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Deciphering the Interactions of Bromelain with Carbon Nanotubes: Role of Protein as Well as Carboxylated Multiwalled Carbon Nanotubes in a Complexation Mechanism Indrani Jha, and Pannuru Venkatesu J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b03547 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on July 6, 2016

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The Journal of Physical Chemistry

Deciphering the Interactions of Bromelain with Carbon Nanotubes: Role of Protein as Well as Carboxylated Multiwalled Carbon Nanotubes in a Complexation Mechanism Indrani Jha and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi-110 007 *e-mail: [email protected]; [email protected]; Tel:+91-11-27666646-142; Fax: +91-11-2766 6605 ABSTRACT The interaction of the carboxylated multiwalled carbon nanotubes (MCNTCOOH) with globular protein bromelain (BM) has been studied using various spectroscopic and biophysical techniques. BM exhibited decreased stability in the presence of MCNTCOOH compared to BM in buffer. Additionally, the morphological changes in BM were decoded using a Field emission scanning electron microscope (FESEM). Additionally, the increase in the size (hydrodynamic radius/dH) of MCNTCOOH as a result of BM adsorption was analyzed using dynamic light scattering (DLS). From this study, it was determined that the interaction of BM with MCNTCOOH results in a complex formation between the fluorophore and the quencher. In addition to hydrophobic and ᴨ-ᴨ interactions, electrostatic interactions a play vital role as the interacting force between BM and MCNTCOOH. Additionally, the DLS results revealed that a high concentration of BM can induce MCNTCOOH aggregations, which are reversible in nature. These results have helped in understanding the mechanism by which BM binds to MCNTCOOH and in interpreting the roles of both the nanomaterial and protein in the complexation mechanism. From this study, we have found that the adsorption of BM on the MCNTCOOH is strongly dependent on the protein concentration because a high protein concentration can cause nanotube aggregation.

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INTRODUCTION In recent years, carbon nanotubes (CNTs) have attracted a great deal of attention because of their broad range of applications in the fields of nanoelectronics, molecular transporters, targeted drug delivery and other applications.1-6 The extraordinary mechanical, unique optical, distinctive electronic properties of CNTs have made all of the above-mentioned applications possible. The main driving force for the interaction of nanoparticles (NPs) with biomolecules, such as lipids, proteins and biological metabolites, is their nano-size and large surface to mass ratios.7 Considering the biological effects of CNTs, the interaction between proteins and CNTs are supposed to play a crucial role. Recent developments in nanotechnology have drawn attention to the relevance of merging proteins and CNTs. A clear picture representing the detailed depiction of the structural changes in proteins caused by the CNTs would certainly lead to valuable information that can be utilized in drug delivery and other related fields.1-6 Different proteins bind to the surface of the NPs through non-covalent interactions when these NPs enter the blood stream or tissue interstitial fluids.7 Thus, one can see the proteins allied with the nanoparticle as a ‘corona”. Several forces, such as hydrogen bonding, solvation forces, and van der Waals interactions, play major roles in the adsorption of protein at the nano-bio interface.8 In particular, studies related to the interaction between proteins and NPs have grown rapidly because once NPs are in contact with biological media, the interaction with proteins can affect the way the cells interact with, recognize and process NPs.7 To use NPs for therapeutic purposes, the first step will be to assess the toxic risk of their introduction into the blood streams of humans and other animals. There are studies related to protein-NPs interactions available in the literature. Ge et al.9 studied the interactions between single-wall carbon nanotubes (SWCNTs) and human serum proteins using both theoretical and experimental approaches and showed that ᴨ-ᴨ stacking interactions between the SWCNTs 2 ACS Paragon Plus Environment

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and aromatic residues play an imperative part in determining their capacity for adsorption. Another study by Paoli et al.10 suggested that the CNT surface property is the main driving force behind protein binding. Furthermore, it showed that protein binding onto carboxylated multiwalled carbon nanotubes (MCNTCOOH) affected the agglomeration state and charge of the MCNTCOOH. Atomic force microscopy (AFM) was used to demonstrate that type, arrangement model, size and surface modifications of the CNTs played a major role in its adsorption capacity for proteins.11 Detailed interaction between Bovine serum albumin (BSA) and amidated SWCNTs showed that hydrophobic forces had a leading contribution in binding of BSA on amidated SWCNTs, and their interaction affected the secondary conformation of the protein.12 The depiction of the details of the structural changes brought about by the MCNTCOOH will be most helpful in further studies in this field. The studies of NPs-protein interactions have been continuously increasing and considered important for biomedical applications, particularly in drug delivery. Once intravenous administration is carried out, the nanomaterial is exposed to blood as the first physiological environment. Therefore, the studies that are available in the literature are mainly focused on the interaction of NPs with plasma proteins.13 Another major application of NPs can be as potential oral delivery systems for proteins because upon oral administration, peptides and proteins may remain poorly bioavailable. One of the strategies that can be applied is their association with NPs, which can be used to improve the proteins’ bioavailability. Therefore, apart from plasma proteins, other proteins, such as bromelain (BM), which has antidematous, antiflammatory, antithrombotic, fibrinolytic, antitumoral, immunomodulator, antimetastic, antiinvasive properties and can be administered orally, needs to be studied in terms of its interactions with NPs are concerned. Basically, the therapeutic importance of BM encouraged us to carry out studies based on its interaction with CNTs. Additionally, in spite of the studies available in the literature that consider 3 ACS Paragon Plus Environment


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nanomaterials and proteins, very little is known about the interactions that occurs between functionalized CNTs, particularly with carboxylic groups and fine details of the type of interaction. In addition, significantly less knowledge is available on the structural perturbation in proteins on account of their interactions with nanomaterials. Therefore, a deeper understanding of the mechanism governing the adsorption of protein on MCNTCOOH is required. Additionally, compared to non-functionalized or amine-modified NPs, carboxylated NPs are capable of attracting more proteins from plasma.13 We have divided the experimental part into three sections. In the first part, we have determined the type of interaction between BM and MCNTCOOH and type of complex formed as well as the thermal stability of BM in the presence of MCNTCOOH using UV-vis and fluorescence spectroscopy. Additionally, CD analysis was further utilized to verify the changes in the secondary and tertiary structures of BM as a consequence of the interaction. In addition, we observed morphological changes in the BM by using Field emission scanning electron microscopy (FESEM). For these experiments, we kept the protein concentration constant and varied the concentration of MCNTCOOH. Second, we demonstrated whether the enzymatic activity of BM is influenced by the nanoscale environment of the MCNTCOOH. Through this assay, we analyzed whether the MCNTCOOH promoted the retention or loss of native protein properties using the proteolytic enzyme assay. Finally, to confirm our results, we performed dynamic light scattering (DLS) measurements to monitor the adsorption of BM and understand the specific binding and non-specific adsorption of BM on MCNTCOOH. For this, we varied the BM concentration while keeping the MCNTCOOH constant. We carried out two types of experiments: in one, we kept the BM concentration fixed, and in DLS, we varied the BM concentration. By applying this approach, we were able to interpret the roles of both the protein and the MCNTCOOH in the complexation mechanism. We performed a detailed 4 ACS Paragon Plus Environment

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analysis of the interaction MCNTCOOH with BM protein. We used carboxylic acid functionalized CNTs because we required a water soluble counterpart to carry out the experimental procedures. MATERIALS AND METHODS Materials Bromelain (BM), lyophilized powder (molecular weight: 23.8 kDa) was obtained from SigmaAldrich. Carboxylated multiwalled carbon nanotubes (MCNTCOOH) with outer diameters of 15 nm and lengths of 10-30 µm were purchased from Global Nanotech (India) with a purity higher than 97%. Sodium phosphate buffer (10 mM) at pH 7.0 was prepared using distilled deionized water (dd water) with a resistivity of 18.3 Ωcm. Throughout the experimental procedure, 500 mg/L protein solution in sodium phosphate buffer was used, except in the DLS experiments, in which we used 3000 mg/L of BM. MCNTCOOH were dispersed using an ultra-sonicator bath for 3 hrs in the sodium phosphate buffer prior to the experiment. Methods The bioconjugate solutions were prepared by mixing BM and MCNTCOOH in the Sodium phosphate buffer (10 mM) at pH 7.0. Then, the solutions were incubated at 4 °C for at least 30 min before the spectra were obtained. Series of the bioconjugates with different concentration (mg/L) ratios of BM/MCNTCOOH were prepared, where the concentration of BM was kept constant while the concentration of MCNTCOOH was varied. No separation techniques (i.e., centrifugation) were employed in the experiment to retain the original conformational structure in the solution and to prevent shear forces from disrupting the structure of the bioconjugates. All measurements in the following discussion were obtained at room temperature (25 °C). Steady-state fluorescence experiments Fluorescence emission spectra measurements of the BM were measured with a Cary Eclipse fluorescence spectrofluorimeter (Varian optical spectroscopy instruments, Mulgrave, Victoria, 5 ACS Paragon Plus Environment


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Australia) equipped with an intense Xenon flash lamp as the light source. The interaction study between MCNTCOOH and BM was carried out by mixing a fixed concentration of BM (500 mg/L) and increasing concentrations of MCNTCOOH (0.5-5.0 mg/L at 0.5 mg/L intervals) at a constant temperature of 25 °C. The excitation wavelength was set at 295 nm to calculate the contribution of the tryptophan (Trp) residues to the overall fluorescence emission. Excitation wavelength was reported from 300-450 nm. Absorption spectroscopy Absorption spectra for BM and for BM in the presence of various concentrations of MCNTCOOH were recorded on a Shimadzu UV-1800 (Japan) spectrophotometer with the highest resolution (1 nm) using matched 1 cm path length quartz cuvettes. We varied the concentration of MCNTCOOH (0.5- 4.0 mg/L at 0.5 mg/L intervals). Thermal fluorescence analysis The thermal stability of BM was assessed using fluorescence spectroscopy by following the changes in the spectra of BM with increasing temperature. Thermal denaturation measurements of the BM in both the absence and presence of MCNTCOOH were taken at a heating rate of 1 °C.min-1. We varied the concentration of MCNTCOOH (0.5-4.0 mg/L at 0.5 mg/L intervals). Circular dichroism Spectroscopy (CD) CD spectroscopic studies were performed using a PiStar-180 spectrophotometer (Applied Photophysics, U.K.) equipped with a Peltier system for temperature control. The protein concentration was 500 mg/L for the far and near UV-CD, and each spectrum was collected by averaging six spectra. CD spectra of BM were measured in the absence and presence of MCNTCOOH, in which the BM and MCNTCOOH concentrations were 500 mg/L and 0.5, 2.5 and 4 mg/L, respectively. Here, we report the results for only three concentrations of MCNTCOOH for the clarity of presentation. Field emission scanning electron microscope (FESEM)


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FESEM studies were carried out using a MIRA3 TESCAN electron microscope operating at 10 kV. For the FESEM images, carbon nanotubes–protein solutions were prepared in purified distilled water, placed into liquid nitrogen and dried in a freeze dryer. Then, the samples were loaded onto the surface of copper substrates and sputter coated with a thin layer of gold before observation. All of the FESEM micrographs were taken at a resolution of 5µwith an accelerating voltage that was optimized to obtain the best images. Dynamic light scattering (DLS) measurement The hydrodynamic diameters (dH) of the MCNTCOOH in the absence and presence of different concentrations of BM were measured using the Zetasizer Nano ZS90 dynamic light scattering (DLS) instrument (Malvern Instruments Ltd, UK). A fixed scattering angle of 90° was used for the size measurement. The instrument is equipped with 4 mW HeNe laser with a fixed wavelength, λ = 633 nm, as a light source to characterize the obtained aggregate sizes. We carried out all the DLS experiments constant temperature of 25 °C. A filtered, bubble-free sample of approximately 1.5 mL was transferred into a quartz sample cell, which was sealed with a Teflon-coated screw cap to protect it from dust. Then, the air-tight sample was introduced into the sample holder of the sample chamber of the DLS instrument. The Brownian motion of particles was detected by the DLS and was correlated to the particle size. The relationship between the size of a particle and its speed due to Brownian motion is defined by the Stokes-Einstein equation. All data obtained were analyzed using the Malvern Zetasizer Software version 7.01. For the DLS measurements, we incubated MCNTCOOH with BM. The samples were prepared by adding 30, 60, 90, 120 or 150 µL of protein solution or dd water to 1500 µL of MCNTCOOH solution. Additionally, DLS measurements of MCNTCOOH at various concentrations, such as (0.54.0 mg/L), are provided in the supplementary information as Table 2S. RESULTS 7 ACS Paragon Plus Environment


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Fluorescence measurements and quenching analysis The study of the structure and conformation of proteins by the application of fluorescence spectroscopy has proven to be prolific. The fluorescence quenching analysis can reveal a great deal of knowledge about the accessibility of a quencher to the fluorophore present in the protein and can aid in the understanding of the mechanism by which the fluorophore and the quencher bind.14,15 BM contains five Trp residues in the entire polypeptide chain.16 Among the five Trp residues, three may be buried in the hydrophobic core, and two are located near the surface.17 We monitored the fluorescence spectra of BM with varying concentrations of MCNTCOOH. Figure 1a shows the emission spectra of BM in buffer and in the presence of different concentrations of CNTs. The emission maximum (λmax) was found to be 347 nm for BM in the presence of the buffer (shown by the black trace) in Figure 1a. The fluorescence intensity of BM decreased gradually with the increase in the concentration of MCNTCOOH with no shift in λmax. This shows the quenching effect as a result of the direct interaction between MCNTCOOH and the chromophore of the proteins, which, in this case, is Trp. This decrease in the fluorescence intensity can be attributed to the quenching of the fluorophore with the changes occurring in the solution conditions, such as temperature, pH or the addition of any substance.18 There are many types of molecular interactions that embrace molecular rearrangement, including ground state complex formation, excited state reactions, energy transfer and collision quenching.19,20 The observed quenching of fluorescence with no significant shift in the λmax may have ultimately resulted in complex formation between fluorophore and the quencher. We monitored the quenching of BM in the presence of 0.5-5.0 mg/L of MCNTCOOH. However, we found that BM obeyed the Stern-Volmer relationship only up to 3.0 mg/L. Therefore, we focused on the concentration range only up to 3.0 mg/L of


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MCNTCOOH for our calculation. Further, the Stern-Volmer equation21 was used to study the mechanism of quenching.

= 1 + []

(1)

where I0 and I are the fluorescence intensities in the absence and the presence of quencher, respectively. [] is the concentration of the quencher, and is the SternVolmer constant. The MCNTCOOH sample does not have a definite molecular weight, so its molar concentration cannot be determined correctly. In this work, we use a weight concentration multiplied by Y as a pseudo molar concentration,22 where the value of Y is not more 1×10-4. The Stern-Volmer plot has been provided in Figure 1b, where the derived value is found to be 0.659/A Lmol-1 (Table 1S). By using equation 2, Log ( − )/ = log K + n log []

(2)

the graph of log ( − )/ vs. log [] yields the number of binding sites (n), which is 1.19247 (Figure 1c). The approximate value of n is 1, which suggests the presence of a preferable binding site on BM.23 There is linear correlation between I0 / I and the concentration of MCNTCOOH, which indicates a single mechanism of quenching between the fluorophore and the quencher for up to 3.0 mg/L of MCNTCOOH. As has already been explained, the quenching of the fluorescence of the protein by the MCNTCOOH could be through excited-state reaction, collision quenching, and ground state complex formation. The mechanisms of quenching can be static, dynamic or both. Static quenching can occur due to the formation of a nonfluorescent complex between the fluorophore in the ground state and the quencher. However, dynamic quenching can occur due to the collision of the fluorophore in the excited state with the quenching agent. The absorption spectra of BM in the presence of MCNTCOOH can give valuable information regarding the mechanism of quenching. A 9 ACS Paragon Plus Environment


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careful examination of the absorption spectra of the fluorophore can provide us with an easy method for distinguishing static and dynamic quenching. In collisional quenching, only excited states of the fluorophores are affected, so no changes in the absorption spectra are expected. Generally, the absorption spectra of the fluorophore are altered due to formation of a non-fluorescent complex between the fluorophore in the ground state and the quencher, which occurs in the case of static quenching. 24 Upon further increasing the concentration of MCNTCOOH, there was no absolute linear correlation between I0 / I and the concentration of MCNTCOOH, which indicated a different quenching mechanism. The Stern-Volmer equation shows an upward curvature that is concave towards the y-axis when higher concentrations of MCNTCOOH are included (Figure 1S). In such a case, it might be a possibility that the quenching of fluorescence of the fluorophore occurs by collisions and by complex formation with the same quencher. The main interactions between the proteins and CNTs include hydrophobic interactions, ᴨ-ᴨ stacking interactions and electrostatic forces.25-27 Because CNTs have a broad absorption in the UV-Vis region, there is the possibility that a decrease in the intensity of fluorescence can be attributed to the CNTs, even if there is no direct interaction between the CNTs and BM. Therefore, to clarify the fact that a decrease in the intensity of fluorescence is not caused by CNTs themselves but is instead due to the interaction between BM and CNTs, Figure 2S has been provided. Figure 2S shows the fluorescence emission of CNTs at its different concentrations (0, 0.5-5.0 mg/L at 0.5 mg/L intervals). This figure clearly shows that CNTs at the specified concentration are not producing any fluorescence emission. UV-visible investigation on the conformational changes of BM Furthermore, to distinguish between the type of mechanism of binding between MCNTCOOH and BM and to explore the structural changes occurring in BM due to its


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complex formation with MCNTCOOH, UV-absorption spectroscopy can be applied. The peak emerging at 200 nm is mainly because of the peptide bonds. The changes in the absorption spectra with the variations in the structure of protein, particularly in the microenvironment of chromophores such as Trp and tyrosine (Tyr), can be observed as shifts in the wavelength or increases and decreases of the absorbance intensity approximately 280 nm.28 For Tyr in aqueous buffer, the absorption maximum occurs at approximately 275 nm, and it is approximately 277-278 nm in proteins.29 There are fourteen Tyr residues in the BM protein.30 As Figure 2 shows, at all concentrations of MCNTCOOH, the absorption intensity is higher than for pure BM. This suggests complex formation between them, which is consistent with our fluorescence results. The absorbance at 279 nm increased with the addition of MCNTCOOH. This clearly indicates that MCNTCOOH particles interact with the BM surface and gradually increase with an increasing MCNTCOOH concentration. In general, the absorption maximum is slightly shifted towards higher wavelength when transferred from a polar to non-polar environment. This occurs because such movement in the interior of a globular protein may perturb the energy essential for the promotion of electrons to the excited states and results in the positions of absorption bands shifting to higher wavelengths. Thus, the protein under study shows enhanced absorbance in the spectra of BM as a result of interaction with MCNTCOOH. This difference is apparent in the absorption spectra (Figure 2 and 4S), which indicates the probability of a static quenching mechanism up to 3.0 mg/L of MCNTCOOH. In the above text, it has been shown that an increase in the absorption intensity upon the addition of CNTs is possibly due to the absorption of the CNTs themselves. However, as observed in Figure 3S, the CNTs at their different concentrations are absorbing approximately 255 nm. Therefore, it is quite clear from the Figure 3S that the increase in the absorption intensity is due to the interaction between the CNTs and BM only. Further, Figure 4S shows the



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subtracted background spectra of each CNT solution from the respective raw data of the protein in the presence of MCNTCOOH, which also shows an increase in the absorption intensity with increasing concentrations of MCNTCOOH. Thermal Stability of BM in the presence of MCNTCOOH The thermal stability of BM can be quantified by following the changes in the fluorescence spectra of BM with increasing temperature. Here, we applied thermal fluorescence analysis to measure the relative stability of BM in the presence of MCNTCOOH. The results of the thermal stability of BM were analyzed as a two-state equilibrium between the folded state (N) and the unfolded state (U), from which the transition temperature (Tm) of BM in the presence of the same concentrations of MCNTCOOH was calculated to analyze the effect of MCNTCOOH on the thermal stability of BM at these concentrations. The thermal unfolding of BM as a result of increasing temperature indicates a Tm of 66.8 °C. Thermal unfolding of the BM-MCNTCOOH system with increasing concentration of MCNTCOOH shows decreasing Tm values (Figure 3). Although we could not find any direct correlation between the decreased thermal stability and the MCNTCOOH concentration, it was clear from the Tm values that the conjugates had a lower thermal stability than did BM alone. However, there is not much difference in the Tm values, which is consistent with other experimental results, such as UV-vis, CD and fluorescence data. CD analysis of BM in the presence of CNTs CD is prominently used to study the various changes in protein conformation, in which the far-UV region (180-250 nm) provides information regarding the secondary structure of proteins and the near-UV region (250-350 nm) examines the side chain tertiary structure of proteins.31 Hence, we examined the secondary and tertiary structural changes in BM both with and without MCNTCOOH. The BM spectrum shows bands at 208, 215, and 222 nm,


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with a more intense CD signal at 208 than 222 nm, as shown in Figure 4a (the black trace). Therefore, BM is composed of α-helix and β-sheet regions, which are indicative of the typical α+ β class of enzyme.32 There is almost no change in secondary structures at 0.5 mg/l of MCNTCOOH, as shown by the spectra. For other concentrations of MCNTCOOH, there is decrease in secondary structure, as depicted from the ellipticity values. We did not observe any drastic shifts in the bands upon the addition of MCNTCOOH. This suggests that there is no gross structural variation in the native state of BM on binding with MCNTCOOH. It is quite apparent from the far-UV spectra that compared to native protein, there is a slight decrease in the secondary structure contents for concentrations other than 0.5 mg/L of MCNTCOOH, which gives further evidence and confirmation that the MCNTCOOH bind to the residues of the BM polypeptide. Therefore, it can be summarized that there is no drastic change in the secondary structure of BM. The near-UV BM spectrum of BM shows bands a negative band at 300 nm and a broad positive band at 260-280 nm, which are the characteristic bands of aromatic amino acids residues (Figure 4b). Aside from 0.5 mg/L of MCNTCOOH, for all other BMMCNTCOOH conjugates, there was perturbation in the tertiary structure of BM. Therefore, near-UV CD spectra were consistent with the binding of the MCNTCOOH to the aromatic amino acid residues of BM. Morphological changes studied using FESEM measurements The investigation of surface structure can be carried out using the FESEM technique. The structural morphologies of MCNTCOOH, BM and BM-MCNTCOOH conjugates were studied using FESEM. Figure 5 shows the SEM images of MCNTCOOH, BM and BMMCNTCOOH conjugates. The SEM image of MCNTCOOH shows the tubular structure of MCNTCOOH twining around each other (Figure 5a). Figure 5b shows the morphology of



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BM alone. When BM interacted with MCNTCOOH, the image (Figure 5c) became dim, and the protein adsorbed onto the surface of the nanotubes. More adsorption was seen at high concentrations of MCNTCOOH (Figure 5d). The SEM visualization of the BM adsorption on MCNTCOOH showed thicker, darker areas, indicating the formation of aggregates in the presence of high concentrations of MCNTCOOH. DLS analysis of MCNTCOOH-BM interaction DLS is one of the techniques that are routinely used for the analysis of NPs. Generally, the dH values of typical globular proteins are in the range of 1-10 nm. Here, DLS will benefit from the fact that the size of the protein will certainly increase as a result of the adsorption of the protein on the MCNTCOOH. Many research groups have used this technique to show the specific binding and the nonspecific adsorption of protein on NPs.33-35 Generally, a DLS measurement is used to obtain the diameter of particles with a spherical shape; however, it can also provide hydrodynamic diameters for nanotubes.36,37 The size distributions of the MCNTCOOH and MCNTCOOH-BM has been presented in Figure 6a. The MCNTCOOH showed an average dH of 125 nm. As shown in Figure 6a, upon mixing with BM, the MCNTCOOH showed a change in size distribution. The average particle size of the 125 nm peak changed in the presence of BM when compared to the water control solution. There was change in the intensity and size of the peak. We have used different amounts of protein (2-10 % in the intervals of 2), which is to be adsorbed on the surface of the MCNTCOOH. One interesting aspect of the study was that the change in the 125 nm peak was increasingly prominent with increasing amounts of BM. The size of MCNTCOOH increased from 125 nm to 132 nm in the presence of 2% of BM. However, the size of the MCNTCOOH reached 213 and 473 nm at 4 and 6% of BM, respectively. These increases in the size of MCNTCOOH with the addition of increasing amounts of BM clearly show that


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there is an interaction between BM and MCNTCOOH, which is consistent with the results of other studies. Therefore, as the concentration of BM increases, the size of the scattering entities progressively increases (Figure 6b). Upon further increases in the amount of BM, such as 8 and 10 %, we observed some dramatic and interesting changes in the MCNTCOOH-BM solution and in the DLS results. First, with respect to the MCNTCOOHBM solution appearance, we observed that up to 6 % of BM, the solution was almost homogenous in appearance. As presented in Figure 7, the dispersion stability of MCNTCOOH-BM suspension was largely maintained, even after many days. As shown in Figure 7a, b, when the MCNTCOOH-BM conjugates were initially prepared, both of them (2 % and 10 % BM, respectively) appeared to be homogenous in appearance. After one day (Figure 7c,d), there was no change in the suspension appearance with 2 % BM. However, in the suspension containing 10% BM, the MCNTCOOH colloids with adsorbed layers of BM started to settle. Even after many days, we did not observe any noticeable change in the 2% BM suspension, but we did observe aggregation of the MCNTCOOH due to the adsorption of BM with 10 % BM. Additionally, we observed that the peak at 125 nm shifted to 2748 and 2800, respectively, with 8 and 10 % BM in MCNTCOOH. Therefore, two aspects were very clear from the DLS results. First, the particle size increase caused by BM appeared to be dependent on the amount of protein. Second, in the presence of 8 and 10 % BM, the dispersion stability of MCNTCOOH decreased. The dH of MCNTCOOH increased approximately 7 nm upon incubation with 2 % of BM. Figure 6a clearly shows that the effective “size” of the MCNTCOOH increased dramatically as they became coated with the BM protein. The MCNTCOOH-BM complex that formed from this simple mixing process remained stable for many days (only up to 6 % BM in MCNTCOOH). Discussion 15 ACS Paragon Plus Environment


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An understanding of the mechanism of the binding of BM to the surface of NPs will certainly help clarify the use of NPs as molecular transporters and targeted drug delivery, as mentioned above. Studies of protein adsorption on NPs other than plasma proteins are important because of two main reasons. The first is the scarcity of literature on the behavior of proteins other than plasma proteins when adsorbed on NPs. Another main reason is that, considering the therapeutic importance of BM, it is quite essential to study its adsorption behavior on nanomaterials. BM is considered to be fairly absorbable in the body, and it does not lose its proteolytic activity or produce any major side effects. BM has also some anticancerous activities and can play a major role in promoting apoptotic cell death. In a study by Castell et al.,38 it was found that BM can retain its proteolytic activity in plasma and may be linked to the two antiproteinases of blood and to both alpha 2-macroglobulin and alpha1-antichymotrypsin. Peptides and proteins are poorly available biologically on oral administration, but it has been observed that their oral delivery can be improved by the use of carriers such as polymeric NPs. Therefore, there is currently a need to assess the mechanism of the interaction of proteins that have high therapeutic importance by using nanomaterials. Multiple spectroscopic methods were exploited to unveil the mechanism of binding and the structural variations in BM due to its interaction with MCNTCOOH. We found a considerable quenching of fluorescence without a shift in the λmax, which indicated an unperturbed microenvironment around Trp as a result of complex formation. The mechanism of quenching can be static with complex formation between the fluorophore and the quencher. The absorption peak around 280 nm provides information about Trp and Tyr residues. We did not observe any shift in λmax from the fluorescence quenching study, which showed almost no change in the polarity of environment around Trp. Therefore, the fluorescence results clearly indicate the existence of a binding interaction between 16 ACS Paragon Plus Environment

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MCNTCOOH and BM, which in turn indicates complex formation between the MCNTCOOH and BM with a retention of polarity. This, in summary, implies that the presence of MCNTCOOH does not result in a drastic change in the structure of BM. It is quite clear from the thermal stability measurements of BM that upon interaction with MCNTCOOH, thermal stability is reduced only nominally. The comparison of the unfolding of BM in the presence of different concentrations of MCNTCOOH clearly indicates that the protein does not seem to alter its thermal stability drastically upon interaction with MCNTCOOH. Therefore, the thermal stability of the protein has been reduced slightly, and it exists in a somewhat more flexible folded structure compared to native BM (Figure 3). Secondary and tertiary CD analyses show slightly perturbed BM structures after the addition of MCNTCOOH. Therefore, the slight structural disturbance of BM due to its interaction with MCNTCOOH is clearly witnessed from CD and from the spectroscopic results. Furthermore, FESEM showed a considerable adsorption of protein on the surface of BM, which further increased with increasing MCNTCOOH concentration. Therefore, from the results, it is quite clear that MCNTCOOH are able to perturb the structure of BM. Nevertheless, the perturbation is not very drastic either structurally or thermally. Samples of MCNTCOOH pre-incubated with BM exhibited larger dH values than did MCNTCOOH alone, which is consistent with the adsorption of BM onto the surface of MCNTCOOH. One important aspect to note in the experiment was the lack of any particular trend in the values of Tm and uniform protein adsorption along the MCNTCOOH in the FESEM pictures with increasing concentrations of MCNTCOOH. Additionally, we did not observe. The possible reason can be that the functionalization of CNTs may not be homogenous and may contain areas in which more COOH groups bind more protein than the less functionalized areas.13



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The studies of Ge et al.9 revealed that among the hydrophobic residues such as Tyr, Trp and Phe, only Tyr and Phe were observed in the contact with the surface of SWCNT directly, whereas Trp residues made little contribution. Therefore, this finding indicates that the BM-MCNTCOOH interactions may be governed by hydrophobic residues. The adsorption of MCNTCOOH on the hydrophobic amino acid residues is through ᴨ-ᴨ stacking interactions. Zuo et al.26,

27

used molecular dynamics (MD) simulations to study the

interactions between several proteins and an unmodified carbon nanotube. Their MD simulations indicated that CNTs can enter into the hydrophobic core of proteins and form stable complexes by hydrophobic and ᴨ-ᴨ stacking interactions. In the present study, we used MCNTCOOH, where the COOH group present in the CNT may play a role in the binding interaction between BM and the CNT. Therefore, the basic forces involved in the binding of proteins to non-functionalized CNTs are ᴨ-ᴨ stacking interactions and hydrophobic interactions, whereas electrostatic interactions are also present in functionalized CNTs. Our results are consistent with the studies of Wijaya et al.,39 who showed that in carboxylated multiwalled carbon nanotubes (MWCNTs), other interactions, such as electrostatic and hydrogen bonding, are assumed to contribute to a stronger protein-nanotube interaction. We used the pH 7.0, which is lower than the isoelectric point (P.I.) of BM, 10.1. In such a scenario, apart from hydrophobic and ᴨ-ᴨ interactions, there can be electrostatic interactions through the protonated amino moieties with the carboxylic group present in the functionalized nanotube. The P.I. of BM lies in the basic range because the interaction pH is a physiological pH. Therefore, the BM molecule has a significant amount of positively charged functional groups attached to the surface, which can result in attractive columbic interaction between the negatively charged MCNTCOOH and BM. Therefore, the presence of MCNTCOOH provides a slightly destabilizing environment where the stability of protein may be slightly 18 ACS Paragon Plus Environment

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compromised. Moreover, it is quite clear from the DLS measurements that the size of the scattering entities progressively increased as the BM concentration is increased. There was a drastic change in the dH values at high BM concentrations. One possibility is that at neutral pH, BM (which can exist as a polycation) can adsorb on the MCNTCOOH and can progressively neutralize the negative surface charge. At higher BM concentrations, it may eventually promote flocculation. The increase in the concentration of adsorbed BM may have induced the aggregation of MCNTCOOH (Scheme 1). The possible reason for this aggregation may be protein-protein interactions among the proteins adsorbed on the surface of MCNTCOOH. We do not have exact proof of this; therefore, this is our assumption based on the literature results. The interaction of nanotubes with proteins might result in the alteration of protein conformation and the exposure of new epitopes on the protein surface or the perturbation of the normal protein function.40,41 This is of particular interest and requires a separate set of experiments to further understand the mechanism, which is our future plan. Conclusion The interaction of CNTs with proteins needs to be explored to design new functions and applications for carbon nanotubes, especially for drug delivery. Our current study provides a systematic spectroscopic analysis of BM interaction with MCNTCOOH, which shows that hydrophobic and ᴨ-ᴨ interactions as well as electrostatic interactions exist between residues of BM and the nanotube. Several key issues applicable to carbon nanotube and biomolecular interactions have been highlighted in our present study. First, it clearly establishes the different types of interactions possible between biomolecules and functionalized CNTs. Additionally, it has quite clearly revealed the role of both the protein and nanotubes in the complex formation process. Interestingly, we could not find any direct correlation between concentration of MCNTCOOH and the stability of BM. Additionally, DLS provided some 19 ACS Paragon Plus Environment


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interesting results regarding the role of the concentration of protein in the aggregation of MCNTCOOH. Our further studies on these lines will certainly be helpful in providing a deep understanding of the various factors contributing to protein and CNTs interactions. ACKNOWLEDGEMENT We are grateful for the financial support from the Department of Biotechnology (DBT), New Delhi, through the grant ref./file no. BT/PR5287/BRB/10/1068/2012. I J is grateful to CSIR, New Delhi for awarding the Senior Research Fellowship (SRF). Supporting Information Available: This supporting information is available free of charge via the Internet at http://pubs.acs.org. Figure 1S: Stern-Volmer plot of fluorescence quenching for BM in the presence of MCNTCOOH. Figure 2S: Fluorescence intensity of only MCNTCOOH, Figure 3S: The UV–visible spectra of only the CNTs. Figure 4S: The background subtracted UV–visible spectra of each CNT solution from the respective raw data of BM. Table 1S: Stern-Volmer quenching constants of the MCNTCOOH-BM. Table 2S: Hydrodynamic diameter (dH) values of the MCNTCOOH at various concentrations.


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FIGURE CAPTIONS Figure 1. (a) Fluorescence spectra of BM showing the quenching of fluorescence intensity with increasing concentration (0, 0.5-5.0 mg/L in the intervals of 0.5) of MCNTCOOH at 25 °C. Spectra obeyed Stern–Volmer rule up to 3.0 mg/L, after which the quenching percentage seems to be higher. (b) Stern-Volmer plot of fluorescence quenching for BM in the presence of MCNTCOOH (0, 0.5-3.0 mg/L) at 25 °C. (c) Double logarithm regression curve of MCNTCOOH-BM at 25 °C. Figure 2. The UV–visible spectra of BM before and after interaction with increasing concentrations of MCNTCOOH (0, 0.5-4.0 mg/L at 0.5 mg/L intervals) 25 °C. Enhanced absorbance of BM in the presence of MCNTCOOH during interaction is illustrated. Figure 3. The variation in Tm values of BM in buffer (black) and in the presence of various concentrations of MCNTCOOH, which is obtained from fluorescence analysis with only BM (black) 0.5 mg/L (red), 1.0 mg/L (green), 1.5 mg/L (blue), 2.0 mg/L (cyan), 2.5 mg/L (magenta), 3.0 mg/L (yellow), 3.5 mg/L (dark yellow) and 4.0 mg/L (navy). Figure 4. (a) Far-UV CD spectra of BM in the absence as well as in the presence of MCNTCOOH. The BM concentration was 500 mg/L. The concentrations of MCNTCOOH were 0 (black), 0.5 (red), 2.5 (green) and 4 mg/L (blue). (b) Near-UV CD spectra of BM in the absence as well as in the presence of MCNTCOOH. The BM concentration was 500 mg/L. The concentrations of MCNTCOOH were 0 (black), 0.5 (red), 2.5 (green) and 4 mg/L (blue). Figure 5. (a) FESEM image of MCNTCOOH (b) BM alone (c) MCNTCOOH-BM conjugate (low concentration

of

MCNTCOOH)

(d) MCNTCOOH-BM conjugate (high

concentration of

MCNTCOOH). Figure 6. (a) Plot of the intensity distribution of MCNTCOOH in the presence of different concentrations of BM. (b) DLS correlation plots relative to mg/L dispersions of MCNTCOOH with added BM, at 25 °C. All samples were prepared by adding 30 (Red), 60 (green), 90 (blue), 120 (cyan) and 150 (magenta) µL of BM solution or dd water (black) into 1500 µL of MCNTCOOH solution. 26 ACS Paragon Plus Environment

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Figure 7. Dispersion state of MCNTCOOH in the presence of (a) a low concentration of BM (30 µL of BM in 1500 µL of MCNTCOOH solution) and (b) a high concentration of BM (150 µL of BM in 1500 µL of MCNTCOOH solution) immediately after preparation. Dispersion state of MCNTCOOH in the presence of (c) a low concentration of BM (30 µL of BM in 1500 µL of MCNTCOOH solution) and (d) a high concentration of BM (150 µL of BM in 1500 µL of MCNTCOOH solution) after 1 day of preparation. Dispersion state of MCNTCOOH in the presence of (e) a low concentration of BM (30 µL of BM in 1500 µL of MCNTCOOH solution) and (f) a high concentration of BM (150 µL of BM in 1500 µL of MCNTCOOH solution) after many days of preparation. Scheme 1. Schematic depiction of the influence of protein concentration on the MCNTCOOH aggregation as a result of protein adsorption.



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600

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(b)

Increasing MCNTCOOH concentration

3.0

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400

2.0 200

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1.0 0 350

400

450

0.0

0.5

Wavelength (nm)

1.0

1.5

2.0

2.5

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Concentration (mg/L)

0.4

(c) 0.2

0.0

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-0.4

-0.6 -0.4

-0.2

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0.6

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Figure 1


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1.2

1.0

0.8

0.6

0.4

0.2 250

300

350

Figure 2



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70

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Figure 3

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-6

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240

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Figure 4


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(a)

(c)

(b)

(d)

Figure 5



19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

(a)

100

1000

dH (nm) (b)

dH 1.0(nm) 0.9 0.8

Increasing BM concentration 150 µL of BM 120 µL of BM

0.7 Correlation Coefficient

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NO BM 0.6

30 µL of BM 0.5

60 µL of BM

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0.2 0.1 0.0 0.1

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Figure 6


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Figure 7 33 ACS Paragon Plus Environment


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Scheme 1


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Table of Contents Deciphering the Interactions of Bromelain with Carbon Nanotubes: Role of Protein as Well as Carboxylated Multiwalled Carbon Nanotubes in a Complexation Mechanism Indrani Jha and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi-110 007



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Table of Content Deciphering the Interactions of Bromelain with Carbon Nanotubes: Role of Protein as Well as Caboxylated Multiwalled Carbon Nanotubes in Complexation Mechanism Indrani Jha and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi-110 007

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Deciphering the Interactions of Bromelain with ... - ACS Publications

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