Investigation of the Interaction of Pepsin with Ionic Liquids by Using Fluorescence Spectroscopy

Abstract

The molecular mechanism of the interaction between pepsin and two typical ionic liquids (ILs), 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) and 1-octyl-3-methylimidazolium chloride ([C₈mim]Cl), was investigated with fluorescence spectroscopy, ultraviolet absorption, and circular dichroism spectroscopy at a pH value of 1.6. The results suggest that ILs could quench the intrinsic fluorescence of pepsin, probably via a dynamic quenching mechanism. The fluorescence quenching constants were determined by employing the classic Stern-Volmer equation. The constant values are very small, indicating that only a very weak interaction between ILs and pepsin exists. The Gibbs free-energy change, enthalpy change (ΔH), and entropy change (ΔS) during the interaction of pepsin and ILs were estimated. Positive values of ΔH and ΔS indicate that the interaction between ILs and pepsin is mainly driven by hydrophobic interaction. Synchronous and three-dimensional fluorescence spectra demonstrate that the addition of ILs (0–0.20 mol L⁻¹ for each IL) does not bring apparent changes to the microenvironments of tyrosine and tryptophan residues. Activity experiments show that the activity of pepsin is concentration dependent; higher concentrations of ILs (>0.22 mol L⁻¹ for [C₈mim]Cl and >0.30 mol L⁻¹ for [C₄mim]Cl) cause the remarkable reduction of enzyme activity. The presence of ILs also does not improve the thermal stability of pepsin.

Keywords

Pepsin Fluorescence spectroscopy Ionic liquids (ILs)Interaction Three-dimensional fluorescence Enzyme activity

INTRODUCTION

Ionic liquids (ILs) have attracted extensive attention mainly due to their negligible vapor pressure and low degree of flammability. Environmental and safety problems arising through the use of volatile organic solvents can be avoided by the use of these innovative materials. Therefore, ILs have been widely used as novel media for applications in organic synthesis,¹ electrochemistry,² and separation science.³

Although the non-volatility of ILs likely means little atmospheric pollution if released, either intentionally after industrial waste processing or accidentally during a spill, they would accumulate in the environment because of their high solubility in water and high stability, thereby increasing the potential for contamination of aquatic ecosystems.⁴ It is therefore important to obtain enough information on the environmental impact of ILs prior to intensive use in industrial settings. Recently, efforts have been made to understand the biological effects of each family of ILs, and a growing body of evidence suggests that they can be toxic to aquatic organisms.^5–7

Molecular mechanisms of the interaction between ILs and biomacromolecules are fundamental to our understanding of the harmful effects of this kind of chemical on the environment and their more direct effects on human health.⁸ Therefore, the effects of ILs on proteins and DNA have been reported during the past few years,^9–19 mainly by investigation of their catalytic activity and stability in IL-containing systems. Baker's group⁹ recently reported the extreme thermodynamic stabilization of monellin, offered by the IL 1-butyl-1-methylpyrrolidinium bis(trifluoromethane sulfonyl)imide ([C₄mpy][Tf₂N]); the unfolding temperature of monellin is about 105 °C in [C₄mpy] [Tf₂N] compared with 40 °C in bulk water. However, IL 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) is a denaturant for green fluorescent protein,¹⁰ human serum albumin,¹¹ and cytochrome c.¹² Akdogan's group¹³ studied the effects of imidazolium-based ILs on the functional solution structure of human serum albumin (HSA) by using continuous-wave electron paramagnetic resonance spectroscopy and electron-electron resonance spectroscopy. Because competitive binding of imidazolium cations in HSA decreases the number of bound fatty acids (FAs), conformational changes of HSA after addition of ILs could be obtained by measurements of the spatial distribution of the binding sites of spin-labeled FAs to HSA. It is found that addition of imidazolium-based ILs to an aqueous solution of HAS-FA conjugates is accompanied by significant destabilization and unfolding of the protein's tertiary structure; however, the thermodynamics, which can provide information on the nature of the binding of ILs to HSA, are not mentioned in their work.

In this work, the interaction between imidazolium-based ILs and pepsin was investigated by spectroscopy including fluorescence spectroscopy, UV absorption, and circular dichroism (CD) spectroscopy. Great attempts were made to explore the interaction mechanism at a molecular level and the effect of imidazolium-based ILs on the enzymatic activity of pepsin. Fluorescence techniques are good aids in this study because of their high sensitivity, rapidity, and ease of implementation. From fluorescence quenching measurements, valuable information such as quenching mechanism, binding constants, and the thermodynamic parameters of the interaction process can be obtained. Two widely used ILs, 1-octyl-3-methylimidazolium chloride ([C₈mim]Cl) and [C₄mim]Cl, were selected, because this choice enabled us to investigate how the nature of the alkyl chain length of the ILs affects the interaction. Pepsin was chosen for study because of its well-characterized molecular structure and wide applications.

EXPERIMENTAL

Reagents. 1-Chlorooctane (99%), 1-chlorobutane (99.8%), and pepsin (activity: 1:15 000) from hog stomach were obtained from the Aladdin Reagent Co. (Shanghai, China). 1-Methylimidazole (99%) was purchased from the Energy Chemical Co. (Shanghai). Casein was obtained from the Xiya Reagent Co. (Chengdu, China). The two ILs [C₄mim]Cl and [C₈mim]Cl were synthesized and purified as described in our previous work.²⁰ All the other chemicals were analytical grade unless stated otherwise and used without further purification. Ultrapure water (18.2 MΩ·cm), produced by a GW-UP purification system (Purkinje General Instrument Co., Ltd., China), was used throughout the experiments.

Since pepsin is most active in acidic environments (pH 1.5–2.0),²¹ all the pH values of aqueous media were fixed at 1.6. Therefore, stock solutions of pepsin (1.0 g L⁻¹), NaCl (0.5 mol L⁻¹), [Ctmim]Cl (2.0 mol L⁻¹), [C₈mim]Cl (2.0 mol L⁻¹), and casein (0.5 g L⁻¹) were prepared by dissolving a desired amount of that substance in HCl solution (pH 1.6). All stock solutions were stored in the dark at 0–4 °C.

Apparatus. Fluorescence spectroscopic analysis was carried out on a Cary Eclipse fluorescence spectrophotometer (Agilent, Santa Clara, CA) equipped with 1.0 cm quartz cells and a thermostat bath. Measurements of fluorescence lifetime were conducted on a TemPro-01 time-resolved spectrofluorometer (Horiba Jobin Yvon S.A.S., Longjumeau, France). All pH values were tested with a pHS-3B digital pH meter (Shanghai Leici Instrument Factory, Shanghai, China) equipped with a combined glass electrode. The ultraviolet (UV)-visible spectra and enzymatic activity measurements were performed on a UV-1900 spectrophotometer (Purkinje General Instrument Co., Ltd., China). Scanning intervals of UV absorption spectra were set at 1 nm.

Fluorescence Spectroscopic and Lifetime Measurements. For the fluorescence measurement, 2.0 mL of NaCl solution, 4.0 mL of pepsin solution, and an appropriate amount of IL were added to a 10.0 mL standard flask and diluted with HCl solution (pH 1.6) to volume; the final pH of the mixture was 1.6. Fluorescence quenching spectra of pepsin were obtained at excitation (Λ_ex) and emission (Λ_em) wavelengths of 280 and 290–500 nm, respectively. Under the same conditions, the synchronous fluorescence spectra were recorded from 240 to 350 nm at ΔΛ = 60 and 15 nm (scanning interval: Δk = Λ_em – Λ_ex). The slit widths for both excitation and emission were 5 nm.

Three-dimensional fluorescence spectra were obtained under the following conditions: The emission wavelengths were recorded from 240 to 500 nm; the excitation wavelengths ranged from 220 nm to 310 nm, with an increment of 5 nm, and the number of scanning curves was 19. The slit widths for both excitation and emission were also set at 5 nm. The concentration of pepsin was 0.4 g L⁻¹, and the concentrations of [C₄mim]Cl and [C₈mim]Cl were both 5.0 × 10⁻³ mol L⁻¹.

For the fluorescence lifetime measurements, samples were excited at 280 nm; the emission wavelength was set at 342 nm. The slit widths were both 4 nm for excitation and emission. Concentration of pepsin was 0.4 g L⁻¹, and the concentrations of [C₄mim]Cl and [C₈mim]Cl were both 0.04 mol L⁻¹.

Circular Dichroism Spectroscopy. Circular dichroism spectra were recorded on a J-815 spectropolarimeter (Jasco, Tokyo, Japan) in HCl aqueous solution (pH 1.6) containing 0.4 g L⁻¹ of pepsin and for the same solution containing 5 × 10⁻³ mol L⁻¹ of an IL ([C₄mim]Cl or [C₈mim]Cl). The wavelength range was 190–250 nm, performed with a CD cuvette (18 mm pathlength). The spectra were corrected by subtracting the corresponding solvent blank. The relative amounts of secondary structures of pepsin were calculated from corresponding CD spectra data with the online DichroWeb software.^22,23 More information about the software is available at the following Web site: http://dichroweb.cryst.bbk.ac.uk/html/home.shtml.

Pepsin Activity Measurements. The activity of pepsin, with casein as substrate, was measured per the Standardization Administration of China method,²⁴ with minor modification. In brief, 0.2 mL of pepsin (0.5 g L⁻¹, pH 1.6) and 12.0 mL of casein (0.5 g L⁻¹, pH 1.6) were added to a glass-stoppered tube. Before the addition, both the substrate and enzyme solutions were incubated at 40 °C for 20 min. After incubation at 40 °C for 10 min, the mixture was inactivated by adding 3.0 mL of trichloroacetic acid (0.8 mol L⁻¹) and diluted with water to 20 mL. The resulting suspension was centrifuged for 3 min, and the UV absorbance of the supernatant was measured at 275 nm.

The blank solution was prepared in the same manner: 0.2 mL of pepsin (0.5 g L⁻¹, pH 1.6) and 3.0 mL of trichloroacetic acid (0.8 mol L⁻¹) were added to a glass-stoppered tube and incubated at 40 °C for 10 min. Then, 12.0 mL of casein (0.5 g L⁻¹, pH 1.6) was added to the mixture, and the final volume of the mixture was adjusted with water to 20 mL. After centrifuging, the UV absorbance of the supernatant was also measured at 275 nm.

To study the IL effects, a specified amount of each IL was added after the addition of enzyme solution to reach a final concentration of 0–1.0 mol L⁻¹. The results are reported as the ratio between the activity of the enzyme with and without the presence of each IL (A:A₀).

RESULTS AND DISCUSSION

Fluorescence Spectra Analysis. Fluorescence quenching processes in solution fall into two general types,^25,26 (i) static quenching through the formation of a ground state complex and (ii) dynamic quenching (collisional quenching) due to diffusive collisions between the fluorophore and the quencher. They can be differentiated by their dissimilar dependence on temperature and viscosity, or preferably, by lifetime measurements.²⁷ Dynamic quenching is diffusion controlled, because the quencher must diffuse to the fluorophore during the lifetime of the excited state. Since high temperature results in faster diffusion, a larger extent of collisional quenching is expected with increasing temperature, but a reversed effect is observed for the static quenching, because high temperatures will also result in the dissociation of weakly bound complexes.

Fluorescence emission spectra of pepsin with the addition of different levels of [C₄mim]Cl and [C₈mim]Cl are shown in Fig. 1. Pepsin shows a strong fluorescence emission, with a peak at 342 nm at Λ_ex = 280 nm, while ILs were almost nonfluorescent under the present experiment conditions. Obviously, the fluorescence of pepsin is effectively quenched by the addition of ILs, which indicates that interaction between ILs and pepsin occurred. However, the shape of the fluorescence spectra and peak position do not alter, suggesting that the interaction of pepsin with ILs is weak and there might be no complexes formation between ILs and pepsin.^27–29 However, additional information is required to distinguish between dynamic or static processes, for example, the temperature dependence of the quenching rate constants, UV absorption spectra, and fluorescence lifetime measurements.

Fig. 1.

Fluorescence emission spectra of pepsin in the presence of various concentrations of [C₄mim]Cl (a) and [C₈mim]Cl (b), with pepsin at 0.4 g L⁻¹. From (a) to (f), respectively: 0, 0.04, 0.08, 0.12, 0.16, and 0.2 mol L⁻¹ for each IL.

In order to judge the possible fluorescence quenching mechanism, the process of fluorescence quenching was first assumed to be dynamic. For dynamic quenching, the mechanism can be described by the Stern-Volmer equation:²⁸

\frac{F_{0}}{F} = 1 + k_{q} τ_{0} [Q] = 1 + K_{SV} [Q]

(1)

where F₀ and F are the fluorescence intensities in the absence and presence of quencher, respectively; k_q, K_SV, τ₀, and [Q] are the bimolecular quenching rate constant, the Stern-Volmer quenching constant, the average lifetime of molecule without quencher, and concentration of quencher, respectively. The Stern-Volmer plots of quenching of pepsin by different ILs and at different temperatures are displayed in Fig. 2. Based on the experimental data in Fig. 2, the dynamic quenching constants at different temperatures are shown in Table I.

TABLE I.

The dynamic quenching parameters between ILs and pepsin.

IL	T (K)	K_SV (L mol⁻¹)	r ^a
[C₄mim]Cl	288	1.50	0.9990
	298	1.79	0.9991
	308	2.09	0.9991
[C₈mim]Cl	288	2.17	0.9996
	298	2.85	0.9997
	308	3.55	0.9992

r = correlation coefficient.

Fig. 2.

Stern-Volmer plots of pepsin (0.4 g L⁻¹). (a) [C₄mim]Cl, (b) [C₈mim]Cl. F₀ and F are the fluorescence intensities in the absence and presence of ILs, respectively.

The results shown in Fig. 2 and Table I suggest that the Stern-Volmer plots are linear, and the slope increases with increasing temperature, indicating that the fluorescence quenching is initiated by dynamic collision.

Analysis of Ultraviolet Spectra. Ultraviolet absorption measurement is a very simple method and applicable to explore the structure and the complex formation.²⁹ The UV absorption spectra of pepsin in the absence and presence of IL are shown in Fig. 3. Obviously, pepsin has strong absorbance, with a peak at 275 nm, and the absorbance of pepsin remains nearly constant, with no changes in the maximum absorption wavelength after the addition of ILs, which also rules out the formation of ground-state complexes between the ILs and pepsin.

Fig. 3.

The UV absorption spectra of pepsin in the presence of different concentrations of [C₄mim]Cl (a) and [C₈mim]Cl (b), with pepsin at 0.4 g L⁻¹. From (a) to (f), respectively: 0, 0.04, 0.08, 0.12, 0.16, and 0.2 mol L⁻¹ for each IL.

Assay of Fluorescence Lifetime. The measurement of fluorescence lifetimes is the most definitive method to distinguish static and dynamic quenching. Static quenching will not reduce the lifetime of the sample, because static quenching merely reduces the apparent concentration of the fluorophore; those fluorophores that are not complexed will have normal excited-state properties and hence able to emit fluorescence after excitation, while dynamic quenching reduces the apparent fluorescent lifetime due to the collision between the fluorophore and a quencher.³⁰ From Table II, it is apparent that with the addition of ILs, the lifetimes of pepsin decrease, suggesting that ILs quench the fluorescence of pepsin by a dynamic process.

TABLE II.

Fluorescence lifetimes (τ) of pepsin, without and with the addition of ILs.

	Lifetime (ns)
System	τ₁	τ₂	τ₃
Pepsin	2.1	6.2	0.79
Pepsin + [C₄mim]Cl system	1.4	5.8	0.50
Pepsin + [C₈mim]Cl system	1.0	5.8	—^a

______ = not detectable.

Thermodynamic Analysis. The interactive forces between molecules can include electrostatic interaction, hydrogen bonds, van der Waals forces, and hydrophobic interaction. The thermodynamic parameters such as enthalpy change (ΔH), Gibbs free-energy change (ΔG), and entropy change (ΔS) are the main evidence to determine the interactive forces.^31,32 From the temperature dependence of quenching constants, it is possible to calculate values for the thermodynamic functions involved in the interactive process.

Free-energy change can be estimated from the following equation, based on the quenching constants at different temperatures:³³

Δ G = - R T ln K

(2)

where K is the Stern–Volmer quenching constant at corresponding temperature, T is the experimental temperature, and R is the gas constant. If ΔH and ΔS values do not vary significantly over the temperature range studied, then they can be determined by plotting the binding constants according to the van't Hoff equation:³⁴

ln K = - \frac{Δ H}{R T} + \frac{Δ S}{R}

(3)

The van't Hoff plots are shown in Fig. 4, and the results are presented in Table III.

TABLE III.

Thermodynamic parameters between ILs and pepsin.

IL	T (K)	ΔG (kJ mol⁻¹)	ΔH (kJ mol⁻¹)	ΔS (J mol⁻¹ K⁻¹)
[C₄mim]Cl	288	−0.97	12.24	45.86
	298	−1.44
	308	−1.89
[C₈mim]Cl	288	−1.86	18.17	69.56
	298	−2.60
	308	−3.24

Fig. 4.

The van't Hoff plots for [C₄mim]Cl (a) and [C₈mim]Cl (b). K is the Stern-Volmer quenching constant, T is the experimental temperature.

As described in Table III, the negative change in free energy means that the binding process between ILs and pepsin is spontaneous; both ΔH and ΔS have positive values, indicating that hydrophobic interaction could play a major role in the interaction process.^34,35 Recently, several research groups reported that the hydrophobicity of ILs increases with alkyl chain length increase.^36–38 Since the alkyl chain length of the two ILs increases in the order [C₈mim]Cl > [C₄mim]Cl, it would be easy to understand why the interaction of pepsin with [C₈mim]Cl is stronger than that with [C₄mim]Cl.

Synchronous Fluorescence Spectra. The synchronous fluorescence spectra give information about the molecular environment in the vicinity of the chromophores. ΔΛ, representing the value of difference between excitation and emission wavelengths (ΔΛ = Λ_em – Λ_ex), is an important parameter. As ΔΛ is 15 nm, synchronous fluorescence offers characteristics of tyrosine residues, while when ΔΛ is 60 nm, it provides the characteristic information of tryptophan residues.³⁹ Synchronous fluorescence spectral changes of pepsin on addition of [C₈mim]Cl are presented in Figs. 5 and 6 (synchronous fluorescence spectra of [C₄mim]Cl are not shown due to their close similarity to that of [C₈mim]Cl). It is evident that the fluorescence of pepsin mainly comes from tryptophan residues. It is also can be found that with the addition of ILs, fluorescence intensities of tryptophan and tyrosine residues remarkably decrease; however, no shifts in the synchronous fluorescence spectral peaks on the addition of ILs suggest that microenvironments around tyrosine and tryptophan residues have no obvious changes.²⁹

Fig. 5.

Synchronous fluorescence spectra of pepsin (0.4 g L⁻¹) in the presence of different concentrations of [C₈mim]Cl, with ΔΛ = 15 nm. From (a) to (f), respectively: 0, 0.04, 0.08, 0.12, 0.16, and 0.2 mol L⁻¹ for each IL.

Fig. 6.

Synchronous fluorescence spectra of pepsin (0.4 g L⁻¹) in the presence of different concentrations of [C₈mim]Cl, with ΔΛ = 60 nm. From (a) to (f), respectively: 0, 0.04, 0.08, 0.12, 0.16, and 0.2 mol L⁻¹ for each IL.

Three-Dimensional Fluorescence Spectra. Three-dimensional fluorescence spectroscopy has proven a useful tool for the investigation of conformational changes of proteins, because it can comprehensively exhibit the fluorescence information of the chromophores.^40,41 The three-dimensional fluorescence spectra of pepsin before and after the addition of the two ILs were measured. It is seen that the three-dimensional fluorescence spectra of pepsin contain two fluorescence peaks. One (peak 1) is located at 280–342 nm (Λ_ex-Λ_em), and another (peak 2) is found at 235–340 nm (Λ_ex-Λ_em). Peak 1 is the fluorescence peak and primarily shows the spectral characteristic of tryptophan and tyrosine residues, the maximum emission wavelength, and the fluorescence intensity of the residues are in close correlation with the polarity of the microenvironment.⁴⁰ Peak 2 mainly exhibits the fluorescence characteristic of polypeptide backbone structures C=O of proteins; conformational changes of proteins usually cause the shift in the maximum emission wavelength of peak 2.^41–43 The corresponding characteristic parameters of peaks 1 and 2 are listed in Table IV. Obviously, after the addition of ILs, the fluorescence intensity of peak 1 decreases, with no discernible shift in the maximum emission wavelength, indicating that the microenvironments around the tyrosine and tryptophan residues do not noticeably change during the interaction process, which is in agreement with the results of synchronous fluorescence spectra. Meanwhile, the fluorescence intensity of peak 2 also decreases, with no apparent change in the maximum emission wavelength and peak shape, suggesting that the conformation of pepsin does not change remarkably in the interaction process between ILs and pepsin at the tested concentration range.

TABLE IV.

Three-dimensional fluorescence spectral characteristic parameters of pepsin in the absence and presence of the two ILs.

	System
	Pepsin		Pepsin + [C₄mim]Cl		Pepsin + [C₈mim]Cl
Peak	Peak position range (nm)^a	_F ^b	Peak position range (nm)	F	Peak position range (nm)	F
1	280–342	370.5	280–342	369.5	280–342	354.6
2	235–340	180.1	235–339	128.3	235–339	114.5

Peak position range measured from the excitation wavelength to the emission wavelength.

F = intensity.

Circular Dichroism Spectra. Circular dichroism spectroscopy is an important technique in structural biology for examining the folding, conformational changes, and especially secondary structures of proteins. The CD spectra of pepsin before and after the addition of ILs were measured, and the relative contents of secondary structure forms of pepsin including helix, strand, turn, and unordered structure were calculated and are presented in Table V. Obviously, at the studied level (5 × 10⁻³ mol L⁻¹ for each IL), the addition of ILs does not induce remarkable changes in the secondary structure of pepsin.

TABLE V.

Fractions of different secondary structures of pepsin.

	Secondary structure (%)
System	Helix	Strand	Turn	Unordered
Pepsin	6.6	41.5	12.7	39.1
Pepsin + [C₄mim]Cl	6.2	42.3	12.5	38.9
Pepsin + [C₈mim]Cl	6.2	42.0	12.9	39.0

Effect of Ionic Liquids on the Activity of Pepsin. To ascertain whether the ILs cause any alternation in the enzyme activity, the effects of different concentrations of the two ILs on the activity of pepsin were studied. As shown in Fig. 7, the activity of pepsin is almost constant with addition of the two ILs at lower levels. However, further increase in the concentration of ILs (> 0.22 mol L⁻¹ for [C₈mim]Cl and >0.30 mol L⁻¹ for [C₄mim]Cl) causes the remarkable reduction of enzyme activity. Additionally, the activity of pepsin decreases in the order of NaCl > [C₄mim]Cl > [C₈mim]Cl, indicating the IL cation's dominating effect. In fact, several articles have reported that there exists significant hydrophobic interaction between an IL cation and the inner hydrophobic moieties of the enzyme molecule.^44,45 Additionally, the abovementioned fluorescence spectra and thermodynamic analyses also suggest that hydrophobic interaction play a major role in the interaction pepsin with ILs. Therefore, it would be reasonable to assume that the loss of enzymatic activity after the addition of higher concentration of ILs can also be attributed to the hydrophobic interaction between the IL cation and the inner hydrophobic moieties of pepsin. Furthermore, as mentioned above, [C₈mim]Cl is more hydrophobic than [C₄mim]Cl, which explains why [C₈mim]Cl has greater depressing effect on the enzymatic activity of pepsin as compared with [C₄mim]Cl.

Fig. 7.

Activity of pepsin in the presence of different concentrations of NaCl (a), [C₄mim]Cl (b), and [C₈mim]Cl (c). A₀ and A are the UV absorbances of the reaction products before and after the addition of an IL at 275 nm, respectively.

Thermal Stability. To better understand the effect of ILs on pepsin, its thermal stability in aqueous solutions was also investigated. It was found that without the presence of ILs, the activity of pepsin decreases remarkably when the temperature is above 50 °C. However, the addition of ILs has no effect on the activity of pepsin, i.e., the presence of [C₈mim]Cl or [C₄mim]Cl (< 0.2 mol L⁻¹ for each IL) does not improve the thermal stability. It is clear that [C₈mim]Cl-water and [C₄mim]Cl-water mixtures are poor candidate solvents for the pepsin-catalyzed hydrolysis.

CONCLUSION

This work demonstrates the interactive nature of ILs and pepsin by using fluorescence, CD, and UV spectroscopic techniques. The results show that the Stern-Volmer quenching constants, K_SV, in all cases are directly proportional to temperature, which indicates that the possible quenching mechanism is initiated by dynamic quenching. Low KSV values and no changes in the shapes of fluorescence and UV spectra demonstrate that the interaction between pepsin and ILs is weak, and no pepsin-IL complexes are formed within the studied concentration range (0–0.2 mol L⁻¹ for each IL). Thermodynamic parameters (ΔH > 0 and ΔS > 0) suggest that hydrophobic interaction is the main driving force in the interaction of pepsin with ILs. The IL [C₈mim]Cl has stronger quenching ability with pepsin than that of [C₄mim]Cl due to the former having stronger hydrophobicity. Synchronous and three-dimensional fluorescence spectra indicate that the microenvironments of tryptophan and tyrosine residues do not change; enzyme activity measurements suggest that at lower concentration levels (<0.22 mol L⁻¹ for [C₈mim]Cl and <0.30 mol L⁻¹ for [C₄mim]Cl), the activity of pepsin remains constant; however, the enzymatic activity decreases remarkably with further increasing the IL concentrations. Finally, the presence of [C₈mim]Cl or [C₄mim]Cl does not improve the thermal stability of pepsin, indicating that dialkylimidazolium chloride-water mixtures are poor solvents for biocatalytic reactions. However, it remains possible that other water-miscible ILs can provide better enzyme activity for use in biocatalytic processes, and continued research is required.

Footnotes

ACKNOWLEDGMENTS

This work was financially supported by the Natural Science Foundation of the Education Department of Henan Province (nos. 12B150017 and 12A150010) and the Foundation of Henan Polytechnic University (No. B2010–14).

References

Sowmiah

Cheng

C.I.

Chu

Y.H.

. “Ionic Liquids for Green Organic Synthesis”. Curr. Org. Syn. 2012. 9(1): 74–95.

Silvester

D.S.

. “Recent Advances in the Use of Ionic Liquids for Electrochemical Sensing”. Analyst. 2011. 136(23): 4871–4882.

T.D.

Canestraro

A.J.

Anderson

J.L.

. “Ionic Liquids in Solid-Phase Microextraction: A Review”. Anal. Chim. Acta. 2011. 695(1, 2): 18–43.

Pham

T.P.T.

Cho

C.W.

Yun

Y.S.

. “Environmental Fate and Toxicity of Ionic Liquids: A Review”. Water Res. 2010. 44(2): 352–372.

Petkovic

Seddon

K.R.

Rebelo

L.P.N.

Pereira

C.S.

. “Ionic liquids: A Pathway to Environmental Acceptability”. Chem. Soc. Rev. 2011. 40(3): 1383–1403.

J.M.

Cai

L.L.

Zhang

B.J.

L.W.

X.Y.

Wang

J.J.

. “Acute Toxicity and Effects of 1-Alkyl-3-Methylimidazolium Bromide Ionic Liquids on Green Algae”. Ecotox. Environ. Safe. 2010. 73(6): 1465–1469.

Alvarez-Guerra

Irabien

. “Design of Ionic Liquids: An Ecotoxicity (Vibrio fischeri) Discrimination Approach”. Green Chem. 2011. 13(6): 1507–1516.

McKinney

J.D.

. “The Molecular Basis of Chemical Toxicity”. Environ. Health Perspect. 1985. 61: 5–10.

Baker

S.N.

McCleskey

T.M.

Pandey

Baker

G.A.

. “Fluorescence Studies of Protein Thermostability in Ionic Liquids”. Chem. Commun. 2004. 4(8): 940–941.

10.

Heller

W.T.

O'Neill

H.M.

Zhang

Baker

G.A.

. “Characterization of the Influence of the Ionic Liquid 1-Butyl-3-Methylimidazolium Chloride on the Structure and Thermal Stability of Green Fluorescent Protein”. J. Phys. Chem. B. 2010. 114(43): 13866–13871.

11.

Baker

G.A.

Heller

W.T.

. “Small-Angle Neutron Scattering Studies of Model Protein Denaturation in Aqueous Solutions of the Ionic Liquid 1-Butyl-3-Methylimidazolium Chloride”. Chem. Eng. J. 2009. 147(1): 6–12.

12.

Baker

S.N.

Zhao

Pandey

Heller

W.T.

Bright

F.V.

Baker

G.A.

. “Fluorescence Energy Transfer Efficiency in Labeled Yeast Cytochrome c: A Rapid Screen for Ion Biocompatibility in Aqueous Ionic Liquids”. Phys. Chem. Chem. Phys. 2011. 13(9): 3642–3644.

13.

Akdogan

Junk

M.J.N.

Hinderberger

. “Effect of Ionic Liquids on the Solution Structure of Human Serum Albumin”. Biomacromolecules. 2011. 12(4): 1072–1079.

14.

Rodrigues

J.V.

Prosinecki

Marrucho

Rebelo

L.P.N.

Gomes

C.M.

. “Protein Stability in an Ionic Liquid Milieu: On the Use of Differential Scanning Fluorimetry”. Phys. Chem. Chem. Phys. 2011. 13(30): 13614–13616.

15.

Weingärtner

Cabrele

Herrmann

. “How Ionic Liquids Can Help to Stabilize Native Proteins”. Phys. Chem. Chem. Phys. 2012. 14(2): 415–426.

16.

Ding

Zhang

Xie

Guo

. “Binding Characteristics and Molecular Mechanism of Interaction Between Ionic Liquid and DNA”. J. Phys. Chem. B. 2010. 114(5): 2033–2043.

17.

Wang

Zhang

. “Binding Gibbs Energy of Ionic Liquids to Calf Thymus DNA: A Fluorescence Spectroscopy Study”. Phys. Chem. Chem. Phys. 2011. 13(9): 3906–3910.

18.

Cardoso

Micaelo

N.M.

. “DNA Molecular Solvation in Neat Ionic Liquids”. Chem. Phys. Chem. 2011. 12(2): 275–277.

19.

Shi

Liu

Y.L.

Lai

P.Y.

Tseng

M.C.

Tseng

M.J.

Chu

Y.H.

. “Ionic Liquids Promote PCR Amplification of DNA”. Chem. Commun. 2012. 48(43): 5325–5327.

20.

Fan

Pei

Wang

Fan

. “Solvent Extraction of Selected Endocrine-Disrupting Phenols Using Ionic Liquids”. Sep. Purif. Technol. 2008. 61(3): 324–331.

21.

Yoshino

Sakai

Mizuha

Shimizuike

Yamamoto

. “Peptic Digestibility of Raw and Heat-Coagulated Hen's Egg White Proteins at Acidic pH Range”. Int. J. Food Sci. Nutr. 2004. 55(8): 635–640.

22.

Whitmore

Wallace

B.A.

. “Protein Secondary Structure Analyses from Circular Dichroism Spectroscopy: Methods and Reference Databases”. Biopolymers. 2008. 89(5): 392–400.

23.

Lees

J.G.

Miles

A.J.

Wien

Wallace

B.A.

. “A Reference Database for Circular Dichroism Spectroscopy Covering Fold and Secondary Structure Space”. Bioinformatics. 2006. 22(16): 1955–1962.

24.

Standardization Administration of China. 2009. “GB/T 23527-2009. Protease Preparations”. Beijing.

25.

Hossain

Khan

A.Y.

Kumar

G.S.

. “Study on the Thermodynamics of the Binding of Iminium and Alkanolamine Forms of the Anticancer Agent Sanguinarine to Human Serum Albumin”. J. Chem. Thermodyn. 2012. 47: 90–99.

26.

Fan

Zhang

Kong

Miao

. “Study on the Interaction Between an Ionic Liquid and L-Tryptophan by Fluorescence Spectroscopic Technique”. Microchem. J. 2011. 99(2): 439–442.

27.

Liu

E.H.

L.W.

. “Structural Relationship and Binding Mechanisms of Five Flavonoids with Bovine Serum Albumin”. Molecules. 2010. 15(12): 9092–9103.

28.

Zhang

Zhao

Wang

. “Fluorescence Spectrometric Studies on the Binding of Puerarin to Human Serum Albumin Using Warfarin, Ibuprofen, and Digitoxin as Site Markers with the Aid of Chemometrics”. J. Lumin. 2011. 131(12): 2716–2724.

29.

Cui

Qin

Zhang

Liu

Yao

Lei

. “Interaction of Anthracycline Disaccharide with Human Serum Albumin: Investigation by Fluorescence Spectroscopic Technique and Modeling Studies”. J. Pharmaceut. Biomed. Anal. 2008. 48(3): 1029–1036.

30.

Lakowicz

J.R.

. “Quenching of Fluorescence”. In: Lakowicz

J.R.

, editor. Principles of Fluorescence Spectroscopy. New York: Springer, 2006. Pp. 282.

31.

Zhang

L.N.

F.Y.

Liu

A.H.

. “Study of the Interaction between 2,5-di-[2-(4-Hydroxy-Phenyl)Ethylene]-Terephthalonitril and Bovine Serum Albumin by Fluorescence Spectroscopy”. Spectrochim. Acta A. 2011. 79(1): 97–103.

32.

Zhang

Tang

. “Synthesis and Fluorescence Properties of Tb^III Complex with a Novel β-Diketone Ligand as well as Spectroscopic Studies on the Interaction between Tb^III Complex and Bovine Serum Albumin”. J. Mol. Struct. 2012. 1010: 116–122.

33.

Feng

Cui

Xing

Zhang

Yao

. “Interaction of 3′- Azido-3′-Deamino Daunorubicin with Human Serum Albumin: Investigation by Fluorescence Spectroscopy and Molecular Modeling Methods”. Bioorg. Med. Chem. Lett. 2010. 20(23): 6899–6904.

34.

Ross

P.D.

Subramanian

. “Thermodynamics of Protein Association Reactions: Forces Contributing to Stability”. Biochemistry. 1981. 20(11): 3096–3102.

35.

Bakkialakshmi

Chandrakala

. “Investigation of the Fluorescence Quenching of Bovine Serum Albumin by Certain Substituted Uracils”. J. Mol. Liq. 2012. 168: 1–6.

36.

Freire

M.G.

Neves

C.M.S.S.

Carvalho

P.J.

Gardas

R.L.

Fernandes

A.M.

Marrucho

I. M.

Santos

L.M.N.B.F.

Coutinho

J.A.P.

. “Mutual Solubilities of Water and Hydrophobic Ionic Liquids”. J. Phys. Chem. B. 2007. 111(45): 13082–13089.

37.

Huddleston

J.G.

Visser

A.E.

Reichert

W.M.

Willauer

H.D.

Broker

G.A.

Rogers

R.D.

. “Characterization and Comparison of Hydrophilic and Hydrophobic Room-sTemperature Ionic Liquids Incorporating the Imidazolium Cation”. Green Chem. 2001. 3(4): 156–164.

38.

Freire

M.G.

Santos

L.M.N.B.F.

Fernandes

A.M.

Coutinho

J.A.P.

Marrucho

I.M.

. “An Overview of the Mutual Solubilities of Water-Imidazolium-Based Ionic Liquids Systems”. Fluid Phase Equilib. 2007. 261(1-2): 449–454.

39.

Xiao

Wei

Wang

Liu

. “Fluorescence Resonance Energy-Transfer Affects the Determination of the Affinity Between Ligand and Proteins Obtained by Fluorescence Quenching Method”. Spectrochim. Acta A. 2009. 74(4): 977–982.

40.

Ding

Liu

Z.Y.

Sun

. “A Study of the Interaction Between Malachite Green and Lysozyme by Steady-State Fluorescence”. J. Fluoresc. 2009. 19(5): 783–791.

41.

Zhang

Y.Z.

Xiang

Mei

Dai

Zhang

L.L.

Liu

. “Spectroscopic Studies on the Interaction of Congo Red with Bovine Serum Albumin”. Spectrochim. Acta A. 2009. 72(4): 907–914.

42.

Xiang

. “Study of the Interaction between a New Schiff-Base Complex and Bovine Serum Albumin by Fluorescence Spectroscopy”. Spectrochim. Acta A. 2010. 77(2): 430–436.

43.

Liu

Ding

Sun

. “Characterization of Phenosafranine-Hemoglobin Interactions in Aqueous Solution”. J. Solution Chem. 2011. 40(2): 231–246.

44.

Lai

J.Q.

Lü

Y.H.

Yang

. “Specific Ion Effects of Ionic Liquids on Enzyme Activity and Stability”. Green Chem. 2011. 13(7): 1860–1868.

45.

Yang

. “Hofmeister Effects: an Explanation for the Impact of Ionic Liquids on Biocatalysis”. J Biotechnol. 2009. 144(1): 12–22.