J BIOCHEM MOLECULAR TOXICOLOGY Volume 29, Number 9, 2015
New Insights into the Toxicity of n-Butanol to Trypsin: Spectroscopic and Molecular Docking Descriptions Rui Zhang, Tao Sun, Chunguang Liu, Wei Song, Zhaozhen Cao, and Rutao Liu School of Environmental Science and Engineering, Shandong University, China -America CRC for Environment & Health, Jinan 250100, People’s Republic of China Received 6 January 2015; revised 27 March 2015; accepted 1 April 2015
ABSTRACT: n-Butanol has been widely used and its residue exists extensively in the environment. It could lead to conformational and functional changes of trypsin by forming a complex with it. Docking method and spectrographic technique were employed to the study of the complex of trypsin and n-butanol. The fluorescence results indicated that n-butanol can form a complex with trypsin and change the distance between tryptophan and fluorescence quenchers. The conformational changes of trypsin were proved by UV–visible absorption and synchronous fluorescence spectroscopy indicating that n-butanol had little effect on the conformation of trypsin at a low concentration while denatured and coagulated the trypsin at a high concentration. The binding site was displayed by molecular modeling, which gave information about distances and binding forces between n-butanol and trypsin. The results were in accordance with spectroscopic experiments. Besides, enzyme activity assay gave the doseC response relationship of n-butanol with trypsin. 2015 Wiley Periodicals, Inc. J. Biochem. Mol. Toxicol. 29:418–425, 2015; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21711
Trypsin; n-Butanol; Spectroscopic Studies; Molecular Docking; Toxicity Assessment
KEYWORDS:
Correspondence to: Rutao Liu. Contract Grant Sponsor: NSFC. Contract Grant Numbers: 20875055, 21277081, 21477067. Contract Grant Sponsor: Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education, Ministry of Education of China. Contract Grant Numbers: 708058, 20130131110016. Contract Grant Sponsor: Science and Technology Development Plan of Shandong Province. Contract Grant Number: 2014GSF117027. Supporting Information is available in the online issue at www.wileyonlinelibrary.com. (), (), () are also acknowledged. C 2015 Wiley Periodicals, Inc.
INTRODUCTION Proteases are verified to control various pathological and physiological processes [1]. The interaction of small molecules such as surfactants, dyestuffs, drugs with proteases has been studied for many years to elucidate details of the binding mechanism and the proteases conformation [2–10]. Such research has been carried out by scientists of various fields and applied to the fields of cosmetic laboratories, biological, toxicology, pharmaceutical, etc. Trypsin, the most important digestive enzyme extracted from the pancreatic acinar cells, is a water-soluble serine protease, which has been used as a typical protein for diverse attentions [11]. It cleaves peptide bonds on the terminal side of lysine and arginine acid residues of the carbon skeleton [12, 13]. Trypsin has 223 amino acid residues and its molecular weight is about 23,300 Da. Six disulfide bridges hold the individual chains together. A trypsin molecule is composed of two domains, which are in the nearly same size. Six antiparallel strands of polypeptide chain were connected together into a β-sheet unit through a network of H-bonds, which is the major constituent of each domain. Catalytic triad— His 57, Asp 102, and Ser 195 are the active sites of trypsin which locate between the two domains [14, 15]. n-Butanol (1-butanol or normal butyl alcohol) is a colorless, volatile solvent. With an energy density closer to gasoline, n-butanol is capable of being used in existing engines with little or no modification [16]. In addition, due to its relatively lower vapor pressure, the storage and transportation become simple by existing infrastructure [17, 18]. Therefore, it can be widely encountered in industrial and scientific environments. Unfortunately, n-butanol has an odor that can exist for a long time. n-Butanol is one of the butanol isomers, and others are sec-n-butanol, iso-n-butanol, and tert-nbutanol. The production of n-butanol via biochemical processes from renewable feedstock has been reported [19]. Furthermore, Atsumi et al. [20] found a new way 418
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to yield n-butanol from glucose by Escherichia coli. The toxicity of n-butanol has been extensively studied with organisms by lots of scientists and has proven toxic to microbial cells [21–23]. As n-butanol is used extensively, a lot of research work on it has been carried out. However, most of the researches are focused on the n-butanol’s toxicity to organisms as just stated. Little is reported about its toxic effects on biomacromolecules such as DNA and proteins. This article will investigate the relevance between the addition of n-butanol and the changes of the trypsin’s structure. Spectroscopic methods including UV–visible absorption spectroscopy, circular dichroism spectroscopy (CD), fluorescence spectroscopy, and enzyme activity assay. Furthermore, molecular modeling method was used to investigate the binding mode and binding sites of trypsin and n-butanol. Such processes have been performed to better understand the effect of n-butanol on the conformation and function of trypsin and help us to know some information about the biological impact of n-butanol on the human body.
MATERIALS AND METHODS
UV–Visible Absorption Spectra We recorded UV–visible spectra data in the range of 190–350 nm with 1.0 cm × 1.0 cm quartz cuvettes holding sample at 298 K. We added 1 mL of trypsin solution, 1 mL PB (0.2 mol L−1 ), and various amounts of n-butanol to a 10-mL volumetric flask in which a buffer was used to contain the equivalent concentration of n-butanol as the reference solution.
Fluorescence Measurements We recorded the fluorescence spectra with a scanning speed of 240 nm min−1 within the wavelength range of 290–450 nm. The excitation wavelength was kept at 278 nm and the voltage of scanning was 700 V. We added 1.0 mL of 1.0 × 10−4 mol L−1 trypsin and 1.0 mL of 0.2 mol L−1 phosphate buffer (pH 8.0) into each of a series of 10 mL tubes; Ultra-pure water and a series of volumes of n-butanol were added to dilute the mixture to the scale mark. Finally, we equilibrated all the solution systems for 30 min at 298 K. Synchronous fluorescence spectra were measured at λex = 250 nm, λ = 0 nm, λ = 15 nm, and λ = 60 nm.
Circular Dichroism Spectra
Reagents Trypsin (from bovine pancreas, AMRESCO, No.0458) was dissolved in ultrapure water to form a 1 × 10−4 mol L−1 solution. A phosphate buffer (0.2 mol L−1 , a mixture of NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O) was used to control the pH at about 8.0. NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O were of analytical reagent grade, obtained from Tianjin Damao Chemical Reagent Factory. BAEE (N-R-benzoyl-L-arginine ethyl ester, from Sinopharm Chemical Reagent, BR) was dissolved in ultrapure water to prepare a 1.5 × 10−3 mol L−1 solution. n-Butanol was of analytical reagent grade. Throughout the whole experiments, the ultrapure water was used as solvent with the resistivity 18.25 M.
Apparatus and Methods We recorded all the fluorescence spectra data on an F-4600 spectrofluorimeter (Hitachi, Japan) equipped with a 10 mm quartz cell and a 150 W Xenon lamp. And we set the excitation and emission slit widths at 5 nm. UV–vis absorption spectra were measured on a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). CD spectra were recorded on a J-810 CD spectrometer (JASCO). The pH measurements were performed with a pHs-3C acidity meter (Pengshun, Shanghai, China). J Biochem Molecular Toxicology
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In order to understand the conformational changes of trypsin comprehensively, the CD measurements were carried between 190 and 260 nm using a temperature-controlled cell of 1 mm path length with a data pitch of 0.2 nm and accumulation of 3 scans. We set the bandwidth, response time, and scan rate at 1 nm, 1 s, and 200 nm min−1 , respectively. The concentration of the phosphate buffer solution was 0.2 mol L−1 . We estimated the secondary structural changes of trypsin through the SELON3 program stocked in the CDPro software package.
Molecular Docking Studies We carried out the docking simulations experiment through the Lamarckian genetic algorithm (LGA) method to find the optimal binding site between trypsin and n-butanol. AutoDock 4.2 was used to evaluate conformations of the complex generated from the reaction between these two materials. The structure of trypsin was obtained from RCSB Protein Data Bank and the crystal structure of n-butanol was taken from ZINC database.
Trypsin Activity Measurement The way to measure trypsin activity has been described in many other papers [24]. We measured the
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trypsin activity using 1 mM BAEE as the substrate with a Shimadzu UV-2450 spectrophotometer. BAEE can be catalyzed by trypsin into N-benzoyl-L-arginine (BA). The absorption of BA is much intense than that of BAEE at 253 nm. Then as the BAEE was catalyzed by trypsin, the absorption intensity of the system became stronger. We mixed the BAEE (1.0 × 10−3 mol L−1 ) with phosphate buffer (0.2 mol L−1 ) in 3-mL quartz cells, which was used as the reference solution. The absorption of the system was measured at 253 nm to determine the trypsin activity before and after the addition of n-butanol. The hydrolysis of BAEE was recorded after 30 s at room temperature.
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As a simple but effective technique, UV–visible absorption spectroscopy has been widely used to monitor structural changes in protein initiated by toxic substances and to demonstrate the complex formation [25–27]. We measured the UV–vis spectra of trypsin with increasing concentrations of n-butanol to reconfirm the conformational changes of trypsin. Trypsin gives two absorption peaks, between which the weak absorption peak at around 280 nm may due to the absorption of aromatic amino acids (Trp, Tyr, and Phe) and the strong one at about 208 nm was due to the transition of π →π * of C=O. Figure 1 displays the UV– vis absorption values of trypsin-n-butanol mixture at 280 and 208 nm. The spectrum profile shows an initial decrease of absorbance when the concentration of n-butanol is low and an increase at a high concentration of n-butanol. The low concentration of n-butanol can make a marginal effect on the structure of trypsin and leads to the loosening and unfolding of the trypsin conformation, so the absorption intensity decreased [28]. The high concentration of n-butanol can denature and coagulate the protein, which makes the absorption increase.
Investigations on Fluorescence Spectra of Trypsin Fluorescence Spectroscopy The fluorescence detection has a high sensitivity and selectivity, which can be used to investigate micromolecules effectively [29]. In this work, we studied the toxic effects of n-butanol on trypsin by using fluorescence spectra technique. The fluorescence intensity of
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FIGURE 1. UV–visible absorption spectra of trypsin with different concentrations of butanol. The concentration of trypsin was at 1.0 × 10−5 mol L−1 . (A) Cn-butanol (v/v) 1–3: 0, 1, 4%, respectively; (B) Cn-butanol (v/v) 3–5: 4, 5, 7%, respectively; pH 8.0, T = 293 K. C: Cn-butanol (v/v) 1–5: 0, 1, 4, 5, 7%, respectively.
trypsin at a fixed concentration (1 × 10−5 mol L−1 ) increased gradually with the increasing concentration of n-butanol at 290 K (Figure 2A) without any shift on the absorption peak. The increased fluorescence intensity indicates that n-butanol interacted with trypsin and enhanced its intrinsic fluorescence. Trypsin has four tryptophans (Trp 51, Trp 141, Trp 215, and Trp 237), which can be used as intrinsic fluorophores [30]. Trp is highly sensible to its local environmental changes, which is a J Biochem Molecular Toxicology
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and instrument geometry [37]. The signal of the protein can be quenched by inner-filter effect. The magnitude of the inner-filter effect depends on the wavelength range, path length, and concentrations of quenching components and must be measured for each instrumental configuration if accurate fluorescence measurements are to be obtained [38]. The effect is important already at low absorbance. An absorption coefficient of 11.5 m−1 (corresponding to 0.05 absorbance in a 1 cm cuvette) in the sample reduces the fluorescence by 5% [39]. When we set the concentration of n-butanol at 8% (v/v), the fluorescence intensity at the excitation or emission wavelength of trypsin was less than 0.01 (Figure S1 in the Supporting Information). Therefore the inner-filter effect can be neglected [40]. Figure 2 shows the fluorescence intensity changes with different concentrations of n-butanol. The maximum excitation wavelength of trypsin is 278 nm and a strong fluorescence emission peak appears at about 330 nm. The fluorescence intensity of trypsin increased gradually as we raised the concentration of n-butanol, while no shift was found on the absorption peak. The weakening of tryptophan quenching leads to the increase in fluorescence intensity. It is probably the addition of n-butanol that makes the structure of the trypsin change in such way that the position of tryptophan is further away from fluorescence quenchers [41]. The secondary and tertiary structures of trypsin were studied to confer the inference above.
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FIGURE 2. (A) Fluorescence spectra of trypsin-n-butanol system. Experimental conditions: T = 293 K, λex = 278 nm, pH 8.0; Ctrypsin = 1 × 10−5 mol L−1 , Cn-butanol (v/v) 1–6: 0, 1, 3, 4, 5, 7%. (B) CD spectra of trypsin and trypsin–butanol mixtures. Experimental conditions: Ctrypsin = 5 × 10−5 mol L−1 , Cn-butanol (v/v) 1–2: 0, 7%; pH 8.0, T = 293 K.
significant feature of intrinsic fluorescence of protein [31]. Some groups such as cysteine and disulfide bonds that contain sulfur can quench tryptophan fluorescence a lot [32]. Accordingly, in the native state tryptophan neighboring to sulfur-containing groups do not significantly contribute to the overall fluorescence emission [33]. In order to investigate the effect of the inner-filter exists in the protein–ligand system, we carried out relevant experiment primarily. Inner-filter effect has been observed and studied for a long time [34–36]. Recorded fluorescence intensity is in general not proportional to the sample concentration owing to the absorption of the incident and emitted light passing through the sample to and from the point inside the cell where the emission is detected. This well-known phenomenon is called inner-filter effect, which depends on sample absorption J Biochem Molecular Toxicology
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Synchronous Fluorescence Synchronous mode of fluorescence spectroscopy, which was introduced by Lloyd in 1971, has become a popular method for studying micromolecules [42]. The measurements are performed by the scanning of the emission and excitation monochromators simultaneously while maintaining a constant wavelength interval (λ) between them. Trp is highly sensible to the changes of its local environment. The synchronous fluorescence spectroscopy gives information about the molecular environment in the vicinity of chromophore and can avoid different perturbing effects [43]. When the scanning interval λ ( λ= λem -λex ) is fixed at 0, 15, and 60 nm, the results of synchronous fluorescence give characteristic information of tyrosine residues or tryptophan residues, respectively [44]. Figures 3A and 3B show the results of the synchronous fluorescence spectra of trypsin. In this work, the synchronous fluorescence peaks of trypsin with various amounts of n-butanol have no shift in Figure 3A, indicating that the interaction of n-butanol with trypsin has no effect on the conformation of Tyr micro-region. Instead, in Figure 3B, the maximum emission of tryptophan residues showed a slight redshift, which illustrates that
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TABLE 1. The Influence of n-Butanol on the Secondary Structure of Trypsin
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Circular dichroism (CD) is a simple and sensitive technique that can provide information about the conformational changes in protein [46]. As is shown in Figure 2B, two negative bands are exhibited in the CD spectrum of n-butanol in the ultraviolet region at about 208 nm and 220 nm, which are the characteristic of α-helix and β-sheet, respectively [47–49]. CDPro software was employed to calculate the secondary structural elements and the results are listed in Table 1. When n-butanol was added into the trypsin solution, the secondary structure content of trypsin fluctuated in different trends. The content of α-helix increased and the content of β-sheet decreased, which is consistent with previous spectroscopy experiments. It is implied that n-butanol changed the secondary structure of trypsin.
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FIGURE 3. Synchronous fluorescence spectroscopy of trypsin. (A) Tryptophan (λ = 60 nm); (B) Tyrosine (λ = 15 nm); (C) (λ = 0 nm). Experimental conditions: Ctrypsin = 1 × 10−5 mol L−1 ; PH 8.0; Cn-butanol (v/v) = 0, 4, 5, 7%.
the polarity around tryptophan increased and the hydrophobicity decreased [45]. Figure 3C shows a sharp increase in the fluorescence peak of trypsin when the concentration of n-butanol is 4%, which indicates that n-butanol may have denatured trypsin. This is in accordance with the results of UV–visible absorption.
Molecular Docking As a molecular modeling method that was originally used to investigate the binding information between proteins and ligands, AutoDock 4.2 software was applied to simulate the binding mode of trypsin and n-butanol in this work. As a serine protease, the active sites of trypsin are the catalytic triad Asp 102, Ser 195 and His 57 [50, 51]. The best ranked results are presented in Figure 4. Figure 4 shows the docked structure with the minimum energy. As can be observed in Figure 4A, n-butanol binds in the trypsin central cavity. Figure 4B shows that two hydrogen bonds exist between the n˚ is formed between butanol and trypsin: one (2.48 A) an oxygen atom in the n-butanol and a hydrogen atom ˚ is formed from Lys 230 residue, and the other (1.87 A) between an oxygen atom in the Met 180 residue and the hydrogen atom from n-butanol. In addition, Figure 4C shows that the n-butanol molecule located in a hydrophobic cavity on the surface of the protein, which indicates that hydrophobic interaction is one of the interaction forces between them. J Biochem Molecular Toxicology
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FIGURE 4. The molecule docking results of trypsin–butanol complex. (A) The binding site location of n-butanol to trypsin. (B) The relative residues (Gly 142 and Asp 194) of the hydrogen bonds. (C) N-butanol binds to a hydrophobic cavity of trypsin, blue sticks symbolize n-butanol.
Effect of n-Butanol on Trypsin Activity
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We have investigated the interaction between trypsin and n-butanol through the experiments above and confirmed the conformational changes in trypsin. But how the enzyme activity of trypsin changes when it is exposed to n-butanol is what fascinates us most, because it is significant for us to research proteins. Figure 5 displays the effects of various amounts of n-butanol on the activity of trypsin in vitro. We set the activity of trypsin that is without n-butanol to be 1 and other values of trypsin’s activity are calculated based on it. As shown in Figure 5, the protein activity raised when the concentration of n-butanol was low and decreased at a high concentration of n-butanol. When the concentration of n-butanol was low, the n-butanol molecule entered into the active site cavity of trypsin and made the catalytic activity center more exposed but it did not inactivate the enzyme. This was conductive to substrate binding for the enzyme. As the concentration of n-butanol increased by 5% which was near the precipitation concentration of n-butanol, the
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trypsin was denatured by n-butanol and the activity of trypsin decreased (consistent with the above results of UV–vis spectroscopy).
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DISCUSSION
and molecular docking calculations. Spectrochim Acta A Mol Biomol Spectrosc 2013;115:629-635. Neamtu S, Tosa N, Bogdan M. Spectroscopic investigation of tolmetin interaction with human serum albumin. J Pharm Biomed Anal 2013;85:277-282. Ozmen M, Maltas E, Patir IH, Bayrakci M. Combined voltammetric and spectroscopic investigation of binding interaction between nifedipine and human serum albumin on polyelectrolyte modified ITO electrode. Electrochim Acta 2013;111:535-542. Shahabadi N, Khorshidi A, Moghadam NH. Study on the interaction of the epilepsy drug, zonisamide with human serum albumin (HSA) by spectroscopic and molecular docking techniques. Spectrochim Acta A Mol Biomol Spectrosc 2013;114:627-632. Tabassum S, Al-Asbahy WM, Afzal M, Arjmand F. Synthesis, characterization and interaction studies of copper based drug with human serum albumin (HSA): spectroscopic and molecular docking investigations. J Photochem Photobiol B 2012;114:132-139. 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. Chi Z, Liu R. Phenotypic characterization of the binding of tetracycline to human serum albumin. Biomacromolecules 2011;12(1):203-209. Wang Y, Luo W, Reiser G. Trypsin and trypsin-like proteases in the brain: proteolysis and cellular functions. Cell Mol Life Sci 2008;65(2):237-252. Hirota M, Ohmuraya M, Baba H. The role of trypsin, trypsin inhibitor, and trypsin receptor in the onset and aggravation of pancreatitis. J Gastroenterol 2006;41(9):832836. Li X, Zhu S, Xu B, Ma K, Zhang J, Yang B, Tian W. Self-assembled graphene quantum dots induced by cytochrome c: a novel biosensor for trypsin with remarkable fluorescence enhancement. Nanoscale 2013;5(17):77767779. Ibarz A, Garvin A, Garza S, Pagan J. Toxic effect of melanoidins from glucose-asparagine on trypsin activity. Food Chem Toxicol 2009;47(8):2071-2075. Wang J, Liu R, Qin P. Toxic interaction between acid yellow 23 and trypsin: spectroscopic methods coupled with molecular docking. J. Biochem Mol Toxicol 2012;26(9):360-367. Yang B, Oßwald P, Li Y, Wang J, Wei L, Tian Z, Qi ¨ F, Kohse-Hoinghaus K. Identification of combustion intermediates in isomeric fuel-rich premixed butanol– oxygen flames at low pressure. Combustion Flame 2007;148(4):198-209. Veloo PS, Wang YL, Egolfopoulos FN, Westbrook CK. A comparative experimental and computational study of methanol, ethanol, and n-butanol flames. Combustion Flame 2010;157(10):1989-2004. Grana R, Frassoldati A, Faravelli T, Niemann U, Ranzi E, Seiser R, Cattolica R, Seshadri K. An experimental and kinetic modeling study of combustion of isomers of butanol. Combustion Flame 2010;157(11):2137-2154. Lin YL, Blaschek HP. Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth. Appl Environ Microbiol 1983;45(3):966-973. Veloo PS, Egolfopoulos FN. Flame propagation of butanol isomers/air mixtures. Proceedings of the Combustion Institute 2011;33(1):987–993.
In this work, the interaction mechanism of trypsin and n-butanol has been studied by spectroscopic techniques and molecular docking method under simulated physiological conditions in vitro. The fluorescence spectroscopy results revealed the changes of microenvironment of Trp residues in trypsin molecules. We found that the polarity around tryptophan increased and the hydrophobicity decreased in trypsin through the results of synchronous fluorescence (λ= 60 or 15 nm). The results of CD spectra indicate that n-butanol could bind to trypsin and change its secondary structure by increasing the content of α-helix and decreasing the content of β-sheet. The skeletal structure of trypsin is loosened and its active sites are more exposed. The enzyme activity assay, UV–visible absorption spectroscopy, and the synchronous fluorescence (λ = 0 nm) indicated that n-butanol increased the activity of trypsin when the concentration of n-butanol was low and denatured it when the concentration of n-butanol was higher than 4% (v/v). Besides, the molecular docking results detailed the information of hydrophobic interaction and revealed that hydrogen bond is the main force of the n-butanol-trypsin complex. Our work focused on the potential toxicity of nbutanol to trypsin at the molecular level. The results of these experiments gave some important information about the structure and function of protein and may contribute to comprehend the enzyme toxicity of n-butanol and human health risks in daily life.
SUPPORTING INFORMATION Fig. S1 The UV-visible absorption spectra of n-butanol. Experimental conditions: Cn-butanol (v/v): 7%, Ctrypsin = 1×10-5 mol-1L
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36. Guan J, Dai J, Zhao X, Liu C, Gao C, Liu R. Spectroscopic investigations on the interaction between carbon nanotubes and catalase on molecular level. J Biochem Mol Toxicol 2014;28(5):211-216. ¨ 37. Kubista M, Sjoback R, Eriksson S, Albinsson B. Experimental correction for the inner-filter effect in fluorescence spectra. Analyst 1994;119(3):417-419. 38. Liu Y, Kati W, Chen C-M, Tripathi R, Molla A, Kohlbrenner W. Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction. Anal Biochem 1999;267(2):331-335. 39. Larsson T, Wedborg M, Turner D. Correction of innerfilter effect in fluorescence excitation-emission matrix spectrometry using Raman scatter. Anal Chim Acta 2007;583(2):357-363. 40. Van de Weert M, Stella L. Fluorescence quenching and ligand binding: a critical discussion of a popular methodology. J Mol Struct 2011;998(1):144-150. 41. Koutsopoulos S, Patzsch K, Bosker WT, Norde W. Adsorption of trypsin on hydrophilic and hydrophobic surfaces. Langmuir 2007;23(4):2000-2006. 42. Tong C, Zhuo X, Liu W, Wu J. Synchronous fluorescence measurement of enrofloxacin in the pharmaceutical formulation and its residue in milks based on the yttrium (III)-perturbed luminescence. Talanta 2010;82(5):18581863. 43. Hu Y-J, Liu Y, Pi Z-B, Qu S-S. Interaction of cromolyn sodium with human serum albumin: a fluorescence quenching study. Bioorg Med Chem 2005;13(24):66096614. 44. He W, Li Y, Xue C, Hu Z, Chen X, Sheng F. Effect of Chinese medicine alpinetin on the structure of human serum albumin. Bioorg Med Chem 2005;13(5):18371845. 45. Klajnert B, Bryszewska M. Fluorescence studies on PAMAM dendrimers interactions with bovine serum albumin. Bioelectrochemistry 2002;55(1):33-35. 46. Kelly SM, Jess TJ, Price NC. How to study proteins by circular dichroism. Biochim et Biophys Acta (BBA)-Proteins Proteomics 2005;1751(2):119-139. 47. Li D, Wang Y, Chen J, Ji B. Characterization of the interaction between farrerol and bovine serum albumin by fluorescence and circular dichroism. Spectrochim Acta Part A: Mol Biomol Spectrosc 2011;79(3):680-686. 48. Sandhya B, Hegde AH, Kalanur SS, Katrahalli U, Seetharamappa J. Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes. J Pharm Biomed Anal 2011;54(5):1180-1186. 49. Zhang H-M, Chen J, Zhou Q-H, Shi Y-Q, Wang Y-Q. Study on the interaction between cinnamic acid and lysozyme. J Mol Struct 2011;987(1):7-12. 50. Chakraborty B, Roy AS, Dasgupta S, Basu S. Magnetic field effect corroborated with docking study to explore photoinduced electron transfer in drug–protein interaction. J Phys Chem A 2010;114(51):13313-13325. 51. Roy D, Dutta S, Maity SS, Ghosh S, Singha Roy A, Ghosh KS, Dasgupta S. Spectroscopic and docking studies of the binding of two stereoisomeric antioxidant catechins to serum albumins. J Luminescence 2012;132(6):13641375.
New Insights into the Toxicity of n-Butanol to Trypsin: Spectroscopic and Molecular Docking Descriptions Rui Zhang, Tao Sun, Chunguang Liu, Wei Song, Zhaozhen Cao, and Rutao Liu School of Environmental Science and Engineering, Shandong University, China -America CRC for Environment & Health, Jinan 250100, People’s Republic of China Received 6 January 2015; revised 27 March 2015; accepted 1 April 2015
ABSTRACT: n-Butanol has been widely used and its residue exists extensively in the environment. It could lead to conformational and functional changes of trypsin by forming a complex with it. Docking method and spectrographic technique were employed to the study of the complex of trypsin and n-butanol. The fluorescence results indicated that n-butanol can form a complex with trypsin and change the distance between tryptophan and fluorescence quenchers. The conformational changes of trypsin were proved by UV–visible absorption and synchronous fluorescence spectroscopy indicating that n-butanol had little effect on the conformation of trypsin at a low concentration while denatured and coagulated the trypsin at a high concentration. The binding site was displayed by molecular modeling, which gave information about distances and binding forces between n-butanol and trypsin. The results were in accordance with spectroscopic experiments. Besides, enzyme activity assay gave the doseC response relationship of n-butanol with trypsin. 2015 Wiley Periodicals, Inc. J. Biochem. Mol. Toxicol. 29:418–425, 2015; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21711
Trypsin; n-Butanol; Spectroscopic Studies; Molecular Docking; Toxicity Assessment
KEYWORDS:
Correspondence to: Rutao Liu. Contract Grant Sponsor: NSFC. Contract Grant Numbers: 20875055, 21277081, 21477067. Contract Grant Sponsor: Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education, Ministry of Education of China. Contract Grant Numbers: 708058, 20130131110016. Contract Grant Sponsor: Science and Technology Development Plan of Shandong Province. Contract Grant Number: 2014GSF117027. Supporting Information is available in the online issue at www.wileyonlinelibrary.com. (), (), () are also acknowledged. C 2015 Wiley Periodicals, Inc.
INTRODUCTION Proteases are verified to control various pathological and physiological processes [1]. The interaction of small molecules such as surfactants, dyestuffs, drugs with proteases has been studied for many years to elucidate details of the binding mechanism and the proteases conformation [2–10]. Such research has been carried out by scientists of various fields and applied to the fields of cosmetic laboratories, biological, toxicology, pharmaceutical, etc. Trypsin, the most important digestive enzyme extracted from the pancreatic acinar cells, is a water-soluble serine protease, which has been used as a typical protein for diverse attentions [11]. It cleaves peptide bonds on the terminal side of lysine and arginine acid residues of the carbon skeleton [12, 13]. Trypsin has 223 amino acid residues and its molecular weight is about 23,300 Da. Six disulfide bridges hold the individual chains together. A trypsin molecule is composed of two domains, which are in the nearly same size. Six antiparallel strands of polypeptide chain were connected together into a β-sheet unit through a network of H-bonds, which is the major constituent of each domain. Catalytic triad— His 57, Asp 102, and Ser 195 are the active sites of trypsin which locate between the two domains [14, 15]. n-Butanol (1-butanol or normal butyl alcohol) is a colorless, volatile solvent. With an energy density closer to gasoline, n-butanol is capable of being used in existing engines with little or no modification [16]. In addition, due to its relatively lower vapor pressure, the storage and transportation become simple by existing infrastructure [17, 18]. Therefore, it can be widely encountered in industrial and scientific environments. Unfortunately, n-butanol has an odor that can exist for a long time. n-Butanol is one of the butanol isomers, and others are sec-n-butanol, iso-n-butanol, and tert-nbutanol. The production of n-butanol via biochemical processes from renewable feedstock has been reported [19]. Furthermore, Atsumi et al. [20] found a new way 418
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to yield n-butanol from glucose by Escherichia coli. The toxicity of n-butanol has been extensively studied with organisms by lots of scientists and has proven toxic to microbial cells [21–23]. As n-butanol is used extensively, a lot of research work on it has been carried out. However, most of the researches are focused on the n-butanol’s toxicity to organisms as just stated. Little is reported about its toxic effects on biomacromolecules such as DNA and proteins. This article will investigate the relevance between the addition of n-butanol and the changes of the trypsin’s structure. Spectroscopic methods including UV–visible absorption spectroscopy, circular dichroism spectroscopy (CD), fluorescence spectroscopy, and enzyme activity assay. Furthermore, molecular modeling method was used to investigate the binding mode and binding sites of trypsin and n-butanol. Such processes have been performed to better understand the effect of n-butanol on the conformation and function of trypsin and help us to know some information about the biological impact of n-butanol on the human body.
MATERIALS AND METHODS
UV–Visible Absorption Spectra We recorded UV–visible spectra data in the range of 190–350 nm with 1.0 cm × 1.0 cm quartz cuvettes holding sample at 298 K. We added 1 mL of trypsin solution, 1 mL PB (0.2 mol L−1 ), and various amounts of n-butanol to a 10-mL volumetric flask in which a buffer was used to contain the equivalent concentration of n-butanol as the reference solution.
Fluorescence Measurements We recorded the fluorescence spectra with a scanning speed of 240 nm min−1 within the wavelength range of 290–450 nm. The excitation wavelength was kept at 278 nm and the voltage of scanning was 700 V. We added 1.0 mL of 1.0 × 10−4 mol L−1 trypsin and 1.0 mL of 0.2 mol L−1 phosphate buffer (pH 8.0) into each of a series of 10 mL tubes; Ultra-pure water and a series of volumes of n-butanol were added to dilute the mixture to the scale mark. Finally, we equilibrated all the solution systems for 30 min at 298 K. Synchronous fluorescence spectra were measured at λex = 250 nm, λ = 0 nm, λ = 15 nm, and λ = 60 nm.
Circular Dichroism Spectra
Reagents Trypsin (from bovine pancreas, AMRESCO, No.0458) was dissolved in ultrapure water to form a 1 × 10−4 mol L−1 solution. A phosphate buffer (0.2 mol L−1 , a mixture of NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O) was used to control the pH at about 8.0. NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O were of analytical reagent grade, obtained from Tianjin Damao Chemical Reagent Factory. BAEE (N-R-benzoyl-L-arginine ethyl ester, from Sinopharm Chemical Reagent, BR) was dissolved in ultrapure water to prepare a 1.5 × 10−3 mol L−1 solution. n-Butanol was of analytical reagent grade. Throughout the whole experiments, the ultrapure water was used as solvent with the resistivity 18.25 M.
Apparatus and Methods We recorded all the fluorescence spectra data on an F-4600 spectrofluorimeter (Hitachi, Japan) equipped with a 10 mm quartz cell and a 150 W Xenon lamp. And we set the excitation and emission slit widths at 5 nm. UV–vis absorption spectra were measured on a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). CD spectra were recorded on a J-810 CD spectrometer (JASCO). The pH measurements were performed with a pHs-3C acidity meter (Pengshun, Shanghai, China). J Biochem Molecular Toxicology
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In order to understand the conformational changes of trypsin comprehensively, the CD measurements were carried between 190 and 260 nm using a temperature-controlled cell of 1 mm path length with a data pitch of 0.2 nm and accumulation of 3 scans. We set the bandwidth, response time, and scan rate at 1 nm, 1 s, and 200 nm min−1 , respectively. The concentration of the phosphate buffer solution was 0.2 mol L−1 . We estimated the secondary structural changes of trypsin through the SELON3 program stocked in the CDPro software package.
Molecular Docking Studies We carried out the docking simulations experiment through the Lamarckian genetic algorithm (LGA) method to find the optimal binding site between trypsin and n-butanol. AutoDock 4.2 was used to evaluate conformations of the complex generated from the reaction between these two materials. The structure of trypsin was obtained from RCSB Protein Data Bank and the crystal structure of n-butanol was taken from ZINC database.
Trypsin Activity Measurement The way to measure trypsin activity has been described in many other papers [24]. We measured the
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trypsin activity using 1 mM BAEE as the substrate with a Shimadzu UV-2450 spectrophotometer. BAEE can be catalyzed by trypsin into N-benzoyl-L-arginine (BA). The absorption of BA is much intense than that of BAEE at 253 nm. Then as the BAEE was catalyzed by trypsin, the absorption intensity of the system became stronger. We mixed the BAEE (1.0 × 10−3 mol L−1 ) with phosphate buffer (0.2 mol L−1 ) in 3-mL quartz cells, which was used as the reference solution. The absorption of the system was measured at 253 nm to determine the trypsin activity before and after the addition of n-butanol. The hydrolysis of BAEE was recorded after 30 s at room temperature.
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As a simple but effective technique, UV–visible absorption spectroscopy has been widely used to monitor structural changes in protein initiated by toxic substances and to demonstrate the complex formation [25–27]. We measured the UV–vis spectra of trypsin with increasing concentrations of n-butanol to reconfirm the conformational changes of trypsin. Trypsin gives two absorption peaks, between which the weak absorption peak at around 280 nm may due to the absorption of aromatic amino acids (Trp, Tyr, and Phe) and the strong one at about 208 nm was due to the transition of π →π * of C=O. Figure 1 displays the UV– vis absorption values of trypsin-n-butanol mixture at 280 and 208 nm. The spectrum profile shows an initial decrease of absorbance when the concentration of n-butanol is low and an increase at a high concentration of n-butanol. The low concentration of n-butanol can make a marginal effect on the structure of trypsin and leads to the loosening and unfolding of the trypsin conformation, so the absorption intensity decreased [28]. The high concentration of n-butanol can denature and coagulate the protein, which makes the absorption increase.
Investigations on Fluorescence Spectra of Trypsin Fluorescence Spectroscopy The fluorescence detection has a high sensitivity and selectivity, which can be used to investigate micromolecules effectively [29]. In this work, we studied the toxic effects of n-butanol on trypsin by using fluorescence spectra technique. The fluorescence intensity of
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FIGURE 1. UV–visible absorption spectra of trypsin with different concentrations of butanol. The concentration of trypsin was at 1.0 × 10−5 mol L−1 . (A) Cn-butanol (v/v) 1–3: 0, 1, 4%, respectively; (B) Cn-butanol (v/v) 3–5: 4, 5, 7%, respectively; pH 8.0, T = 293 K. C: Cn-butanol (v/v) 1–5: 0, 1, 4, 5, 7%, respectively.
trypsin at a fixed concentration (1 × 10−5 mol L−1 ) increased gradually with the increasing concentration of n-butanol at 290 K (Figure 2A) without any shift on the absorption peak. The increased fluorescence intensity indicates that n-butanol interacted with trypsin and enhanced its intrinsic fluorescence. Trypsin has four tryptophans (Trp 51, Trp 141, Trp 215, and Trp 237), which can be used as intrinsic fluorophores [30]. Trp is highly sensible to its local environmental changes, which is a J Biochem Molecular Toxicology
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and instrument geometry [37]. The signal of the protein can be quenched by inner-filter effect. The magnitude of the inner-filter effect depends on the wavelength range, path length, and concentrations of quenching components and must be measured for each instrumental configuration if accurate fluorescence measurements are to be obtained [38]. The effect is important already at low absorbance. An absorption coefficient of 11.5 m−1 (corresponding to 0.05 absorbance in a 1 cm cuvette) in the sample reduces the fluorescence by 5% [39]. When we set the concentration of n-butanol at 8% (v/v), the fluorescence intensity at the excitation or emission wavelength of trypsin was less than 0.01 (Figure S1 in the Supporting Information). Therefore the inner-filter effect can be neglected [40]. Figure 2 shows the fluorescence intensity changes with different concentrations of n-butanol. The maximum excitation wavelength of trypsin is 278 nm and a strong fluorescence emission peak appears at about 330 nm. The fluorescence intensity of trypsin increased gradually as we raised the concentration of n-butanol, while no shift was found on the absorption peak. The weakening of tryptophan quenching leads to the increase in fluorescence intensity. It is probably the addition of n-butanol that makes the structure of the trypsin change in such way that the position of tryptophan is further away from fluorescence quenchers [41]. The secondary and tertiary structures of trypsin were studied to confer the inference above.
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FIGURE 2. (A) Fluorescence spectra of trypsin-n-butanol system. Experimental conditions: T = 293 K, λex = 278 nm, pH 8.0; Ctrypsin = 1 × 10−5 mol L−1 , Cn-butanol (v/v) 1–6: 0, 1, 3, 4, 5, 7%. (B) CD spectra of trypsin and trypsin–butanol mixtures. Experimental conditions: Ctrypsin = 5 × 10−5 mol L−1 , Cn-butanol (v/v) 1–2: 0, 7%; pH 8.0, T = 293 K.
significant feature of intrinsic fluorescence of protein [31]. Some groups such as cysteine and disulfide bonds that contain sulfur can quench tryptophan fluorescence a lot [32]. Accordingly, in the native state tryptophan neighboring to sulfur-containing groups do not significantly contribute to the overall fluorescence emission [33]. In order to investigate the effect of the inner-filter exists in the protein–ligand system, we carried out relevant experiment primarily. Inner-filter effect has been observed and studied for a long time [34–36]. Recorded fluorescence intensity is in general not proportional to the sample concentration owing to the absorption of the incident and emitted light passing through the sample to and from the point inside the cell where the emission is detected. This well-known phenomenon is called inner-filter effect, which depends on sample absorption J Biochem Molecular Toxicology
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Synchronous Fluorescence Synchronous mode of fluorescence spectroscopy, which was introduced by Lloyd in 1971, has become a popular method for studying micromolecules [42]. The measurements are performed by the scanning of the emission and excitation monochromators simultaneously while maintaining a constant wavelength interval (λ) between them. Trp is highly sensible to the changes of its local environment. The synchronous fluorescence spectroscopy gives information about the molecular environment in the vicinity of chromophore and can avoid different perturbing effects [43]. When the scanning interval λ ( λ= λem -λex ) is fixed at 0, 15, and 60 nm, the results of synchronous fluorescence give characteristic information of tyrosine residues or tryptophan residues, respectively [44]. Figures 3A and 3B show the results of the synchronous fluorescence spectra of trypsin. In this work, the synchronous fluorescence peaks of trypsin with various amounts of n-butanol have no shift in Figure 3A, indicating that the interaction of n-butanol with trypsin has no effect on the conformation of Tyr micro-region. Instead, in Figure 3B, the maximum emission of tryptophan residues showed a slight redshift, which illustrates that
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TABLE 1. The Influence of n-Butanol on the Secondary Structure of Trypsin
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Circular dichroism (CD) is a simple and sensitive technique that can provide information about the conformational changes in protein [46]. As is shown in Figure 2B, two negative bands are exhibited in the CD spectrum of n-butanol in the ultraviolet region at about 208 nm and 220 nm, which are the characteristic of α-helix and β-sheet, respectively [47–49]. CDPro software was employed to calculate the secondary structural elements and the results are listed in Table 1. When n-butanol was added into the trypsin solution, the secondary structure content of trypsin fluctuated in different trends. The content of α-helix increased and the content of β-sheet decreased, which is consistent with previous spectroscopy experiments. It is implied that n-butanol changed the secondary structure of trypsin.
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the polarity around tryptophan increased and the hydrophobicity decreased [45]. Figure 3C shows a sharp increase in the fluorescence peak of trypsin when the concentration of n-butanol is 4%, which indicates that n-butanol may have denatured trypsin. This is in accordance with the results of UV–visible absorption.
Molecular Docking As a molecular modeling method that was originally used to investigate the binding information between proteins and ligands, AutoDock 4.2 software was applied to simulate the binding mode of trypsin and n-butanol in this work. As a serine protease, the active sites of trypsin are the catalytic triad Asp 102, Ser 195 and His 57 [50, 51]. The best ranked results are presented in Figure 4. Figure 4 shows the docked structure with the minimum energy. As can be observed in Figure 4A, n-butanol binds in the trypsin central cavity. Figure 4B shows that two hydrogen bonds exist between the n˚ is formed between butanol and trypsin: one (2.48 A) an oxygen atom in the n-butanol and a hydrogen atom ˚ is formed from Lys 230 residue, and the other (1.87 A) between an oxygen atom in the Met 180 residue and the hydrogen atom from n-butanol. In addition, Figure 4C shows that the n-butanol molecule located in a hydrophobic cavity on the surface of the protein, which indicates that hydrophobic interaction is one of the interaction forces between them. J Biochem Molecular Toxicology
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FIGURE 4. The molecule docking results of trypsin–butanol complex. (A) The binding site location of n-butanol to trypsin. (B) The relative residues (Gly 142 and Asp 194) of the hydrogen bonds. (C) N-butanol binds to a hydrophobic cavity of trypsin, blue sticks symbolize n-butanol.
Effect of n-Butanol on Trypsin Activity
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We have investigated the interaction between trypsin and n-butanol through the experiments above and confirmed the conformational changes in trypsin. But how the enzyme activity of trypsin changes when it is exposed to n-butanol is what fascinates us most, because it is significant for us to research proteins. Figure 5 displays the effects of various amounts of n-butanol on the activity of trypsin in vitro. We set the activity of trypsin that is without n-butanol to be 1 and other values of trypsin’s activity are calculated based on it. As shown in Figure 5, the protein activity raised when the concentration of n-butanol was low and decreased at a high concentration of n-butanol. When the concentration of n-butanol was low, the n-butanol molecule entered into the active site cavity of trypsin and made the catalytic activity center more exposed but it did not inactivate the enzyme. This was conductive to substrate binding for the enzyme. As the concentration of n-butanol increased by 5% which was near the precipitation concentration of n-butanol, the
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trypsin was denatured by n-butanol and the activity of trypsin decreased (consistent with the above results of UV–vis spectroscopy).
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DISCUSSION
and molecular docking calculations. Spectrochim Acta A Mol Biomol Spectrosc 2013;115:629-635. Neamtu S, Tosa N, Bogdan M. Spectroscopic investigation of tolmetin interaction with human serum albumin. J Pharm Biomed Anal 2013;85:277-282. Ozmen M, Maltas E, Patir IH, Bayrakci M. Combined voltammetric and spectroscopic investigation of binding interaction between nifedipine and human serum albumin on polyelectrolyte modified ITO electrode. Electrochim Acta 2013;111:535-542. Shahabadi N, Khorshidi A, Moghadam NH. Study on the interaction of the epilepsy drug, zonisamide with human serum albumin (HSA) by spectroscopic and molecular docking techniques. Spectrochim Acta A Mol Biomol Spectrosc 2013;114:627-632. Tabassum S, Al-Asbahy WM, Afzal M, Arjmand F. Synthesis, characterization and interaction studies of copper based drug with human serum albumin (HSA): spectroscopic and molecular docking investigations. J Photochem Photobiol B 2012;114:132-139. 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. Chi Z, Liu R. Phenotypic characterization of the binding of tetracycline to human serum albumin. Biomacromolecules 2011;12(1):203-209. Wang Y, Luo W, Reiser G. Trypsin and trypsin-like proteases in the brain: proteolysis and cellular functions. Cell Mol Life Sci 2008;65(2):237-252. Hirota M, Ohmuraya M, Baba H. The role of trypsin, trypsin inhibitor, and trypsin receptor in the onset and aggravation of pancreatitis. J Gastroenterol 2006;41(9):832836. Li X, Zhu S, Xu B, Ma K, Zhang J, Yang B, Tian W. Self-assembled graphene quantum dots induced by cytochrome c: a novel biosensor for trypsin with remarkable fluorescence enhancement. Nanoscale 2013;5(17):77767779. Ibarz A, Garvin A, Garza S, Pagan J. Toxic effect of melanoidins from glucose-asparagine on trypsin activity. Food Chem Toxicol 2009;47(8):2071-2075. Wang J, Liu R, Qin P. Toxic interaction between acid yellow 23 and trypsin: spectroscopic methods coupled with molecular docking. J. Biochem Mol Toxicol 2012;26(9):360-367. Yang B, Oßwald P, Li Y, Wang J, Wei L, Tian Z, Qi ¨ F, Kohse-Hoinghaus K. Identification of combustion intermediates in isomeric fuel-rich premixed butanol– oxygen flames at low pressure. Combustion Flame 2007;148(4):198-209. Veloo PS, Wang YL, Egolfopoulos FN, Westbrook CK. A comparative experimental and computational study of methanol, ethanol, and n-butanol flames. Combustion Flame 2010;157(10):1989-2004. Grana R, Frassoldati A, Faravelli T, Niemann U, Ranzi E, Seiser R, Cattolica R, Seshadri K. An experimental and kinetic modeling study of combustion of isomers of butanol. Combustion Flame 2010;157(11):2137-2154. Lin YL, Blaschek HP. Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth. Appl Environ Microbiol 1983;45(3):966-973. Veloo PS, Egolfopoulos FN. Flame propagation of butanol isomers/air mixtures. Proceedings of the Combustion Institute 2011;33(1):987–993.
In this work, the interaction mechanism of trypsin and n-butanol has been studied by spectroscopic techniques and molecular docking method under simulated physiological conditions in vitro. The fluorescence spectroscopy results revealed the changes of microenvironment of Trp residues in trypsin molecules. We found that the polarity around tryptophan increased and the hydrophobicity decreased in trypsin through the results of synchronous fluorescence (λ= 60 or 15 nm). The results of CD spectra indicate that n-butanol could bind to trypsin and change its secondary structure by increasing the content of α-helix and decreasing the content of β-sheet. The skeletal structure of trypsin is loosened and its active sites are more exposed. The enzyme activity assay, UV–visible absorption spectroscopy, and the synchronous fluorescence (λ = 0 nm) indicated that n-butanol increased the activity of trypsin when the concentration of n-butanol was low and denatured it when the concentration of n-butanol was higher than 4% (v/v). Besides, the molecular docking results detailed the information of hydrophobic interaction and revealed that hydrogen bond is the main force of the n-butanol-trypsin complex. Our work focused on the potential toxicity of nbutanol to trypsin at the molecular level. The results of these experiments gave some important information about the structure and function of protein and may contribute to comprehend the enzyme toxicity of n-butanol and human health risks in daily life.
SUPPORTING INFORMATION Fig. S1 The UV-visible absorption spectra of n-butanol. Experimental conditions: Cn-butanol (v/v): 7%, Ctrypsin = 1×10-5 mol-1L
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