they can be obtained by plotting the binding constant versus 1/T based on the van't Hoff equation (Fan et al., 2013b): Fig. 3. SterneVolmer plots of trypsin in the presence of various concentrations of [C4mim]Cl.
(Figs. S12eS22). As shown in Table 2, the slopes of SterneVolmer plots are inversely correlated with temperature, indicating that the probable fluorescence quenching mechanism is a static quenching rather than a dynamic one. Therefore, the fluorescence quenching of trypsin by ILs should be analyzed by the double-logarithm formula (Fan et al., 2013b):
lg½ðF0 FÞ=F ¼ lgK þ n lg½Q
(8)
where K denotes the binding constant and n is the number of binding sites per protein. The double-logarithm plots for the trypsine[C4mim]Cl system are shown in Fig. 4, all the other doublelogarithm plots for the remaining ILs are shown in SI
ln K ¼ DH=ðRTÞ þ DS=R
(10)
the resulting DG, DH and DS values are listed in Table 3. Negative DG values suggest that the IL-trypsin interaction is spontaneous; both negative values for DH and DS indicate that hydrogen bonding is the major driven force underlying the IL-trypsin interaction (Fan et al., 2013b). Since hydrogen bonding is the main driven force for the ILtrypsin interaction, the hydrogen bonding parameters, a and b of the ILs used in this work were thus determined. As reported in literature, the product of a with b (ab) was recommended as an indicator of the hydrogen bonding strength of ILs (Lungwitz et al., 2010), this approach was also adopted in this work and the values of a, b and ab are listed in Table 1. As can be seen, [C4mim]Br, [C4mim]NO3, [C4mim]Cl and [C4mim]TfMs have similar hydrogen bonding ability, which explains why they have similar inhibitory ability to the trypsin activity. However, [C4mim]BF4 has weaker hydrogen bonding ability but similar inhibitory ability as compared to the above four ILs. Considering the fact that these five ILs have different hydrophobicity, the role of hydrophobicity should be considered. Furthermore, [C4mim]Ac has the strongest hydrogen bonding ability but weakest inhibitory ability. Actually, there is strong hydrogen bonding between the solvent (water) and ILs in the aqueous phase; for [C4mim]Ac, it easily forms hydrogen bonds with water making its more hydrophilic and thus weakening its hydrogen bonding with trypsin (Ab Rani et al., 2011). This also suggests that the role of hydrophobicity of ILs should not be neglected. As far as the ILs in groups I and II are concerned, all of them have the similar hydrogen bonding ability but different hydrophobicity and inhibitory ability, once again suggesting that it should simultaneously consider the roles of hydrophobicity and hydrogen bonding for comprehensive understanding of the effects of ILs on the trypsin activity. 3.3. Development of regression based model
Fig. 4. The double logarithm regression curves of trypsin with [C4mim]Cl.
As mentioned above, the inhibitory ability of ILs depends on
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Table 3 Binding constants (K), the number of the binding sites (n) and thermodynamic parameters for the IL-trypsin interaction. T (K) 288
303
318
DS
[C4mim] [C4mim]Br [C4mim]TfMs [C4mim]BF4 [C4mim]Ac [C4mim]NO3 [C6mim]Cl [C6mim]Br [C8mim]Cl [C8mim]Br [C10mim]Cl [C10mim]Br Cl Ka n DGb K n DG K n DG
(J mol1 K1) DH (kJ mol1) a b
1.85 0.90 1.47 1.50 0.93 1.03 1.11 0.94 0.29 39.1
1.69 0.81 1.26 1.37 0.82 0.79 1.16 0.89 0.39 28.9
1.42 0.94 0.84 1.27 1.04 0.60 1.13 1.15 0.32 16.8
2.35 1.04 2.05 1.95 1.05 1.68 1.55 1.01 1.16 29.6
6.23 0.95 4.38 5.30 0.94 4.20 4.85 0.98 4.17 7.17
12.8
9.58
5.67
10.6
6.42
36.7 1.07 8.63 28.4 1.05 8.43 24.6 1.05 8.47 5.58
10.2
1.52 0.96 1.00 1.34 0.97 0.74 1.25 1.00 0.59 14.0
3.53 0.92 3.02 3.02 0.90 2.78 2.78 0.90 2.70 10.7
3.02 0.901 2.65 2.60 0.95 2.41 2.30 0.98 2.20 14.6
16.2 1.08 6.67 13.1 1.08 6.48 11.5 1.08 6.46 6.88
11.3 0.97 5.81 9.81 1.02 5.75 8.18 1.06 5.56 7.81
13.5 0.99 6.23 11.3 0.99 6.11 10.3 1.03 6.17 2.16
5.02
6.08
6.85
8.62
8.07
6.83
K: M1. DG: kJ mol1.
their hydrogen bonding ability and hydrophobicity. Therefore, in this work, a new parameter H was defined as: H ¼ 4.0 ab e logP. The relationship of IC50 and H was then regressed and the following equation can be obtained:
IC50 ¼ 0:2113H0:2652 ; correlation coefficient; r ¼ 0:9647 (11) this result suggests that the toxicity of ILs can be simply described by using their hydrophobicity and hydrogen bonding parameters. 4. Conclusions In this work, the effects of twelve ILs on the trypsin activity were measured and the interaction nature of IL-trypsin was investigated. Experiments indicated that the presence of ILs inhibited the trypsin activity; the chemical structures of both anion and cation affected the trypsin activity. Thermodynamic analysis suggested that hydrogen bonding was the major driven force ruling the IL-trypsin interaction; meanwhile, hydrophobic interactions between ILs and trypsin should not be ignored. A regression based model was developed to describe the relationship of the inhibitory ability, hydrophobicity and hydrogen bonding ability of ILs and satisfactory results were obtained. The studies of the intermolecular interactions between ILs and biomacromolecules are crucial for better understanding the toxicity of this kind chemical on the environment. We hope this study provides some clues to these questions. Acknowledgments This research was financially supported by the National Natural Science Foundation of China (Grant 21307028), Projects of Henan Province (Grants 132300410293, 122300410004, 142102210049 and 14B150026) and Young Backbone Teachers in Colleges and Universities of Henan Province (Grant 2013GGJS-053). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.01.033. References Ab Rani, M.A., Brant, A., Crowhurst, L., Dolan, A., Lui, M., Hassan, N.H., Hallett, J.P.,
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Evaluation of the toxicity of ionic liquids on trypsin: A mechanism study Yunchang Fan, Xing Dong, Lingling Yan*, Dandan Li, Shaofeng Hua, Chaobing Hu, Chengcheng Pan College of Physics and Chemistry, Henan Polytechnic University, Jiaozuo 454003, 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
The presence of ionic liquids (ILs) inhibited the trypsin activity. The IL-trypsin interaction was mainly driven by hydrogen bonding. Besides hydrogen bonding, hydrophobicity was also an important parameter. Relationship of IC50, hydrogen bonding ability and hydrophobicity was established.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2015 Received in revised form 7 January 2016 Accepted 8 January 2016 Available online xxx
The toxicity of ionic liquids (ILs) was evaluated by using trypsin as biomarker. Experimental results indicated that the trypsin activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. Primary analysis illustrated that hydrophobicity of ILs was one of the driven forces ruling the ILs-trypsin interaction. Thermodynamic parameters, Gibbs free energy change (DG), enthalpy change (DH) and entropy change (DS) were obtained by analyzing the fluorescence behavior of trypsin in the presence of ILs. Both negative DH and DS suggested hydrogen bonding was the major driven force underlying the IL-trypsin interaction. To assess the toxicity of ILs, it should be considered the combination of the hydrogen bonding ability and hydrophobicity of ILs. A regression based model was established to correlate the relationship of the inhibitory ability, hydrophobicity and hydrogen bonding ability of ILs. © 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Jim Lazorchak Keywords: Ionic liquids (ILs) Fluorescence spectroscopy Trypsin Hydrogen bonding Hydrophobicity
1. Introduction Over the past decade, ionic liquids (ILs) have attracted much attention because of their unique physicochemical properties such
* Corresponding author. E-mail address: [email protected] (L. Yan). http://dx.doi.org/10.1016/j.chemosphere.2016.01.033 0045-6535/© 2016 Elsevier Ltd. All rights reserved.
as extremely low vapor pressure, excellent thermal and chemical stability and good solubility for a wide range of organic and inorndez-Fern ganic compounds (Herna andez, 2015). Therefore, ILs have been regarded as a good alternative to conventional solvents and widely used in chemical engineering, biocatalysis and nanotechnology fields (Ajloo et al., 2013). As the environment grew in importance, people come to realize that the use of ILs on a large scale inevitably causes the release of these chemicals into
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environment. The high chemical stability of ILs may lead to their accumulation in the circumstance, thus causing damage to the ecosystem and affecting living organisms. Therefore, it is worth investigating the toxicity and ecotoxicity of ILs systematically to understand their impact on human life comprehensively. Up to now, many works have reported the biological effects of ILs on aquatic organisms like fishes (Dong et al., 2013; Wang et al., 2010; Li et al., 2012), algae (Ma et al., 2010; Das and Roy, 2014; Chen at al., 2014), and marine invertebrates (Shi et al., 2013; Kumar et al., 2011; Pretti et al., 2009; Costello et al., 2009). The obtained results indicate that ILs do exhibit some toxicity. Furthermore, there are many other testing models used to evaluate the toxicity of ILs, such as bacteria (Yan et al., 2015; Costa et al., 2015), crops (Liu et al., 2015, 2013, 2014), mice/rats (Dumitrescu et al., 2014; JodynisLiebert et al., 2010) and cell lines (Samori et al., 2010; Radosevic et al., 2013; Kaushik et al., 2012; Mikkola et al., 2015). Enzymatic inhibition assay was another popular method for evaluating the biological toxicity and environmental impact of the most commonly encountered ILs. In this context, enzymes like acetylcholinesterase (Stolte et al., 2012; Schaffran et al., 2009; €hn et al., 2011), carboxylesterase Torrecilla et al., 2009), lipase (Kla (Laicure et al., 2013; Costa et al., 2014), catalase (Pinto et al., 2011; Saadeh et al., 2009), cellulase (Wang et al., 2011; Adsul et al., 2009; Ilmberger et al., 2012; Bose et al., 2012), and adenosine deaminase (Ajloo et al., 2013) are usually used. It is demonstrated that the alkyl chain length on the IL cation and the types of IL anions are the key parameters determining the toxicity of ILs (Pinto et al., 2011). An IL with a longer alkyl chain is more toxic; anions species have little effect on the enzyme activity (Pham et al., 2010). Despite the progress made in this field, knowledge about the molecular interaction mechanisms between ILs and enzymes are still limited. Generally, the active sites of enzymes usually locate in their hydrophobic regions. Hydrogen bonding is also obligatory for maintaining the enzyme activity. The hydrophobic and hydrogen bonding interactions between ILs and enzymes may have negative influence on the enzyme stability (Fan et al., 2013a, 2012). These findings suggest that studies on the molecular mechanism of ILenzyme interaction are extremely important to better understand the toxicity of ILs. Trypsin is a common digestive protease excreted by the pancreas and takes part in the digestion of food proteins and other biological processes. If the pollutants enter gastrointestinal tract of humans or animals, they may interact with trypsin and affect the trypsin activity, causing pathological changes. Therefore, trypsin can be used as a biomarker to evaluate the toxicity of pollutants. Just recently, two papers have reported the use of trypsin as biomarker to evaluate the potential toxicity of dimethyl phthalate (Wang et al., 2015) and an azo dye (acid yellow) (Wang et al., 2012). The aims of this work were therefore to investigate the molecular mechanism of IL-trypsin interaction with the use of fluorescence technique. The thermodynamic parameters of IL-trypsin interaction were characterized and a model for predicting the toxicity of ILs on trypsin was established. 2. Experimental 2.1. Materials Trypsin from bovine pancreas (2500 units mg¡1) was purchased from Aladdin Reagent Co. (Shanghai, China). Benzoyl-DLarginine p-nitroanilide (BAPA) was purchased from SigmaeAldrich Co. (St. Louis, USA). 1-Butyl-3-methylimidazolium chloride (99%, [C4mim]Cl), 1-butyl-3-methylimidazolium bromide (99%, [C4mim] Br), 1-butyl-3-methylimidazolium trifluoromethanesulfonate (99%, [C4mim]TfMs), 1-butyl-3-methylimidazolium tetrafluoroborate
(99%, [C4mim]BF4), 1-butyl-3-methylimidazolium acetate (99%, [C4mim]Ac), 1-butyl-3-methylimidazolium nitrate (99%, [C4mim] NO3), 1-hexyl-3-methylimidazolium chloride (99%, [C6mim]Cl), 1hexyl-3-methylimidazolium bromide (99%, [C6mim]Br), 1-octyl-3methylimidazolium chloride (99%, [C8mim]Cl), 1-octyl-3methylimidazolium bromide (99%, [C8mim]Br), 1-decyl-3methylimidazolium chloride (99%, [C10mim]Cl), and 1-decyl-3methylimidazolium bromide (99%, [C10mim]Br) were obtained from Lanzhou Institute of Chemical Physics of the Chinese Academy of Sciences (Lanzhou, China). All the other chemicals are analytical grade unless stated otherwise. Ultrapure water (18.2 MU cm) produced by an Aquapro purification system (Aquapro International Co., Ltd., Dover, DE, USA) was used throughout the experiments. The stock solution of trypsin (0.5 g L¡1) was prepared by 1.0 mM HCl aqueous solution, TriseHCl buffer (50 mM, pH 8.2) containing 0.02 M CaCl2 was prepared by dissolving 6.05 g of tris(hydroxymethyl)aminomethane and 2.94 g of CaCl2$2H2O in 1.0 L of water; the substrate solution of BAPA was prepared as follows: dissolving 40 mg of BAPA in 1.0 mL of dimethyl sulfoxide and then diluting to 100 mL with TriseHCl buffer prewarmed to 37 C. Solutions of ILs (0.10 ¡ 2.5 M) were prepared by TriseHCl buffer and all the stock solutions were stored in the dark at 0e4 C. 2.2. Trypsin activity assays Trypsin activity, with BAPA as substrate, was measured via the method reported in the literature with minor modification (Ministry of Agriculture of the People's Republic of China, 2006). In brief: 0.1 mL of trypsin solution (0.5 g L1), 4.9 mL of TriseHCl buffer and 5 mL of BAPA solution (0.4 g L1) were added into a 10mL glass-stoppered tube. Before addition, all the solutions were incubated at 37 C for 20 min at least. After incubation at 37 C for 10 min, the absorbance of the resulting solution was measured by a spectrophotometer (model TU-1810, Purkinje General Instrument Co., Beijing, China) at 400 nm, wherein the trypsin activity was defined as the absorbance variation for a 10-min duration (DA0 min1). To study the effects of ILs, a specified amount of an IL was added into the above enzymatic reaction system, the trypsin activity in the presence of an IL was registered as DA min1; the relative activity of the enzyme with and without the presence of an IL was thus expressed as DA/DA0. The half maximal inhibitory concentration (IC50), indicating the concentration of each IL needed to inhibit trypsin activity by half was used to quantitatively evaluate the inhibition effectiveness of an IL on the trypsin activity. 2.3. Fluorescence measurements Fluorescence spectroscopic analysis was performed on a Cary Eclipse fluorescence spectrophotometer (Agilent, Santa Clara, CA, USA) equipped with 1.0 cm quartz cells and a thermostatic bath. Typically, 5.0 mL of trypsin solution (0.5 g L1), and a known concentration of an IL were added to a 10.0 mL standard flask and diluted with TriseHCl buffer solution to the volume. Fluorescence emission spectra of trypsin were measured in the range of 292e498 nm with excitation wavelength at 280 nm. The slit widths for both excitation and emission were 5 nm. 2.4. Measurements of the octanolwater partition coefficients (P) of ILs The P values of the twelve ILs, used in this work, were measured per the methods already reported (Fan et al., 2014). Typically, the solutions of ILs (0.1 M for each) were prepared with TriseHCl buffer saturated by octanol; 10.0 mL of the solution of a specific IL and
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10.0 mL of octanol saturated with TriseHCl buffer were mixed under stirring for 30 min at 298 K. After phase separation by centrifugation, the IL concentrations in the octanol and water phases were determined by two methods: (I) high performance liquid chromatography (HPLC). Both of the octanol and water phases were injected into an Agilent 1200 HPLC system (Agilent, Santa Clara, CA, USA) equipped with a variable wavelength detector (VWD) and an autosampler. An Amethyst C18-H column (4.6 mm 150 mm, 5 mm, Sepax Technologies Inc., Newark, DE, USA) was used to separate the ILs, and the column temperature was set at 25 C. The injection volume was 1.0 mL and detection wavelength was 220 nm. The mobile phase was the mixture of acetonitrile and 2.0 mM aqueous sodium 1-heptanesulfonate solution: for the analyses of [C6mim]Cl and [C6mim]Br, 40% (v/v) of acetonitrile was adopted; 50% of acetonitrile was used for determining [C8mim]Cl and [C8mim]Br; 70% of acetonitrile was adopted for analyzing [C10mim]Cl and [C10mim]Br, and 30% of acetonitrile was used to elute all the remaining ILs. The flow rate of mobile phase was set at 0.8 mL min1. (II) UVevis spectrophotometry. The octanol and water phases were properly diluted by anhydrous ethanol, and then the IL concentrations in the two phases were determined by a spectrophotometer at 220 nm. The P value was calculated by the equation:
P ¼ Coctanol =Cwater
(1)
where Coctanol and Cwater are the concentrations of a specific IL in the octanol and aqueous phases, respectively.
2.5. Measurements of KamleteTaft polarity parameters The KamleteTaft polarity parameters, a (hydrogen bond donating ability), b (hydrogen bond accepting ability) and p* (dipolarity/polarisability) of the ILs studied in this work were measured using the three dyes, Reichardt's dye (RD), N,N-diethyl4-nitroaniline (DENA) and 4-nitroaniline (NA). The concentrations of DENA and NA in the ILs were both 5.0 105 M and the concentrations of RD in ILs ranged from 2.2 104 to 1.7 102 M to control the absorbance not exceeding the signal range of the spectrophotometer. The KamleteTaft polarity parameters were calculated by the following equations (Hauru et al., 2012):
. vmax cm1 ¼ 104 lðnmÞ
(2)
ET ð30Þ ¼ 28592=lmax ðRDÞ
(3)
p* ¼ 0:314ð27:52 vmax ðDENAÞÞ
(4)
a ¼ 0:0649ET ð30Þ 2:03 0:72p*
(5)
b ¼ ð1:035vmax ðDENAÞ þ 2:64 vmax ðNAÞÞ=2:8
(6)
detailed information about the experimental temperature, maximum absorption wavelengths (lmax) of the three dyes, ET (30) and p* values is listed in Table S1 in the Supplementary Information (SI). All pH values were tested with a pHS-3B digital pH meter (Shanghai Leici Instrument Factory, Shanghai, China) equipped with a combined glass electrode. All the above experiments were conducted in triplicate and the data presented in this work are an average of the obtained values.
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3. Results and discussion 3.1. Effects of ILs on the trypsin activity To facilitate the evaluation of the effects of the IL chemical structures on the activity of trypsin, the ILs were divided into three groups: I) [C4mim]Cl, [C6mim]Cl, [C8mim]Cl and [C10mim]Cl; II) [C4mim]Br, [C6mim]Br, [C8mim]Br and [C10mim]Br; III) [C4mim]Br, [C4mim]Ac, [C4mim]Cl, [C4mim]BF4, [C4mim]TfMs and [C4mim] NO3. The ILs in groups I and II have the same anion but different cation; ILs in group III have the same cation but different anion. The experimental results are shown in Fig. 1 and the IC50 values for each IL are listed in Table 1. Distinctly, all the ILs indicate weak inhibitory effect on the trypsin activity, the inhibitory abilities of the ILs in groups I, II and III follow the order: [C10mim]Br > [C8mim] Br z [C6mim]Br > [C4mim]Br; [C10mim]Cl > [C8mim]Cl z [C6mim] Cl > [C4mim]Cl; and [C4mim]Br z [C4mim]NO3 ¼ [C4mim] Cl z [C4mim]BF4 z [C4mim]TfMs > [C4mim]Ac. Our previous work showed that the inhibitory abilities of ILs on the enzyme activity were closely related to their hydrophobicity (Fan et al., 2013a). Therefore, the hydrophobicity (logP) of the ILs was measured and the results are listed in Table 1. It should be noted that the logP values of some ILs used in this work have been reported (Jain and Kumar, 2016; Ropel et al., 2005; Ulbert et al., 2004; Kaar et al., 2003). As shown in Table S2 in the SI, the logP values of ILs determined in this work are close to those reported in literature. Furthermore, UVevis spectrophotometry is usually used to measure the logP values of ILs (Jain and Kumar, 2016; Ulbert et al., 2004; Kaar et al., 2003). To further validate the precision of HPLC method proposed in this work, the logP values of the ILs were also determined by the UVevis method. As can be seen from Table S2 in the SI, the logP values determined by HPLC method are in good agreement with those measured by the UVevis method, indicating HPLC is also an accurate method. Data shown in Table 1 indicate that the hydrophobicity of ILs increases with increasing the alkyl chain length in the IL cation. As mentioned above, for the ILs in groups I, their inhibitory ability to the trypsin activity also increases with increasing the alkyl chain length in the IL cation with the exception of [C8mim]Cl (its inhibitory ability is equal to [C6mim]Cl); similar results are also observed for the ILs in group II. These results suggest that hydrophobicity is one of the driven forces underlying the IL-trypsin interaction, but not the only one. As far as the ILs in group III are concerned, [C4mim]Ac has the lowest hydrophobicity but the weakest inhibitory ability. The ILs, [C4mim]Br, [C4mim]NO3, [C4mim]Cl, [C4mim]BF4 and [C4mim]TfMs have the same inhibitory ability but greatly different hydrophobicity, suggesting that hydrophobicity is a parameter but not the major one affecting the trypsin activity. Therefore, it is necessary to further explore the driven forces underlying the IL-trypsin interaction. 3.2. Interaction mechanism Fluorescence is a useful tool for sensing the molecular interaction mechanism (Fan et al., 2013a, 2012). The fluorescence behavior of trypsin in the presence of ILs was thus investigated in this work. As an example, the fluorescence emission spectra of trypsin in the presence of various concentrations of [C4mim]Cl are shown in Fig. 2, the fluorescence spectra of trypsin in the presence of other ILs are shown in the SI (Figs. S1eS11). Obviously, with the successive addition of ILs, the fluorescence of trypsin is effectively quenched, thus denoting that the interaction between ILs and trypsin occurs. Fluorescence quenching can occur by different mechanisms,
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Y. Fan et al. / Chemosphere 148 (2016) 241e247
Fig. 1. Effects of ILs on the trypsin activity.
Table 1 IC50 Values, logP values, hydrogen-bonding parameters and ab values of ILs. IL
[C4mim]Cl
IC50 (M) 0.29 logP 2.2 a 0.41 b 0.82 ab 0.34
[C6mim]Cl
[C8mim]Cl
[C10mim]Cl
[C4mim]Br [C6mim]Br [C8mim]Br [C10mim]Br [C4mim]TfMs
[C4mim]BF4 [C4mim]Ac [C4mim]NO3
0.23 1.2 0.41 0.86 0.35
0.23 0.057 0.41 0.83 0.34
0.15 1.1 0.38 0.88 0.34
0.28 2.0 0.41 0.85 0.35
0.31 1.8 0.63 0.37 0.23
0.22 0.91 0.46 0.75 0.35
0.20 0.25 0.44 0.79 0.35
0.07 1.4 0.46 0.76 0.35
0.30 0.96 0.63 0.48 0.30
0.39 2.2 0.47 1.1 0.52
0.29 2.0 0.48 0.66 0.32
static and dynamic quenching are two general types. They can be distinguished by their differing dependence on temperature. Higher temperature results in faster diffusion and leads to the dissociation of weakly bound complexes. Consequently, the quenching constant decreases as temperature increases for the static quenching, while the reverse effect will be observed for the dynamic quenching (Fan et al., 2013a, 2012). For exploring the fluorescence quenching mechanisms, the quenching mode was first assumed a dynamic process. The dynamic quenching data can be analyzed by the SterneVolmer equation (Fan et al., 2013a, 2012):
F0 =F ¼ 1 þ Ksv ½Q
Fig. 2. Fluorescence spectra of trypsin in the presence of different concentrations of [C4mim]Cl; from 1 to 6: 0, 0.01, 0.02, 0.035, 0.05, 0.065 M, respectively.
(7)
where F0 and F denote the fluorescence intensities of trypsin in the absence and presence of the quencher, respectively; [Q] is the concentration of quencher; Ksv is the SterneVolmer quenching constant, which can be obtained by plotting F0/F versus [Q] (shown in Table 2). The SterneVolmer plots of the quenching of trypsin by [C4mim]Cl at different temperatures are shown in Fig. 3 and the SterneVolmer plots for all the other ILs are shown in SI
Y. Fan et al. / Chemosphere 148 (2016) 241e247
245
Table 2 The dynamic quenching constants of ILs. T (K)
[C4mim]Cl
[C4mim]Br
[C4mim]TfMs
[C4mim]BF4
[C4mim]Ac
[C4mim]NO3
[C6mim]Cl
[C6mim]Br
[C8mim]Cl
[C8mim]Br
[C10mim] Cl
[C10mim]Br
288 303 318
2.22 1.69 1.22
2.45 1.92 1.45
1.61 1.24 0.86
2.28 1.82 1.54
7.23 6.19 5.30
26.8 24.0 20.8
1.72 1.46 1.28
4.07 3.72 3.49
3.99 3.05 2.47
12.0 9.92 8.53
12.3 9.64 7.07
13.9 11.8 9.99
(Figs. S23eS33) and the results are listed in Table 3. It is obvious that the binding constant values are very low, denoting that the affinities between various ILs and trypsin are poor. The molecular interaction forces between biomacromolecules and small molecules are usually composed of hydrophobic interactions, van der Waals forces, hydrogen bonding and electrostatic forces. Thermodynamic parameters, enthalpy change (DH), free energy change (DG), and entropy change (DS), are the main evidence determining binding modes. The values of DG, DH, and DS can be obtained from the temperature dependence of the binding constants (Fan et al., 2013b):
DG ¼ RT ln K
(9)
where R, T and K are gas constant, absolute temperature and the binding constant, respectively. If DH and DS values are constant in the temperature range studied, they can be obtained by plotting the binding constant versus 1/T based on the van't Hoff equation (Fan et al., 2013b): Fig. 3. SterneVolmer plots of trypsin in the presence of various concentrations of [C4mim]Cl.
(Figs. S12eS22). As shown in Table 2, the slopes of SterneVolmer plots are inversely correlated with temperature, indicating that the probable fluorescence quenching mechanism is a static quenching rather than a dynamic one. Therefore, the fluorescence quenching of trypsin by ILs should be analyzed by the double-logarithm formula (Fan et al., 2013b):
lg½ðF0 FÞ=F ¼ lgK þ n lg½Q
(8)
where K denotes the binding constant and n is the number of binding sites per protein. The double-logarithm plots for the trypsine[C4mim]Cl system are shown in Fig. 4, all the other doublelogarithm plots for the remaining ILs are shown in SI
ln K ¼ DH=ðRTÞ þ DS=R
(10)
the resulting DG, DH and DS values are listed in Table 3. Negative DG values suggest that the IL-trypsin interaction is spontaneous; both negative values for DH and DS indicate that hydrogen bonding is the major driven force underlying the IL-trypsin interaction (Fan et al., 2013b). Since hydrogen bonding is the main driven force for the ILtrypsin interaction, the hydrogen bonding parameters, a and b of the ILs used in this work were thus determined. As reported in literature, the product of a with b (ab) was recommended as an indicator of the hydrogen bonding strength of ILs (Lungwitz et al., 2010), this approach was also adopted in this work and the values of a, b and ab are listed in Table 1. As can be seen, [C4mim]Br, [C4mim]NO3, [C4mim]Cl and [C4mim]TfMs have similar hydrogen bonding ability, which explains why they have similar inhibitory ability to the trypsin activity. However, [C4mim]BF4 has weaker hydrogen bonding ability but similar inhibitory ability as compared to the above four ILs. Considering the fact that these five ILs have different hydrophobicity, the role of hydrophobicity should be considered. Furthermore, [C4mim]Ac has the strongest hydrogen bonding ability but weakest inhibitory ability. Actually, there is strong hydrogen bonding between the solvent (water) and ILs in the aqueous phase; for [C4mim]Ac, it easily forms hydrogen bonds with water making its more hydrophilic and thus weakening its hydrogen bonding with trypsin (Ab Rani et al., 2011). This also suggests that the role of hydrophobicity of ILs should not be neglected. As far as the ILs in groups I and II are concerned, all of them have the similar hydrogen bonding ability but different hydrophobicity and inhibitory ability, once again suggesting that it should simultaneously consider the roles of hydrophobicity and hydrogen bonding for comprehensive understanding of the effects of ILs on the trypsin activity. 3.3. Development of regression based model
Fig. 4. The double logarithm regression curves of trypsin with [C4mim]Cl.
As mentioned above, the inhibitory ability of ILs depends on
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Y. Fan et al. / Chemosphere 148 (2016) 241e247
Table 3 Binding constants (K), the number of the binding sites (n) and thermodynamic parameters for the IL-trypsin interaction. T (K) 288
303
318
DS
[C4mim] [C4mim]Br [C4mim]TfMs [C4mim]BF4 [C4mim]Ac [C4mim]NO3 [C6mim]Cl [C6mim]Br [C8mim]Cl [C8mim]Br [C10mim]Cl [C10mim]Br Cl Ka n DGb K n DG K n DG
(J mol1 K1) DH (kJ mol1) a b
1.85 0.90 1.47 1.50 0.93 1.03 1.11 0.94 0.29 39.1
1.69 0.81 1.26 1.37 0.82 0.79 1.16 0.89 0.39 28.9
1.42 0.94 0.84 1.27 1.04 0.60 1.13 1.15 0.32 16.8
2.35 1.04 2.05 1.95 1.05 1.68 1.55 1.01 1.16 29.6
6.23 0.95 4.38 5.30 0.94 4.20 4.85 0.98 4.17 7.17
12.8
9.58
5.67
10.6
6.42
36.7 1.07 8.63 28.4 1.05 8.43 24.6 1.05 8.47 5.58
10.2
1.52 0.96 1.00 1.34 0.97 0.74 1.25 1.00 0.59 14.0
3.53 0.92 3.02 3.02 0.90 2.78 2.78 0.90 2.70 10.7
3.02 0.901 2.65 2.60 0.95 2.41 2.30 0.98 2.20 14.6
16.2 1.08 6.67 13.1 1.08 6.48 11.5 1.08 6.46 6.88
11.3 0.97 5.81 9.81 1.02 5.75 8.18 1.06 5.56 7.81
13.5 0.99 6.23 11.3 0.99 6.11 10.3 1.03 6.17 2.16
5.02
6.08
6.85
8.62
8.07
6.83
K: M1. DG: kJ mol1.
their hydrogen bonding ability and hydrophobicity. Therefore, in this work, a new parameter H was defined as: H ¼ 4.0 ab e logP. The relationship of IC50 and H was then regressed and the following equation can be obtained:
IC50 ¼ 0:2113H0:2652 ; correlation coefficient; r ¼ 0:9647 (11) this result suggests that the toxicity of ILs can be simply described by using their hydrophobicity and hydrogen bonding parameters. 4. Conclusions In this work, the effects of twelve ILs on the trypsin activity were measured and the interaction nature of IL-trypsin was investigated. Experiments indicated that the presence of ILs inhibited the trypsin activity; the chemical structures of both anion and cation affected the trypsin activity. Thermodynamic analysis suggested that hydrogen bonding was the major driven force ruling the IL-trypsin interaction; meanwhile, hydrophobic interactions between ILs and trypsin should not be ignored. A regression based model was developed to describe the relationship of the inhibitory ability, hydrophobicity and hydrogen bonding ability of ILs and satisfactory results were obtained. The studies of the intermolecular interactions between ILs and biomacromolecules are crucial for better understanding the toxicity of this kind chemical on the environment. We hope this study provides some clues to these questions. Acknowledgments This research was financially supported by the National Natural Science Foundation of China (Grant 21307028), Projects of Henan Province (Grants 132300410293, 122300410004, 142102210049 and 14B150026) and Young Backbone Teachers in Colleges and Universities of Henan Province (Grant 2013GGJS-053). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.01.033. References Ab Rani, M.A., Brant, A., Crowhurst, L., Dolan, A., Lui, M., Hassan, N.H., Hallett, J.P.,
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