J BIOCHEM MOLECULAR TOXICOLOGY Volume 28, Number 5, 2014
Spectroscopic Investigations on the Interaction between Carbon Nanotubes and Catalase on Molecular Level Jin Guan,1 Jingping Dai,2 Xingchen Zhao,1 Chunhua Liu,1 Canzhu Gao,1 and Rutao Liu1 1 Shandong
Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, China–America CRC for Environment & Health, Shandong Province, Jinan 250100, People’s Republic of China; E-mail: [email protected] 2 Shandong Environmental Monitoring Centre, Jinan, Shandong 250101, People’s Republic of China Received 27 November 2013; revised 19 January 2014; accepted 1 February 2014
ABSTRACT: The interactions between welldispersed multiwalled carbon nanotubes (MWCNTs) and catalase (CAT) were investigated. The activity of CAT was inhibited with the addition of MWCNTs. After deducting the inner filter effect, the fluorescence spectra revealed that the tryptophan (Trp) residues were exposed and the fluorescence intensities of CAT increased with the increase in the MWCNTs concentration. At the same time, the environment of the Trp residues became more hydrophobic. The results of UV–vis absorption spectroscopy and CD spectra indicated that the secondary structure of CAT had been changed, and the amino acid residues were located in a more hydrophobic environment. Meanwhile, the UV– vis spectra indicated that the conformation of the heme porphyrin rings was changed. The microenvironment of CAT activity sites may be interfered by MWCNTs. This research showed that MWCNTs could not only contribute to the conformational changes of protein C 2014 Wiley but also change the enzyme function. Periodicals, Inc. J. Biochem. Mol. Toxicol. 28:211–216, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21555
KEYWORDS: Catalase; Enzyme Activity; Multiwalled Carbon Nanotubes; Spectrum Technology; Toxic Evaluation
Correspondence to: Rutao Liu. Contract Grant Sponsor: National Natural Science Foundation of China. Contract Grant Numbers: 20875055, 21277081. Contract Grant Sponsor: Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China. Contract Grant Number: 708058. Contract Grant Sponsor: Independent Innovation Foundation of Shandong University Natural Science Projects. Contract Grant Number: 2012DX002. C 2014 Wiley Periodicals, Inc.
INTRODUCTION Carbon nanotubes (CNTs) have attracted human beings’ attention since they were discovered by Iijima owing to their fascinating electronic, optical, thermal, and mechanical properties [1]. The wide application of CNTs is on the increase [2]. Thus, the potential harmful effects of CNTs on human health and the environment have been attracted serious concerns from academia, governments, and our society [3, 4]. In recent years, the most common method to study toxic effects caused by CNTs is biological toxicity experiments. There have been many animal toxicology studies [5]. However, the information about the influence of proteins is very limited. Therefore, it is significant to investigate the mechanism of toxicity of CNTs at a molecular level [5]. Studies on the interactions between CNTs and proteins on the molecular level have been reported. Azamian et al. discovered that ferritin were firmly attached onto the surface of SWCNTs, which dramatically increased the solubility of single-walled carbon nanotubes (SWCNTs) [6]. Horn et al. investigated the noncovalent interaction between lysozyme (LSZ) and SWCNT. They suggested that there was a direct molecular interaction between LSZ and SWCNT through the indole moiety of tryptophan (Trp) and the aromatic sidewall of SWCNT [7], but little work has been focused on the change in protein conformation [5, 8]. Catalase (CAT; H2 O2 :H2 O2 oxidoreductase, EC 1.11.1.6; CAT) is a tetrameric iron porphyrin protein that catalyzes the dismutation of H2 O2 to one molecule of H2 O and a half molecule of O2 [9–11]. CAT is composed of four identical subunits, and each of the four active sites contains a ferriporphyrin IX as a prosthetic group [12]. According to low-angle X-ray scattering, bovine liver catalase was found as an ellipsoidal molecule [12]. Five hundred six amino acid residues 211
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constitute one subunit of CAT [13]. CAT has been widely used in many fields including the food industry and enzymatic oxidation reaction [14]. In this paper, we evaluated the mechanism of multiwalled carbon nanotubes (MWCNTs) interaction with CAT by the fluorescence spectroscopy, UV–vis absorption spectroscopy, and circular dichroism (CD) spectroscopy. This study not only provides the impact of MWCNTs on the structure and function of CAT but also indicates the threat from MWCNTs to the human health in vivo.
MATERIALS AND METHODS
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equipped with a 10-mm quartz cell. UV–vis spectra of CAT in the absence and presence of MWCNTs were recorded in the range of 190–420 nm at 298 K with 1.0 cm × 1.0 cm quartz cells used for samples holding.
CD Measurements CD spectra were operated over the wavelength range of 190–250 nm with a J-810 CD spectrometer (Jasco, Tokyo, Japan) using a quartz cell with a path length of 10 mm. The scanning speed was set at 200 nm min−1 . The each spectrum was the average of two scans. The conformation of protein secondary structure was computed through Yang.jwr software [15].
Reagents CAT (from bovine liver; Sigma Chemical Co., St. Louis, MO, USA) was dissolved in ultrapure water to form a 4 × 10−6 mol L−1 solution. Phosphate buffer (0.1 mol L−1 ; a mixture of NaH2 PO4 and Na2 HPO4 solution) was used to control pH at about 7.4 and stored at 4°C to avoid metamorphosis. NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O were of two analytical reagents and obtained from Tianjin Damao Chemical (Tianjin, China). MWCNTs (Shenzhen Nanotech Port Company Ltd., Shenzhen, China) were dissolved in ultrapure water to form a 0.1 g L−1 solution. Ultrapure water (18.25 M) was used throughout the experiments.
CAT Activity Measurement CAT catalyzes the decomposition of hydrogen peroxide into water and oxygen. The H2 O2 and phosphate buffer were mixed in a 3-mL quartz cell and used as a reference solution. The activity of CAT was measured spectrophotometrically by monitoring the decrease of hydrogen peroxide (49.3 mM) absorption at 240 nm [16, 17]. The data were recorded every 30 s for a period of 10 min. The measurements were conducted at room temperature (about 25°C) and repeated three times.
RESULTS Apparatus and Methods Fluorescence Measurements All fluorescence spectra were recorded on an F-4600 fluorophotometer (Hitachi, Tokyo, Japan) equipped with a 10-mm quartz cell and a 150-W xenon lamp. The photo multiplier tube voltage was 650 V, and the excitation and emission slit widths were set at 5.0 nm. The excitation wavelength was set at 280 nm. The scan speed was 1200 nm min−1 . The fluorescence measurements were performed as follows: 1 mL phosphate buffer, different concentrations of MWCNTs, and 1 mL of the CAT solution was added in a series of 10 mL colorimetric tubes. Ultrapure water was added to dilute the mixture to the scaled mark. Each sample was stabilized at 298 K for 30 min before the measurement. The emission wavelength scans ranged from 290 to 450 nm.
UV–Vis Absorption Measurements All UV–vis absorption spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan)
Influence of MWCNTs Dose on the Fluorescence Intensity of CAT Fluorescence Spectroscopy CAT contains three intrinsic fluorophores: Trp, tyrosine (Tyr), and phenylalanine (Phe). The fluorescence intensity of Trp is the strongest. There are six Trp residues (Trp14, Trp142, Trp182, Trp185, Trp276, and Trp302) in CAT, which can be used as intrinsic fluorophores [19]. The high sensitivity of Trp to its microenvironment is a significant feature of intrinsic fluorescence of proteins. To examine the interactions between MWCNTs and CAT, the fluorescence spectra were measured in PB buffer with pH 7.4. From Figure 1A, a gradual increase in the MWCNTs concentration in the solutions of CAT resulted in a decrease of fluorescence intensity and a slight blueshift at the maximum emission wavelength. However, the absorbance of the system will be increased with the concentration of the protein– MWCNTs complex, which causes the inner filter effect (IFE). The IFE from ligand refers to the absorbance or J Biochem Molecular Toxicology
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7050
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7000 6950 6900 6850 6800 6750 6700 0
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Wavelength (nm) FIGURE 1. Fluorescence spectra of the CAT–MWCNTs system. Conditions : T = 298 K, kex = 280 nm, pH 7.4; CCAT = 4.0 × 10−6 mol/L, CCNTs (mg/L) 1–5: 0, 1.0, 2.0, 3.0, and 4.0. A1 : Fluorescence spectra of the CAT–MWCNTs system. A2 : Fluorescence spectra of the CAT–MWCNTs system after correction of IFE for A1 . B: Plot of fluorescence increasing for CAT at 332 nm after the correction of IFE for part A.
optical dispersion of light at the emission wavelength of the fluorescer [18]. MWCNTs can quench the fluorescence signal of the protein. So, we need to eliminate the influence of the IFE, which could interfere with the results. This effect will lead to the deviation of a fluorescence curve, resulting in a nonlinear relationship between the observed fluorescence intensity and the concentration of the fluorophore. Generally, IFE is composed of two parts: the primary inner filter effect (pIFE) is the absorption of excitation radiation, and the secondary inner filter effect (sIFE) is the absorption of emission radiation. According to Gu and Kenny [19], IFE can be calculated as shown in Eq. (1): Fideal (λex , λem ) = Fobs (λex , λem )C F p (λex )C Fs (λem ) ≈ Fobs (λex , λem )10(Aem +Aex )/2 J Biochem Molecular Toxicology
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(1)
where CFp refers to the correction factor for pIFE, depending on the total absorbance of the sample at λex , whereas CFs refers to the correction factor for sIFE, depending on the total absorbance of the sample at λem . Aex and Aem are the absorbance of samples at the fluorescence excitation and emission wavelengths, respectively. Fobs is the observed fluorescence value, and Fideal is the corrected fluorescence intensity. The corrected plots are shown in Figure 1B. The fluorescence intensity increased as the MWCNTs concentration increased. These results indicate that there are interactions between MWCNTs and CAT. The structure of CAT is destroyed by addition of the MWCNTs, while the more Trp residues are exposed to the aqueous environment. The slight blueshift is shown in Figure 1A, which suggests a more hydrophobic environment of the Trp residues [20, 21].
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0.13
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FIGURE 2. UV–vis absorption spectra of CAT with different concentrations of MWCNTs. Conditions: CCAT = 4 × 10−6 mol L−1 , CMWCNTs (mg/L) 1–5: 0, 1.25, 3.5, 7,5, and 10, respectively; pH 7.4, T = 293 K.
Investigations on the Conformational Changes of CAT UV–Vis Absorption Spectroscopy UV–visible absorption spectroscopy is used to analyze the structural changes in a protein and study the protein–ligand complex formation [22]. CAT has a strong absorption band at around 208 nm, resulting from the π → π * transition in the polypeptide backbone structure C=O [23, 24]. CAT also has an absorption band at 405 nm, which reflects the π → π * transition of heme porphyrin rings [25]. In addition to this, the weak one at about 278 nm can offer us the information about the absorption of aromatic amino acids (Trp, Tyr, and Phe) [26]. As shown in Figure 2, the intensity of the peak at 280 nm increased upon adding the MWCNTs, indicating that the MWCNTs increased the hydrophobicity of the microenvironment of the aromatic amino acids, which corresponded to the results of fluorescence spectra. While the intensity of the peak at 405 nm increased, indicating
that the conformation of the heme porphyrin rings was changed [27]. Moreover, the change may influence the activity of CAT. To further comprehend the process of the aromatic amino acids microenvironment changes, we take an αhelix as an example in Figure 3. In general, the amide moieties that are on the surface of the CAT molecule can only contact with water molecules; the others are in the hydrophobic pocket. When MWCNTs were added to the system, fewer water molecules could touch the amide moieties, which were blocked by MWCNTs. As a result, the hydrophobicity of the microenvironment of the amide moieties was increased, which may lead to the decrease in the activity of CAT. The inference will be proved by the following experiments.
Circular Dichroism CD spectra can be used to provide information about secondary protein conformational changes [28, 29]. There are two kinds of wavelength regions,
FIGURE 3. Schematic process for the microenvironment changes of amide moieties, taking an R-helix as an example.
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FIGURE 4. Far-CD spectra of native CAT and CAT–MWCNTs mixtures. Conditions: CCAT = 4 × 10−6 mol L−1 , CMWCNTs (mg/L) 1–3: 0, 1.25, 3.5, respectively. pH 7.4, T = 293 K.
TABLE 1. Influence of MWCNTs on the Secondary Structure of CAT Curve 1 2 3
α-Helix (%)
β-Sheet (%)
28.0 27.4 23.8
33.6 36.3 39.0
According to Fig. 4. Conditions are CCAT = 4 × 10−6 mol L−1 , CMWCNTs (mg/L) 1–3: 0, 1.25, 3.5; pH 7.4, T = 293 K.
far-UV (200–260 nm) and near-UV (260–320 nm). FarUV CD spectra can be used to determine the secondary structure of a protein, such as α-helix, β-turn, β-pleated sheet, and random coil [30]. CD spectra of far-UV regions are shown in Figure 4. It can be seen from Figure 4 that the two negative double humped peaks at 208 and 222 nm reflect the α-helical structure of protein [31]. The results of the portions of the secondary structures by Jasco secondary structure manager software are presented in Table 1. When MWCNTs are added to the system, it is concluded that the MWCNTs bind to the amino acid residues [31, 32], which suggests a more hydrophobic environment of the amino acid residues. At the same time, MWCNTs decreased the amount of α-helix, leading to an increase in the exposure of the Trp residues [30], which is consistent with the above results of fluorescence spectroscopy and UV absorption spectroscopy. What’s more, it may affect the physiological function of CAT [33].
Effect of MWCNTs on CAT Activity From the above investigations, we have known that MWCNTs can affect the structure of CAT. The structure of an enzyme has something to do with the function. So the structural variation may have an effect on its normal physiological function [34]. To further inJ Biochem Molecular Toxicology
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FIGURE 5. Effects of different concentrations of MWCNTs on the activity of CAT. Conditions: pH 7.4, c (CAT) = 4.0 × 10−7 mol L−1 , c(H2 O2 ) = 49.3 mmol L−1 , c(MWCNTs) (mg L−1 ) , a–c: 0, 10, 20, T = 290 K.
vestigate the effect of physiological function by MWCNTs, we measured the activity of CAT in the presence of different concentrations of MWCNTs (0–20 mg/L). As shown in Figure 5, the slope of the straight line defines the CAT activity [33]. We can see the obvious effects on the activity of CAT caused by MWCNTs. There are some amino acid residues around the CAT activity site. e.g., heme [34, 35]. Based on Figure 3 and above results, the hydrophobicity of the microenvironment of the amide acid residues was increased. The microenvironment of CAT activity site may be changed. As a result, the binding of MWCNTs into the enzyme cavity influenced the microenvironment of CAT activity site, which caused the decrease in the CAT activity [14]. The above results imply that MWCNTs can inhibit the CAT activity.
DISCUSSION In this work, we investigated the interactions between MWCNTs and CAT by using spectroscopic techniques under simulative physiological conditions. The results revealed that MWCNTs could bind to the amino acid residues of CAT, leading to a more hydrophobic environment of the amino acid residues. So, the CAT activity was inhibited after binding with MWCNTs. MWCNTs decreased the amount of α-helix, when MWCNTs were added, leading to increasing the exposure of the internal Trp residues. So, the fluorescence intensities of CAT increased with the increase in the MWCNTs concentration. Besides, the UV–vis absorption spectroscopy results also showed that the conformation of the heme porphyrin rings was changed. Based on the relationship between structure and function, this work mainly offers the basic spectroscopic methods for the toxicological study of MWCNTs
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at the molecular level. The above results may provide essential data for the safe use of nanomaterials and development of environment policies.
REFERENCES 1. Iijima S. Helical microtubules of graphitic carbon. Nature 1991;354:56–58. 2. Magrez A, Kasas S, Salicio V, Pasquier N, Seo JW, Celio M, Catsicas S, Schwaller B, Forro´ L. Cellular toxicity of carbon-based nanomaterials. Nano Lett 2006;6(6):1121– 1125. 3. Robert F. Nanomaterials show signs of toxicity. Science 2003;300(11):243–243. 4. Brumfiel G. Nanotechnology: a little knowledge. Nature 2003;424(6946):246–248. 5. Zhao X, Liu R, Chi Z, Teng Y, Qin P. New insights into the behavior of bovine serum albumin adsorbed onto carbon nanotubes: comprehensive spectroscopic studies. J Phys Chem B 2010;114(16):5625–5631. 6. Azamian BR, Davis JJ, Coleman KS, Bagshaw CB, Green ML. Bioelectrochemical single-walled carbon nanotubes. J Am Chem Soc 2002;124(43):12664–12665. 7. Horn DW, Tracy K, Easley CJ, Davis VA. Lysozyme dispersed single-walled carbon nanotubes: interaction and activity. J Phys Chem C 2012;116(18):10341–10348. 8. Zhao X, Liu R. Recent progress and perspectives on the toxicity of carbon nanotubes at organism, organ, cell, and biomacromolecule levels. Environ Int 2012;40:244–255. 9. Giustarini D, Dalle-Donne I, Tsikas D, Rossi R. Oxidative stress and human diseases: origin, link, measurement, mechanisms, and biomarkers. Crit Rev Clin Lab Sci 2009;46(5–6):241–281. 10. Iannone MF, Rosales EP, Groppa MD, Benavides MP. Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf disks exposed to cadmium. Protoplasma 2010;245(1–4):15–27. 11. Yoshimoto M, Sakamoto H, Yoshimoto N, Kuboi R, Nakao K. Stabilization of quaternary structure and activity of bovine liver catalase through encapsulation in liposomes. Enzyme Microbial Technol 2007;41(6):849–858. 12. Malmon AG. Small angle x-ray scattering studies of the size, shape, and hydration of catalase. Biochim Biophys Acta 1957;26(2):233–240. 13. Chang JY, Schroeder W. Reaction of 3-amino-1:2: 4triazole with bovine liver catalase and human erythrocyte catalase. Arch Biochem Biophys 1972;148(2):505–508. 14. Teng Y, Zhang H, Liu R. Molecular interaction between 4aminoantipyrine and catalase reveals a potentially toxic mechanism of the drug. Molec BioSyst 2011;7(11):3157– 3163. 15. Yang JT, Wu CS, Martinez HM. Calculation of protein conformation from circular dichroism. Meth Enzymol 1986;130:208–269. 16. Naik G, Priyadarsini K, Satav J, Banavalikar M, Sohoni D, Biyani M, Mohan H. Comparative antioxidant activity of individual herbal components used in Ayurvedic medicine. Phytochemistry 2003;63(1):97–104. 17. Jiang Y, Wong J, Pi Z, Ng T, Wang C, Hou J, Chen R, Niu H, Liu F. Stimulatory effect of components of rose flowers on catalytic activity and mRNA expression of superoxide dismutase and catalase in erythrocytes. Environ Toxicol Pharmacol 2009;27(3):396–401.
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18. Zong W, Cao S, Xu Q, Liu R. The use of outer filter effects for Cu2+ quantitation: a unique example for monitoring nonfluorescent molecule with fluorescence. Luminescence 2012;27(4):292–296. 19. Gu Q, Kenny JE. Improvement of inner filter effect correction based on determination of effective geometric parameters using a conventional fluorimeter. Anal Chem 2008;81(1):420–426. 20. Murthy M, Reid III TJ, Sicignano A, Tanaka N, Rossmann MG. Structure of beef liver catalase. J Molec Biol 1981;152(2):465–499. 21. Walton AG, Maenpa FC. Application of fluorescence spectroscopy to the study of proteins at interfaces. J Colloid Interf Sc 1979;72(2):265–278. 22. Wang Y-Q, Chen T-T, Zhang H-M. Investigation of the interactions of lysozyme and trypsin with biphenol A using spectroscopic methods. Spectrochim Acta A 2010;75(3):1130–1137. 23. Zhong J, Song L, Meng J, Gao B, Chu W, Xu H, Luo Y, Guo J, Marcelli A, Xie S. Bio–nano interaction of proteins adsorbed on single-walled carbon nanotubes. Carbon 2009;47(4):967–973. 24. Li Z, Abramavicius D, Zhuang W, Mukamel S. Twodimensional electronic correlation spectroscopy of the nπ * and π π * protein backbone transitions: a simulation study. Chem Phys 2007;341(1):29–36. 25. Myer YP. Conformation of cytochromes II. Comparative study of circular dichroism spectra, optical rotatory dispersion, and absorption spectra of horse heart cytochrome c. J Biol Chem 1968;243(9):2115–2122. 26. Chi Z, Liu R, Zhang H. Noncovalent interaction of oxytetracycline with the enzyme trypsin. Biomacromolecules 2010;11(9):2454–2459. 27. Liu Y, Zhang L, Liu R, Zhang P. Spectroscopic Identification of interactions of Pb2+ with bovine serum albumin. J Fluoresc 2012;22(1):239–245. 28. Kelly SM, Jess TJ, Price NC. How to study proteins by circular dichroism. Biochim Biophys Acta 2005;1751(2):119– 139. 29. Shen X-C, Liang H, He X-W, Wang XS. Recent trends and spectroscopic methods for analysis of the protein conformation with circular dichroism. Chin J Anal Chem 2004;32(3):388–394. 30. Hennecke J, Sillen A, Huber-Wunderlich M, Engelborghs Y, Glockshuber R. Quenching of tryptophan fluorescence by the active-site disulfide bridge in the DsbA protein from Escherichia coli. Biochemistry 1997;36(21):6391– 6400. 31. Lu J-Q, Jin F, Sun T-Q, Zhou X-W. Multi-spectroscopic study on interaction of bovine serum albumin with lomefloxacin–copper (II) complex. Int J Biol Macromole 2007;40(4):299–304. 32. Huang BX, Kim H-Y, Dass C. Probing three-dimensional structure of bovine serum albumin by chemical crosslinking and mass spectrometry. J Am Soc Mass Spectrom 2004;15(8):1237–1247. 33. Yang B, Liu R, Hao X, Wu Y, Du J. The interactions of glutathione-capped CdTe quantum dots with trypsin. Biol Trace Elem Res 2012;146(3):396–401. 34. Lavery R, Sacquin-Mora S. Protein mechanics: a route from structure to function. J Biosci 2007;32(1): 891–898. 35. Montavon P, Kukic KR, Bortlik K. A simple method to measure effective catalase activities: optimization, validation, and application in green coffee. Anal Biochem 2007;360(2):207–215. J Biochem Molecular Toxicology
DOI 10.1002/jbt
Spectroscopic Investigations on the Interaction between Carbon Nanotubes and Catalase on Molecular Level Jin Guan,1 Jingping Dai,2 Xingchen Zhao,1 Chunhua Liu,1 Canzhu Gao,1 and Rutao Liu1 1 Shandong
Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, China–America CRC for Environment & Health, Shandong Province, Jinan 250100, People’s Republic of China; E-mail: [email protected] 2 Shandong Environmental Monitoring Centre, Jinan, Shandong 250101, People’s Republic of China Received 27 November 2013; revised 19 January 2014; accepted 1 February 2014
ABSTRACT: The interactions between welldispersed multiwalled carbon nanotubes (MWCNTs) and catalase (CAT) were investigated. The activity of CAT was inhibited with the addition of MWCNTs. After deducting the inner filter effect, the fluorescence spectra revealed that the tryptophan (Trp) residues were exposed and the fluorescence intensities of CAT increased with the increase in the MWCNTs concentration. At the same time, the environment of the Trp residues became more hydrophobic. The results of UV–vis absorption spectroscopy and CD spectra indicated that the secondary structure of CAT had been changed, and the amino acid residues were located in a more hydrophobic environment. Meanwhile, the UV– vis spectra indicated that the conformation of the heme porphyrin rings was changed. The microenvironment of CAT activity sites may be interfered by MWCNTs. This research showed that MWCNTs could not only contribute to the conformational changes of protein C 2014 Wiley but also change the enzyme function. Periodicals, Inc. J. Biochem. Mol. Toxicol. 28:211–216, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21555
KEYWORDS: Catalase; Enzyme Activity; Multiwalled Carbon Nanotubes; Spectrum Technology; Toxic Evaluation
Correspondence to: Rutao Liu. Contract Grant Sponsor: National Natural Science Foundation of China. Contract Grant Numbers: 20875055, 21277081. Contract Grant Sponsor: Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China. Contract Grant Number: 708058. Contract Grant Sponsor: Independent Innovation Foundation of Shandong University Natural Science Projects. Contract Grant Number: 2012DX002. C 2014 Wiley Periodicals, Inc.
INTRODUCTION Carbon nanotubes (CNTs) have attracted human beings’ attention since they were discovered by Iijima owing to their fascinating electronic, optical, thermal, and mechanical properties [1]. The wide application of CNTs is on the increase [2]. Thus, the potential harmful effects of CNTs on human health and the environment have been attracted serious concerns from academia, governments, and our society [3, 4]. In recent years, the most common method to study toxic effects caused by CNTs is biological toxicity experiments. There have been many animal toxicology studies [5]. However, the information about the influence of proteins is very limited. Therefore, it is significant to investigate the mechanism of toxicity of CNTs at a molecular level [5]. Studies on the interactions between CNTs and proteins on the molecular level have been reported. Azamian et al. discovered that ferritin were firmly attached onto the surface of SWCNTs, which dramatically increased the solubility of single-walled carbon nanotubes (SWCNTs) [6]. Horn et al. investigated the noncovalent interaction between lysozyme (LSZ) and SWCNT. They suggested that there was a direct molecular interaction between LSZ and SWCNT through the indole moiety of tryptophan (Trp) and the aromatic sidewall of SWCNT [7], but little work has been focused on the change in protein conformation [5, 8]. Catalase (CAT; H2 O2 :H2 O2 oxidoreductase, EC 1.11.1.6; CAT) is a tetrameric iron porphyrin protein that catalyzes the dismutation of H2 O2 to one molecule of H2 O and a half molecule of O2 [9–11]. CAT is composed of four identical subunits, and each of the four active sites contains a ferriporphyrin IX as a prosthetic group [12]. According to low-angle X-ray scattering, bovine liver catalase was found as an ellipsoidal molecule [12]. Five hundred six amino acid residues 211
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constitute one subunit of CAT [13]. CAT has been widely used in many fields including the food industry and enzymatic oxidation reaction [14]. In this paper, we evaluated the mechanism of multiwalled carbon nanotubes (MWCNTs) interaction with CAT by the fluorescence spectroscopy, UV–vis absorption spectroscopy, and circular dichroism (CD) spectroscopy. This study not only provides the impact of MWCNTs on the structure and function of CAT but also indicates the threat from MWCNTs to the human health in vivo.
MATERIALS AND METHODS
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equipped with a 10-mm quartz cell. UV–vis spectra of CAT in the absence and presence of MWCNTs were recorded in the range of 190–420 nm at 298 K with 1.0 cm × 1.0 cm quartz cells used for samples holding.
CD Measurements CD spectra were operated over the wavelength range of 190–250 nm with a J-810 CD spectrometer (Jasco, Tokyo, Japan) using a quartz cell with a path length of 10 mm. The scanning speed was set at 200 nm min−1 . The each spectrum was the average of two scans. The conformation of protein secondary structure was computed through Yang.jwr software [15].
Reagents CAT (from bovine liver; Sigma Chemical Co., St. Louis, MO, USA) was dissolved in ultrapure water to form a 4 × 10−6 mol L−1 solution. Phosphate buffer (0.1 mol L−1 ; a mixture of NaH2 PO4 and Na2 HPO4 solution) was used to control pH at about 7.4 and stored at 4°C to avoid metamorphosis. NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O were of two analytical reagents and obtained from Tianjin Damao Chemical (Tianjin, China). MWCNTs (Shenzhen Nanotech Port Company Ltd., Shenzhen, China) were dissolved in ultrapure water to form a 0.1 g L−1 solution. Ultrapure water (18.25 M) was used throughout the experiments.
CAT Activity Measurement CAT catalyzes the decomposition of hydrogen peroxide into water and oxygen. The H2 O2 and phosphate buffer were mixed in a 3-mL quartz cell and used as a reference solution. The activity of CAT was measured spectrophotometrically by monitoring the decrease of hydrogen peroxide (49.3 mM) absorption at 240 nm [16, 17]. The data were recorded every 30 s for a period of 10 min. The measurements were conducted at room temperature (about 25°C) and repeated three times.
RESULTS Apparatus and Methods Fluorescence Measurements All fluorescence spectra were recorded on an F-4600 fluorophotometer (Hitachi, Tokyo, Japan) equipped with a 10-mm quartz cell and a 150-W xenon lamp. The photo multiplier tube voltage was 650 V, and the excitation and emission slit widths were set at 5.0 nm. The excitation wavelength was set at 280 nm. The scan speed was 1200 nm min−1 . The fluorescence measurements were performed as follows: 1 mL phosphate buffer, different concentrations of MWCNTs, and 1 mL of the CAT solution was added in a series of 10 mL colorimetric tubes. Ultrapure water was added to dilute the mixture to the scaled mark. Each sample was stabilized at 298 K for 30 min before the measurement. The emission wavelength scans ranged from 290 to 450 nm.
UV–Vis Absorption Measurements All UV–vis absorption spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan)
Influence of MWCNTs Dose on the Fluorescence Intensity of CAT Fluorescence Spectroscopy CAT contains three intrinsic fluorophores: Trp, tyrosine (Tyr), and phenylalanine (Phe). The fluorescence intensity of Trp is the strongest. There are six Trp residues (Trp14, Trp142, Trp182, Trp185, Trp276, and Trp302) in CAT, which can be used as intrinsic fluorophores [19]. The high sensitivity of Trp to its microenvironment is a significant feature of intrinsic fluorescence of proteins. To examine the interactions between MWCNTs and CAT, the fluorescence spectra were measured in PB buffer with pH 7.4. From Figure 1A, a gradual increase in the MWCNTs concentration in the solutions of CAT resulted in a decrease of fluorescence intensity and a slight blueshift at the maximum emission wavelength. However, the absorbance of the system will be increased with the concentration of the protein– MWCNTs complex, which causes the inner filter effect (IFE). The IFE from ligand refers to the absorbance or J Biochem Molecular Toxicology
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7100
Fluorescence intesity(a.u.)
7050
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7000 6950 6900 6850 6800 6750 6700 0
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Wavelength (nm) FIGURE 1. Fluorescence spectra of the CAT–MWCNTs system. Conditions : T = 298 K, kex = 280 nm, pH 7.4; CCAT = 4.0 × 10−6 mol/L, CCNTs (mg/L) 1–5: 0, 1.0, 2.0, 3.0, and 4.0. A1 : Fluorescence spectra of the CAT–MWCNTs system. A2 : Fluorescence spectra of the CAT–MWCNTs system after correction of IFE for A1 . B: Plot of fluorescence increasing for CAT at 332 nm after the correction of IFE for part A.
optical dispersion of light at the emission wavelength of the fluorescer [18]. MWCNTs can quench the fluorescence signal of the protein. So, we need to eliminate the influence of the IFE, which could interfere with the results. This effect will lead to the deviation of a fluorescence curve, resulting in a nonlinear relationship between the observed fluorescence intensity and the concentration of the fluorophore. Generally, IFE is composed of two parts: the primary inner filter effect (pIFE) is the absorption of excitation radiation, and the secondary inner filter effect (sIFE) is the absorption of emission radiation. According to Gu and Kenny [19], IFE can be calculated as shown in Eq. (1): Fideal (λex , λem ) = Fobs (λex , λem )C F p (λex )C Fs (λem ) ≈ Fobs (λex , λem )10(Aem +Aex )/2 J Biochem Molecular Toxicology
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(1)
where CFp refers to the correction factor for pIFE, depending on the total absorbance of the sample at λex , whereas CFs refers to the correction factor for sIFE, depending on the total absorbance of the sample at λem . Aex and Aem are the absorbance of samples at the fluorescence excitation and emission wavelengths, respectively. Fobs is the observed fluorescence value, and Fideal is the corrected fluorescence intensity. The corrected plots are shown in Figure 1B. The fluorescence intensity increased as the MWCNTs concentration increased. These results indicate that there are interactions between MWCNTs and CAT. The structure of CAT is destroyed by addition of the MWCNTs, while the more Trp residues are exposed to the aqueous environment. The slight blueshift is shown in Figure 1A, which suggests a more hydrophobic environment of the Trp residues [20, 21].
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0.13
0.22 0.20
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FIGURE 2. UV–vis absorption spectra of CAT with different concentrations of MWCNTs. Conditions: CCAT = 4 × 10−6 mol L−1 , CMWCNTs (mg/L) 1–5: 0, 1.25, 3.5, 7,5, and 10, respectively; pH 7.4, T = 293 K.
Investigations on the Conformational Changes of CAT UV–Vis Absorption Spectroscopy UV–visible absorption spectroscopy is used to analyze the structural changes in a protein and study the protein–ligand complex formation [22]. CAT has a strong absorption band at around 208 nm, resulting from the π → π * transition in the polypeptide backbone structure C=O [23, 24]. CAT also has an absorption band at 405 nm, which reflects the π → π * transition of heme porphyrin rings [25]. In addition to this, the weak one at about 278 nm can offer us the information about the absorption of aromatic amino acids (Trp, Tyr, and Phe) [26]. As shown in Figure 2, the intensity of the peak at 280 nm increased upon adding the MWCNTs, indicating that the MWCNTs increased the hydrophobicity of the microenvironment of the aromatic amino acids, which corresponded to the results of fluorescence spectra. While the intensity of the peak at 405 nm increased, indicating
that the conformation of the heme porphyrin rings was changed [27]. Moreover, the change may influence the activity of CAT. To further comprehend the process of the aromatic amino acids microenvironment changes, we take an αhelix as an example in Figure 3. In general, the amide moieties that are on the surface of the CAT molecule can only contact with water molecules; the others are in the hydrophobic pocket. When MWCNTs were added to the system, fewer water molecules could touch the amide moieties, which were blocked by MWCNTs. As a result, the hydrophobicity of the microenvironment of the amide moieties was increased, which may lead to the decrease in the activity of CAT. The inference will be proved by the following experiments.
Circular Dichroism CD spectra can be used to provide information about secondary protein conformational changes [28, 29]. There are two kinds of wavelength regions,
FIGURE 3. Schematic process for the microenvironment changes of amide moieties, taking an R-helix as an example.
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FIGURE 4. Far-CD spectra of native CAT and CAT–MWCNTs mixtures. Conditions: CCAT = 4 × 10−6 mol L−1 , CMWCNTs (mg/L) 1–3: 0, 1.25, 3.5, respectively. pH 7.4, T = 293 K.
TABLE 1. Influence of MWCNTs on the Secondary Structure of CAT Curve 1 2 3
α-Helix (%)
β-Sheet (%)
28.0 27.4 23.8
33.6 36.3 39.0
According to Fig. 4. Conditions are CCAT = 4 × 10−6 mol L−1 , CMWCNTs (mg/L) 1–3: 0, 1.25, 3.5; pH 7.4, T = 293 K.
far-UV (200–260 nm) and near-UV (260–320 nm). FarUV CD spectra can be used to determine the secondary structure of a protein, such as α-helix, β-turn, β-pleated sheet, and random coil [30]. CD spectra of far-UV regions are shown in Figure 4. It can be seen from Figure 4 that the two negative double humped peaks at 208 and 222 nm reflect the α-helical structure of protein [31]. The results of the portions of the secondary structures by Jasco secondary structure manager software are presented in Table 1. When MWCNTs are added to the system, it is concluded that the MWCNTs bind to the amino acid residues [31, 32], which suggests a more hydrophobic environment of the amino acid residues. At the same time, MWCNTs decreased the amount of α-helix, leading to an increase in the exposure of the Trp residues [30], which is consistent with the above results of fluorescence spectroscopy and UV absorption spectroscopy. What’s more, it may affect the physiological function of CAT [33].
Effect of MWCNTs on CAT Activity From the above investigations, we have known that MWCNTs can affect the structure of CAT. The structure of an enzyme has something to do with the function. So the structural variation may have an effect on its normal physiological function [34]. To further inJ Biochem Molecular Toxicology
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FIGURE 5. Effects of different concentrations of MWCNTs on the activity of CAT. Conditions: pH 7.4, c (CAT) = 4.0 × 10−7 mol L−1 , c(H2 O2 ) = 49.3 mmol L−1 , c(MWCNTs) (mg L−1 ) , a–c: 0, 10, 20, T = 290 K.
vestigate the effect of physiological function by MWCNTs, we measured the activity of CAT in the presence of different concentrations of MWCNTs (0–20 mg/L). As shown in Figure 5, the slope of the straight line defines the CAT activity [33]. We can see the obvious effects on the activity of CAT caused by MWCNTs. There are some amino acid residues around the CAT activity site. e.g., heme [34, 35]. Based on Figure 3 and above results, the hydrophobicity of the microenvironment of the amide acid residues was increased. The microenvironment of CAT activity site may be changed. As a result, the binding of MWCNTs into the enzyme cavity influenced the microenvironment of CAT activity site, which caused the decrease in the CAT activity [14]. The above results imply that MWCNTs can inhibit the CAT activity.
DISCUSSION In this work, we investigated the interactions between MWCNTs and CAT by using spectroscopic techniques under simulative physiological conditions. The results revealed that MWCNTs could bind to the amino acid residues of CAT, leading to a more hydrophobic environment of the amino acid residues. So, the CAT activity was inhibited after binding with MWCNTs. MWCNTs decreased the amount of α-helix, when MWCNTs were added, leading to increasing the exposure of the internal Trp residues. So, the fluorescence intensities of CAT increased with the increase in the MWCNTs concentration. Besides, the UV–vis absorption spectroscopy results also showed that the conformation of the heme porphyrin rings was changed. Based on the relationship between structure and function, this work mainly offers the basic spectroscopic methods for the toxicological study of MWCNTs
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at the molecular level. The above results may provide essential data for the safe use of nanomaterials and development of environment policies.
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