Article pubs.acs.org/Langmuir
Probing Enzyme Location in Water-in-Oil Microemulsion Using Enzyme−Carbon Dot Conjugates Krishnendu Das, Subhabrata Maiti, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata 700 032, India S Supporting Information *
ABSTRACT: This article delineates the formation and characterization of different enzyme−carbon dot conjugates in aqueous medium (pH = 7.0). We used soybean peroxidase (SBP), Chromobacterium viscosum (CV) lipase, trypsin, and cytochrome c (cyt c) for the formation of conjugate either with cationic carbon dot (CCD) or anionic carbon dot (ACD) depending on the overall charge of the protein at pH 7.0. These nanobioconjugates were used to probe the location of enzymes in water-in-oil (w/o) microemulsion. The size of the synthesized water-soluble carbon dots were of 2−3 nm with distinctive emission property. The formation of enzyme/protein− carbon dot conjugates in aqueous buffer was confirmed via fluorescence spectroscopy and zeta potential measurement, and the structural alteration of enzyme/protein was monitored by circular dichroism spectroscopy. Biocatalytic activities of protein/enzymes in conjugation with carbon dots were found to be decreased in aqueous phosphate buffer (pH 7.0, 25 mM). Interestingly, the catalytic activity of the nanobioconjugates of SBP, CV lipase, and cyt c did not reduce in cetyltrimethylammonium bromide (CTAB)-based reverse micelle. It indicates different localization of carbon dots and the enzymes inside the reverse micelle. The hydrophilic carbon dots always preferred to be located in the water pool of reverse micelle, and thus, enzyme must be located away from the water pool, which is the interface. However, in case of trypsin−carbon dot conjugate, the enzyme activity notably decreased in reverse micelle in the presence of carbon dot in a similar way that was observed in water. This implies that trypsin and carbon dots both must be located at the same place, which is the water pool of reverse micelle. Carbon dot induced deactivation was not observed for those enzymes which stay away from the water pool and localized at the interfacial domain while deactivation is observed for those enzymes which reside at the water pool. Thus, the location of enzymes in the microdomain of w/o microemulsion can be predicted by comparing the activity profile of enzyme−carbon dot conjugate in water and w/o microemulsion.
1. INTRODUCTION Research investigations on protein−nanoparticle conjugates are finding considerable importance in recent times because of its applications in different field of biology and medicine. Intrinsic properties of nanoparticles such as size and nature of the surface charge have been utilized to bind biomolecules like proteins, enzymes, and nucleic acids on the surface of nanoparticles.1−4 In this context, surface-functionalized carbon dots have gained enormous significance in the domain of biotechnology because of its unique physicochemical and optoelectronic properties.5 The advantage of using carbon dots over the other semiconductor-based nanoparticles lies in their chemical inertness, high stability, versatile surface chemistry, and potentially low cytotoxicity.6−8 Small sized carbon dots with its intrinsic fluorescence property are finding notable importance in biosensing, in-vivo imaging, and other research arenas.8,9 However, use of carbon dots in biocatalysis is still overlooked. In this regard, it is known that enzymes/proteins exhibit their biocatalytic efficiency by localizing itself in the different anisotropic microdomain of cellular environment.10 Thus, determining the position of proteins in such © 2014 American Chemical Society
compartmentalized system is always important to know their operational basis in different cellular processes like hydrolysis of dietary fat by hydrolase lipase, scavenging of reactive oxygen species by peroxidase, signal transduction, selective catalysis, etc.10 To this end, reverse micelle or water-in-oil (w/o) microemulsion (a well-known membrane−mimetic system due to its compartmentalized structure) has been widely used as a host for different biocatalytic reactions.11−19 This selfaggregated macroscopically homogeneous system can entrap both hydrophilic enzymes in its water pool and surface-active or membrane-bound enzymes at the interface.16 Herein, our objective is to exploit the nanobioconjugates of carbon dots in combination with different enzymes to identify their location in a membrane mimetic system like w/o microemulsion. In this context, spectroscopic techniques like fluorescence, dynamic and static light quenching, dynamic light scattering (DLS), and small-angle X-ray scattering experiment (SAXS) are Received: October 4, 2013 Revised: February 12, 2014 Published: February 14, 2014 2448
dx.doi.org/10.1021/la403835h | Langmuir 2014, 30, 2448−2459
Langmuir
Article
Figure 1. Structure, TEM images, and emission spectra of the synthesized CCD and ACD. Spectrochem (India). CTAB was crystallized three times from methanol/diethyl ether, and recrystallized CTAB was without minima in its surface tension plot. The substrate for lipase, p-nitrophenyl-noctanoate, was synthesized following the reported protocol.19 Milli-Q water was used throughout the study. The UV−vis absorption spectra were recorded on a PerkinElmer Lambda 25 spectrophotometer. Fluorescence spectra were recorded in Varian Cary Eclipse luminescence spectrometer. Circular dichroism spectroscopy was carried out in a Jasco J-815 spectropolarimeter. Lyophilization was done in a Virtis 4KBTXL-75 freeze-drier. Sorvall RC 6 was used for centrifugation, and zeta potential was measured in a zetasizer Nano-ZS of Malvern Instruments Limited. 1H NMR spectra were recorded on a Bruker Avance DPX-300 spectrophotometer. FTIR spectra were recorded in a PerkinElmer Spectrum 100 spectrometer. 2.2. Synthesis of Cationic Carbon Dot (CCD). CCD was synthesized by following the reported protocol.6,26 Briefly, 2 g of betaine hydrochloride was dissolved in 5 mL of water, and then to this solution 1.2 g of Tris was added (maintaining 1:1 molar ratio) with shaking until complete dissolution. The water-soluble organic salt was then extracted with 100 mL of isopropanol, and solvent was removed under a rotary evaporator. Addition of isopropanol (100 mL) was done three times to extract more sticky mass. It was then dried at 80 °C for 3 days. This dried material was heated in a furnace at 250 °C for 2 h in a porcelain crucible and then cooled to room temperature. Finally, it was extracted with 25 mL of water and precipitated after addition of acetone at 1:10 volume ratio. This combined liquid mixture was centrifuged at 14 000 rpm for 1 h to precipitate out the cationic carbon dot (Figure 1). The supernatant was removed, and the precipitated brownish-black mass was further dried in a hot oven at 80 °C until it became powder in nature. It was then characterized by IR, NMR, and TEM as reported earlier.26 2.3. Synthesis of Anionic Carbon Dot (ACD). For the synthesis of ACD, 1.1 g of glycine (14 mmol) was converted to its carboxylate salt by the addition of an equivalent amount of NaOH solution (2 mL).7,26 To this, 2 mL of citric acid solution (3 g, 14 mmol) was added by maintaining a 1:1 molar ratio. This water-soluble mixture was evaporated to dryness at 100 °C. The sticky mass was collected and dried in hot oven at 80 °C for 3 days. The solid was crushed into a fine powder and was heated in a furnace at 300 °C for 2 h in a porcelain crucible and then cooled to room temperature. The brownish-black product was extracted with 25 mL of hot water. The deep brown solution was precipitated after addition of acetone at 1:10 volume ratio, and the supernatant was collected. This supernatant was centrifuged at 14 000 rpm for 1 h to precipitate out the anionic carbon dot (Figure 1). The supernatant was removed, and the precipitated brownish-black mass was further dried in a hot oven at 80 °C until it became powder in nature.
used to determine the position of enzymes in reverse micelles.20−23 Moreover, Rotello and co-workers have used different surface-charged gold nanoparticles for protein surface reorganization in aqueous medium through electrostatic interaction between enzyme and nanoparticle.24 However, investigation on determining the location of proteins/enzymes in membrane−mimetic systems using surface-functionalized carbon dots has not been explored. Toward the development of enzyme−nanoparticle conjugate, hydrophobic adsorption or immobilization through electrostatic interaction of enzyme on the nanoparticle surface is preferred over the covalent approach as the former two methods do not alter the intrinsic characteristics of the constituents.25 In the present article, we synthesized both cationic and anionic carbon dots with high aqueous solubility and stability. We have characterized these carbon dots by transmission electron microscopy (TEM), FTIR, and NMR spectroscopy. Surface charge of these carbon dots was used to prepare enzyme−nanoparticle conjugate through electrostatic interaction in aqueous phosphate (pH 7.0, 25 mM) buffer. To elucidate the behavior and function of the enzymes in conjugation with carbon dots, we used following enzymes, soybean peroxidase (SBP), Chromobacterium viscosum lipase (CV lipase), trypsin, and cytochrome c (cyt c). Fluorescence, zeta potential, and circular dichroism (CD) spectroscopy were used to confirm the enzyme−carbon dot conjugate formation and also to observe the change in the structure of enzymes. The biocatalytic efficiency of enzyme−carbon dot conjugate showed either different or analogous activity profile within w/o microemulsion compared to that observed in aqueous medium depending on the nature of enzyme. The characteristics in the activity behavior of different class of enzymes in conjugation with varying surface-charged carbon dots determined their respective region of location in the microheterogeneous system of w/o microemulsion.
2. EXPERIMENTAL SECTION 2.1. Materials. Chromobacterium viscosum Lipase (E.C.3.1.1.3 Type XII), soybean peroxidase (SBP, EC 1.11.1.7), trypsin (EC 3.4.21.4), cytochrome c, N-α-benzyloxycarbonyl-L-lysine-p-nitrophenyl ester, and betaine were purchased from Sigma and were used as received. Hydrogen peroxide (30%, w/v solution) was purchased from Ranbaxy, India. Tris, citric acid, glycine, HPLC-grade isooctane, n-hexanol, isopropanol, and analytical grade CTAB were purchased from 2449
dx.doi.org/10.1021/la403835h | Langmuir 2014, 30, 2448−2459
Langmuir
Article
Scheme 1. Reaction Catalyzed by Different Enzymes and Protein
2.4. Preparation of Enzyme−Carbon Dot Conjugate. Enzymes at a fixed concentration were incubated with different concentrations of both ACD and CCD separately at pH 7.0 (25 mM phosphate buffer) for 1 h to prepare the enzyme−nanoparticle conjugate. Newly developed nanobioconjugates were characterized by analyzing the change in tryptophan emission of enzyme at 350 nm by exciting the conjugate at 280 nm in the corresponding fluorescence study. 2.5. Fluorescence Study. Tryptophan fluorescence intensity (at 350 nm) of enzymes in conjugation with the carbon dots (ACD and CCD) were measured in both reverse micelle and pH 7.0 (25 mM phosphate buffer) by exciting the enzyme and enzyme−nanoparticle conjugate at 280 nm. Excitation and emission slits were kept at 10 nm. 2.6. Transmission Electron Microscopy (TEM). A drop of the prepared carbon dot solutions was cast on 300-mesh Cu-coated TEM grid and dried under vacuum for 4 h before taking the image. TEM images were taken on a JEOL JEM 2010 microscope. 2.7. Preparation of Reverse Micelle or w/o Microemulsion. CTAB (36.4 mg) was dispersed in isooctane in a 2 mL volumetric flask, to which the required amount of n-hexanol was added to attain the desired z ([n-hexanol]/[surfactant]) value, and the system was shaken vigorously. Finally, aqueous buffer (25 mM phosphate buffer, pH = 7.0) solution was added into that mixture to reach the corresponding W0 ([water]/[surfactant]) value, and the whole suspension was vortexed to obtain a clear homogeneous solution of CTAB (50 mM)/isooctane/n-hexanol/water reverse micelle.27 We fixed the microstructural parameters z and W0 at 16 and 20, respectively, to maintain uniform reverse miceller system throughout the experiment for SBP, CV Lipase, and trypsin. Only in the case of cytrochrome c W0 was maintained at 12 as a lower W0 value is necessary for substantial activity of cytrochrome c.13
2.8. Activity Measurement of Soybean Peroxidase (SBP) in Aqueous Medium and w/o Microemulsion. The kinetics of pyrogallol oxidation, catalyzed by peroxidase, was monitored spectrophotometrically in a UV−vis spectrophotometer following the protocol reported earlier.17 In a typical experiment, 1 μL of SBP or SBP−carbon dot conjugate (from 1 mg mL−1 stock solution) and 1 μL of H2O2 (from 50 mM stock solution) were added in 500 μL, pH = 7.0 (phosphate buffer) in a quartz cell to attain the final concentration of SBP and H2O2 0.023 and 100 μM, respectively. Then the required amount of pyrogallol was added from 100 mM stock solution in acetone (concentration of pyrogallol was varied from 20 to 500 μM) to initiate the reaction (Scheme 1). The change in absorbance was monitored immediately after the addition of pyrogallol to follow the catalysis. The progress of the reaction was monitored checking the absorbance at 420 nm due to the formation of purpurogallin, the oxidized product of pyrogallol. The initial rate (Vi) of purpurogallin formation was calculated from the molar absorption coefficient of 4400 M−1 cm−1 at 420 nm.17 The catalytic constant (kcat) was calculated from a Michaelis−Menten plot. In a similar way, peroxidase activity of SBP and SBP−nanoparticle conjugates was measured in CTAB (50 mM)/isooctane/n-hexanol/water reverse micelle at pH 7.0 (25 mM phosphate buffer), z = 16 and W0 = 20. 2.9. Activity Measurement of Lipase in Aqueous Medium and w/o Microemulsion. The second-order rate constant (k2) in CV lipase-catalyzed hydrolysis of p-nitrophenyl-n-octanoate in reverse micelle was determined following a similar protocol as reported earlier.16 4.5 μL of aqueous enzyme stock solution (0.34 mgmL−1) was added to 1.5 mL of reverse micelle having 50 mM CTAB and desired pH (pH refers to the pH of the aqueous buffer used for preparing the w/o microemulsions; pH within the water pool of the reverse micelles does not vary significantly,
