Magnetic Nanoparticles Supported Ionic Liquids Improve Firefly ...

Appl Biochem Biotechnol DOI 10.1007/s12010-014-0730-8

Magnetic Nanoparticles Supported Ionic Liquids Improve Firefly Luciferase Properties Ali Reza Noori & Saman Hosseinkhani & Parisa Ghiasi & Jafar Akbari & Akbar Heydari

Received: 25 October 2012 / Accepted: 6 January 2014 # Springer Science+Business Media New York 2014

Abstract Ionic liquids as neoteric solvents, microwave irradiation, and alternative energy source are becoming as a solvent for many enzymatic reactions. We recently showed that the incubation of firefly luciferase from Photinus pyralis with various ionic liquids increased the activity and stability of luciferase. Magnetic nanoparticles supported ionic liquids have been obtained by covalent bonding of ionic liquids-silane on magnetic silica nanoparticles. In the present study, the effects of [γ-Fe2O3@SiO2][BMImCl] and [γ-Fe2O3@SiO2][BMImI] were investigated on the structural properties and function of luciferase using circular dichroism, fluorescence spectroscopy, and bioluminescence assay. Enzyme activity and structural stability increased in the presence of magnetic nanoparticles supported ionic liquids. Furthermore, the effect of ingredients which were used was not considerable on Km value of luciferase for adenosine-5′-triphosphate and also Km value for luciferin. Keywords Firefly luciferase . Magnetic nanoparticles supported ionic liquid . Bioluminescence

Background Firefly luciferase from North American Photinus pyralis (EC 1.13.12.7) is a monooxygenase that performs ATP-dependent conversion of luciferin into a luciferyl-adenylate, which is oxidized in a multistep reaction to electronically excited oxyluciferin [1–4]. Luciferases have been recently used to study gene delivery [5], real-time imaging of luciferase expression in live animals [6], and gene silencing [7]. All metabolites and enzymes participating in reactions including ATP-converting can be assayed by firefly luciferase [8–13]. Firefly luciferase is significantly unstable and its activity decreases at room temperature. Moreover, irreversible aggregation because of the exposure of its hydrophobic sites followed by structural changes leads to its further inactivation [14–16]. To improve the efficiency of firefly luciferase and A. R. Noori : S. Hosseinkhani (*) : P. Ghiasi Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14175-115, Tehran, Iran e-mail: [email protected] J. Akbari : A. Heydari Department of Chemistry, Faculty of Science, Tarbiat Modares University, Tehran, Iran

Appl Biochem Biotechnol

thermostability, different approaches including site-directed mutagenesis [14–25] and solvent modifications [26, 27] have been recently used. Ionic liquids (ILs) are salts with unique physical properties, such as low viscosity and high thermostability and negligible vapor pressure depending on their structure. They can be seen in the liquid state below 100 °C [28]. ILs have become commonplace in the recent years and can be used as a significant alternative medium for catalytic reactions and the traditional organic synthesis [29]. Although in many reactions the ability of ILs has been demonstrated successfully, their extensive use in chemistry process is still hampered by the following practical drawbacks: catalyst recovery, product isolation, and the use of large amounts of ILs in biphasic systems which is costly and may lead to toxicological concerns [30]. To overcome these drawbacks, a concept of ionic liquid catalysis, which is supported, has recently been established to combine the advantages of heterogeneous supporting materials with those of ILs. Nowadays, different supporting matrices such as organic polymers and inorganic silica, especially porous inorganic materials with high surface areas, have been established [31–33]. Several recent researches have confirmed that ILs is the proper media for enzyme catalysis [34, 35]. Some oxidoreductases, such as chloroperoxidase, laccase, D-amino acid oxidase, and peroxidase are shown in activity in ionic liquids [36, 37]. Magnetic nanoparticles have been recently studied extensively for various medical and biological applications [38]. Varieties of nanoparticles, including metal and oxide nanoparticles have been extensively applied in constructing electrochemical biosensors [39, 40]. Magnetic nanoparticle of iron oxide is one of the famous materials in biotechnology and medicine field [41]. Iron oxide nanoparticles have been widely used as magnetic resonance contrast agents, and important studies such as cancer, cellular trafficking, angiogenesis imaging, and gene expression have been well performed [42–46]. In this study, it has been shown that the use of ionic liquids supported on magnetic nanoparticles improves kinetic and structural properties of firefly luciferase using bioluminescence assay, fluorescence spectroscopy, and circular dichroism (CD).

Experimental Chemicals and Reagents ATP, Tris, and MgSO4, were purchased from Sigma (Sigma Aldrich, St. Louis, MO, USA) glycerol, imidazole and ammonium sulfate were purchased from AppliChem (AppliChem, Darmstadt, Germany). D-luciferin was purchased from RESEM (RESEM, the Netherlands). Affinity column buffers were prepared according to the QIAGEN manual (QIAGEN, Hilden, Germany). Substrate solution: Tris–HCl buffer 50 mM, ATP solution 4 mM, luciferin solution 2 mM, MgSO4 solution 10 mM, pH 7.8. All experiments were carried out in 50-mM Tris+50mM NaCl, pH 7.8. Magnetic nanoparticles supported ionic liquids used in this work are synthesized and provided according to the following procedures. Synthesis of Ionic Liquid Supported on Silica-Encapsulated Magnetic Nanoparticles Synthesis of Magnetic Nanoparticles The magnetite (γ-Fe2O3) nanoparticle was synthesized by a chemical co-precipitation method of ferrous and ferric ions in alkali solution. γ-Fe2O3 nanoparticles were synthesized based on a reported technique with minor modifications. FeCl2.4H2O (1.99 g) and anhydrous FeCl3 (3.25 g) were dissolved in water (20 mL) separately, followed by the two iron salt solutions


being mixed under vigorous stirring (800 rpm). A solution of NH4OH (0.6 M, 200 mL) was then added to the stirring mixture at room temperature, immediately followed by the addition of a concentrated NH4OH solution (25 w/w %, 30 mL) to maintain the reaction pH between 11 and 12. The resulting black dispersion was continuously stirred for 1 h at room temperature and then heated for 1 h to reflux to yield a brown dispersion. The magnetic nanoparticles were then purified by a repeated centrifugation (3,000–6,000 rpm, 20 min), decantation, and redispersion cycle three times, until a stable brown magnetic dispersion (pH 9.4) was obtained. Coating of a layer of silica on the surface of the nanoparticles of γ-Fe2O3 was achieved by premixing (ultrasonic) a dispersion of the purified nanoparticles (8.5 w/w %, 20 mL) obtained with methanol (80 mL) previously for 1 h at 40 °C. Concentrated ammonia solution was added, and the resulting mixture was stirred at 40 °C for 30 min. Subsequently, tetraethyl orthosilicate (1.0 mL) was charged to the reaction vessel, and the mixture was continuously stirred at 40 °C for 24 h. The silica-coated nanoparticles were collected by a permanent magnet, followed by washing three times with EtOH, diethyl ether, respectively, and dried at 100 °C in vacuum for 24 h [47–49]. Synthesis of Ionic Liquid 1-Methylimidazole (50 mmol) and 3-chloropropyl-triethoxy-silane (50 mmol) were stirred at 95 °C for 24 h (yield: 95 %). The product was washed with ether and dried under vacuum. Synthesis of IL Supported on Silica-Encapsulated Magnetic Nanoparticles In order to prepare silica-encapsulated magnetic nanoparticles supported ionic liquids (Fig. 1) 100-mg magnetic silica was dispersed in toluene by ultrasonication. One-gram 1-methyl3-(triethoxysilylpropyl)- imidazolium salt was then added to the system and the mixture was stirred at 90 °C for 24 h. After reaction, the solid product was isolated by a magnet, washed with acetonitrile (100 ml) twice and methanol (100 ml) twice, and dried in vacuum. Luciferase Purification P. pyralis Luciferase gene was previously cloned into pET-16b vector with a His6-tagged and transfected to bacterium Escherichia coli BL21, as a host, and was purified with Ni-NTA Sepharose after over-expression. The luciferase purity was confirmed by SDS-PAGE. Approximate protein concentration was quantified according to Bradford’s method (data not shown) [50, 51].

Fig. 1 Schematic synthesis of [γ-Fe2O3@ SiO2-IL]


Characterization of Kinetic Parameters Prior to any measurements, the effect of various concentrations of magnetic nanoparticles supported ionic liquids (0.01, 0.03, 0.06, 0.12, 0.25, and 0.5 mg/ml) were assessed on luciferase activity and the optimum concentration was chosen in which luciferase activity was maximum. Optimum concentration was 0.03 mg/ml (data not shown). However, at higher concentrations of used compounds (especially at 0.25 mg/ml and higher concentration) significant inhibition was observed, but the appropriate concentration of them was chosen lower than 0.25 mg/ml and so, no luciferase activity inhibition was seen at the concentration at which the assessments were done. The measurement of kinetic parameters of luciferin and ATP were performed at 25 °C. To evaluate LH2 Km, 20 μl of various concentrations of luciferin (from 0.01 to 2 mM) were mixed with 20 μl of buffer assay containing 10-mM MgSO4, 4-mM ATP in 50-mM Tris–HCl, pH 7.8, in the absence and presence of magnetic nanoparticles supported ionic liquids containing 20 μl of 0.03 mg/ml [Fe2O3-SiO2][BMImCl] and[Fe2O3-SiO2][BMImI] in each well of a 96-well plate. The initiation of reaction was with injection of 10 μl of diluted enzyme and light emission measured over 5 s (Sirius tube luminometer, Berthold Detection System). The investigation of Km value for ATP was performed in a similar method. Twenty microliters of different concentrations of ATP solution (from 0.01 to 4 mM) were mixed with 20 μl of buffer assay, including 10-mM MgSO4 and 1-mM luciferin in 50-mM Tris–HCl, pH 7.8 in the absence and presence of mentioned magnetic nanoparticles supported ILs. The reaction was initiated with the injection of 10 μl of diluted enzyme, and light emission recorded over 5 S. Apparent kinetic parameters (Km and Vmax) of luciferase were calculated using Lineweaver-Burk plots. Optimum Temperature To obtain the optimum temperature of activity for enzyme in the absence and presence of ionic liquids supported on magnetic nanoparticles, activities were measured in 10–40 °C for 5 min (in assay reagent) and then, its activity was measured. Remaining Activity Measurements Thermal stability of luciferase were calculated by incubation of the enzyme at different time intervals in a time course of 45 min in the absence and presence of ionic liquids supported on magnetic nanoparticles (0.03 mg/ml) of [γ-Fe 2 O 3 @SiO 2 ][BMImCl] and [γFe2O3@SiO2][BMImI] for 5 min at 35 °C. Samples were removed and cooled on ice (5 min), and the thermal stability was measured. Intrinsic Fluorescence Measurements The purified luciferase was dialyzed in dialysis buffer containing 50-mM Tris–HCl, 1 % glycerol, 50-mM NaCl, 1-mM EDTA, 0.05 % β-mercaptoethanol, and pH 7.8 at 4 °C. Intrinsic fluorescence emission spectroscopy was performed in the absence and two concentrations (0.01, 0.03 mg/ml) of ionic liquids supported on magnetic nanoparticles. Intrinsic fluorescence was determined with an excitation wavelength of 290 nm and using 30 μg/ml protein concentration and emission spectra were recorded between 300 and 400 nm.


Circular Dichroism The far-UV CD spectra of luciferase was recorded in the wavelength range of 200 to 250 nm in Tris-NaCl 50 mM, pH 7.8 buffer in the absence and presence of concentrations (0.01, 0.03 mg/ml) of ionic liquids supported on magnetic nanoparticles and the background was corrected against buffer blank. Results are reported as molar ellipticity [ ] (degrees square centimeter per decimole), based on a mean amino acid residue weight (MRW) assuming the average weight of 112.73. The molar ellipticity [ ] was calculated from the formula [θ]λ =(θ×100 MRW)/(Cl), where l is the light path length in centimeters and C is the protein concentration in milligram per millimeter and is the measured ellipticity at a given wavelength in degrees. The contents of regular secondary structure were analyzed by CD software (AVIV spectropolarimeter, model 215, USA) using protein concentration of about (0.5 mg/ml). All CD measurements were performed at 25 °C with the help of a thermostatically controlled cell holder. The far-UV CD spectra of the enzyme in high concentrations of ionic liquids supported on magnetic nanoparticles did not record.

Results and Discussion Synthesis of Ionic Liquids Supported on Magnetic Nanoparticles The TEM and SEM (Fig. 2a–c) and IR spectra (Fig. 2e) for [γ-Fe2O3@Si-IL] is presented. The high frequency bands from 400 to 650 cm−1 are ascribed to the stretching vibrations of Fe–O groups from γ -phase Fe2O3, and the lower frequency bands at about 450 cm−1 are ascribed to the Si–O–Si stretching vibrations, while the higher frequency bands at about 1,000 cm−1 are assigned to vibrations of Si–O–Fe. Amorphous γ-Fe2O3@SiO2 is subjected to further structural characterization with x-ray diffraction (XRD) (Fig. 2d). Diffraction peaks at around 35.5, 43.1, 62.8, and 54 ° corresponding to the (311), (400), (511), and (440) are readily recognized from the XRD pattern. The observed diffraction peaks agree well with the tetragonal structure of maghemite (1999 JCPDS file no 13-0458). In addition, the amount of loading of ionic liquid on magnetic support was measured by thermo gravimetric analysis (TGA) and there was a weight loss at 250 to 300 °C (Fig. 3). This reduction is related to ionic liquid where Fe2O3@Sio2 is even stable over 600 °C. The amount of the ionic liquid loaded on Fe2O3@Sio2 is calculated to be 2.75 mol%. Study of the Kinetic Parameters To evaluate Km and Vmax values for luciferin (LH2 Km) and ATP (ATP Km) Lineweaver-Burk plots were used. Kinetic constants for luciferin in the presence of magnetic nanoparticles supported ionic liquids were almost the same as that of the luciferase in the absence of these compounds. The Km values of luciferase for ATP in the presence of [γ-Fe2O3@ SiO2][BMImCl] and [γFe2O3@ SiO2][BMImI] decreased 1.12 and 1.4 times (Table 1) So, it seems that the binding tendency of substrate to enzyme increased due to the presence of the magnetic nanoparticles supported ionic liquids in the reaction solution. We previously showed that Km for the substrate ATP in the presence of several ILs decreased while Km for luciferin remained constant [52]. Optimum Temperature of the Luciferase Temperature sensitivity of the activity of enzyme in the absence and presence of mentioned ionic liquids supported on magnetic nanoparticles (0.03 mg/ml) at various temperatures are


Fig. 2 The TEM (a), SEM (b, c), XRD spectra (d), and the IR spectra (e) of [γ-Fe2O3@Si-IL]

determined (Fig. 4). Optimum temperature of luciferase activity was obtained, demonstrating that its activity reached to its maximum at 25 °C and fully inactivated at 45 °C while in the presence of mentioned ionic liquids supported on magnetic nanoparticles, it shifted to 30 °C.

Fig. 3 TGA diagram of [γ-Fe2O3@Si-IL]

Appl Biochem Biotechnol Table 1 Kinetic properties of luciferase in the absence and presence of magnetic nanoparticles supported ionic liquids

Native luciferase [γ-Fe2O3@ SiO2][BMIm][Cl] [γ-Fe2O3@ SiO2][BMIm][I]

LH2 Km (μM)

LH2 Vmax (RLU/s)

ATP Km (μM)

ATP Vmax (RLU/s)

10

1.6×107

10 10

125

1.11×107

7

111

1.25×107

7

92

1.18×107

1.54×10 1.52×10

Values are the averages of three experiments

The results showed that the optimum temperature of enzyme activity increased about 5 °C in the presence of the used compounds. Comparison of Thermal Stability of Luciferases The results of the thermal stability of the enzyme in the presence of used compounds indicate the remaining activity of luciferase after incubation for 5 min at 35 °C up to 45 min have been confirmed. In the absence of ionic liquids supported on magnetic nanoparticles, about 65 % of the activity disappeared within 10 min and the remaining activity was about zero after 40 min at 35 °C. By increasing the concentration of compounds mentioned up to 0.03 mg/ml, the thermal stability was increased and the stabilization effect was closely related to magnetic nanoparticle supported ionic liquids concentration. The thermal stability results showed that their original activity remained approximately 20 and 18 % after incubation at 35 °C for 5 min in the presence of [γ-Fe2O3@ SiO2][BMImCl] and [γ-Fe2O3@ SiO2][BMImI], respectively, whereas the native enzyme lost nearly all of its activity (Fig. 5a, b). It seems that one of the reasons for higher stability of the enzyme in the presence of these compounds is that the structure of luciferase may be more compact which prevents the thermal denaturation of enzyme under the tested conditions. Due to the ionic nature of the IL-component, magnetic nanoparticles of ILs may interact with charged groups of enzymes in the active site or at its

Fig. 4 Optimum temperature of luciferase activity in the absence (filled diamond) and presence of (0.03 mg/ml) [γ-Fe2O3@ SiO2] [BMImI] (filled triangle) [γ-Fe2O3@ SiO2][BMImCl] (filled circle). Luciferase activity was assayed after 5-min incubation at each temperature. For more details, see Section 2


Fig. 5 Comparison of thermal stability of firefly luciferase at 35 °C at different time intervals. From left to right; in the presence and absence of two concentration (0.03, 0.01 mg/ml) of [γ-Fe2O3@ SiO2][BMImCl] (a) and [γFe2O3@ SiO2][BMImI] (b), respectively. For more details, see Section 3

periphery. Therefore, it caused structural changes in the enzyme. One possible explanation for the higher thermal stability in using compounds as an additive in solution may form hydrogen bonds and electrostatic interactions with luciferase in the reaction medium and increase of secondary structures of the enzyme, leading to a more compact enzyme conformation and therefore to higher enzyme activity and stability [53, 54]. Intrinsic Fluorescence of Luciferase The multiphasic nature of the protein molecule during the processes of deactivation strengthens the suggestion that complex internal events take place in its conformational transitions. Intrinsic fluorescence analysis was used to study the protein unfolding process in each of the assayed media in order to get a deeper insight into how the ionic liquids affect luciferase, and to understand conformational transitions. To further discriminate the spectroscopic contributions of the tryptophanyl (Trp) residues, the conformational changes of luciferase in the absence and presence of ILs supported on silica-encapsulated magnetic


nanoparticles was evaluated by measuring the intrinsic fluorescence intensity. Fluorescence is a useful technique for the evaluation of three-dimensional changes in protein structure because the intrinsic fluorescence of Trp residues is particularly sensitive to the polarity of microenvironments. In this study, to eliminate the effects of tyrosine and phenylalanine residues the excitation wavelength was at 295 nm. The results of the enzyme fluorescence show that these ingredients had a positive effect on the fluorescence intensity, especially at low concentrations. The fluorescence spectra of the enzyme exhibit the same λmax (335 nm) in the absence of IL and a buffer containing γ-Fe2O3@ SiO2-ILs. The experiments show that the fluorescence intensity of luciferase in the presence of [γ-Fe2O3@ SiO2][BMImCl] and [γ-Fe2O3@ SiO2][BMImI] were increased (Fig. 6). It may be suggested that the increased intrinsic fluorescence spectra of luciferase are due to structural compactness of protein and exposure of Trp residue in a more non-polar environment [55]. Circular Dichroism Studies of Firefly Luciferase CD is one of the most widely used techniques for distinguishing the proteins structure, making it possible to quantify 3D structure conformational modifications from observed changes in the

Fig. 6 Intrinsic fluorescence spectra of the enzyme in the presence of 0.03 mg/ml (2), 0.01 mg/ml (3) concentrations [γ-Fe2O3@ SiO2][BMImCl] (a) [γ-Fe2O3@ SiO2][BMImI] (b) and without any additive (1) at 35 °C. For more details, see Section 2


Fig. 7 Far-UV CD spectra of firefly luciferase in the absence (1) and presence of two concentration:(0.01,0.03 mg/ml) [γ-Fe2O3@SiO2][BMImCl] (2, 3) and [γ Fe2O3@SiO2][BMImI] (4, 5), respectively. For more details, see Section 2

CD spectrum [56, 57]. To give the content of regular secondary structural features, such as αhelix and β-strand, the CD spectrum can be analyzed. The analysis of the far-UV CD spectra of luciferase in the presence of [γ-Fe2O3@ SiO2][BMImCl] and [γ-Fe2O3@ SiO2][BMImI] after incubation (2 min) at 25 °C show a fall in α-helix and an increase in β-strand secondary structures and a concomitant decrease in the unordered fraction (Fig. 7). The observed increase in β-strand could be ascribed to a loss of hydrogen-bonding interactions between the α-helix and water molecules. In this context, it seems reasonable to expect that before full enzyme deactivation occurs; more than one type of interaction must be broken followed by different conformational changes of the native structure.

Conclusion The exact molecular mechanism of enzyme stabilization by ionic liquids is still remaining elusive in applied biocatalysis [58, 59]. The general desire to improve the enzymes low turnover rate in organic media was a major driving force for the extensive use of ionic liquids. Reactant stabilization are mainly, but not exclusively, led to these low turnover rates (compared with aqueous medium) [60]. This latter effect, which is equivalent with increase in solubility, is translated kinetically into an increase in Km and can be remedied by performing reactions at increased concentrations [61]. The above mentioned results indicate that the optimum temperature and thermal stability exhibited by luciferase in ionic liquids supported on magnetic nanoparticles were more than that observed in the absence of IL supported on silica-encapsulated magnetic nanoparticles, and the results of spectroscopic methods (fluorescence and circular dichroism) suggest that [Fe 2 O 3 -SiO 2 ][BMImCl] and similarly [Fe 2 O 3 SiO2][BMImI] are able to stabilize the enzyme via the formation of a flexible and more compact 3D structure, being related to preservation of the essential water shell [62–65]. Moreover, increasing luciferase thermostability increases sensitivity and reliability of luminescence-based measurements [66].

Appl Biochem Biotechnol Acknowledgments The financial support of this work is provided by the research council of Tarbiat Modares University.

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