Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 601–606

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

The effect of sarafloxacin on Cu/ZnSOD structure and activity Zhaozhen Cao a,b, Rutao Liu a,⇑, Ziliang Dong c, Xinping Yang a, Yadong Chen d a School of Environmental Science and Engineering, China–America CRC for Environment & Health, Shandong University, 27# Shanda South Road, Jinan 250100, Shandong Province, PR China b School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, Shandong Province, PR China c China Research Institute of Daily Chemical Industry, Taiyuan 030001, Shanxi Province, PR China d Laboratory of Molecular Design and Drug Discovery, School of Basic Science, China Pharmaceutical University, Nanjing 210009, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The effects of sarafloxacin on Cu/

The effects of sarafloxacin on Cu/ZnSOD were evaluated via investigating the structure and the structure basis activity changes of Cu/ZnSOD upon sarafloxacin binding using multi-spectroscopic methods, isothermal titration microcalorimetry (ITC) and molecular docking method. Sarafloxacin binds to a hydrophobic area located on the surface of b-barrel and tends to form hydrogen bonds with Tyr 108, Pro 100, Asp 25 and Ser 103 residues around it. The binding of sarafloxacin induces structure change in Cu/ZnSOD but does not affect its activity, which can be attributed to the active site and active site channel of Cu/ZnSOD being far away from the binding site and the microenvironment of them not to be affected.

ZnSOD structure and activity were investigated.  Sarafloxacin can bind to Cu/ZnSOD mainly through hydrogen bond and hydrophobic forces.  The binding process tends to be saturated as the concentration of sarafloxacin reaches 4 times of Cu/ ZnSOD.  The binding induces structure change in Cu/ZnSOD but does not affect its activity.  Sarafloxacin binds to the surface of bbarrel of Cu/ZnSOD far away from the active site and active site channels.

a r t i c l e

i n f o

Article history: Received 16 July 2014 Received in revised form 30 August 2014 Accepted 18 September 2014 Available online 13 October 2014 Keywords: Cu/ZnSOD Sarafloxacin Multi-spectroscopic methods ITC Molecular docking Enzyme activity

a b s t r a c t The effect of sarafloxacin to Cu/ZnSOD was evaluated via investigating the change in Cu/ZnSOD structure and the structure basis activity upon sarafloxacin binding. Multi-spectroscopic methods, isothermal titration microcalorimetry (ITC) and molecular docking method were adopted in this study. Sarafloxacin binds to Cu/ZnSOD mainly through hydrophobic and hydrogen bond forces and tends to be saturated as the molar ratio of sarafloxacin to Cu/ZnSOD reaches 4. The binding changed the microenvironment around Tyr and the secondary structure of Cu/ZnSOD but did not affect the activity of Cu/ZnSOD. Molecular docking study revealed that sarafloxacin binds into a hydrophobic area with possibility to form hydrogen bonds with Tyr 108, Asp 25, Pro 100 and Ser 103 of Cu/ZnSOD. The binding area locates on the surface of b-barrel close to the second Greek key loop (GK2) and V-loop but far away from the active site and active site channel of Cu/ZnSOD. These promoted the understanding of the experiment phenomenons. The binding of sarafloxacin does not affect the activity of Cu/ZnSOD should attribute to the binding not to change the microenvironment of Cu/ZnSOD active site and active site channel. Ó 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Tel./fax: +86 531 88364868. E-mail address: [email protected] (R. Liu). http://dx.doi.org/10.1016/j.saa.2014.09.073 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

602

Z. Cao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 601–606

Introduction Sarafloxacin (Scheme 1) is a fluoroquinolone antibiotic drug which acts by inhibiting the activity of DNA gyrase. As a veterinary medicine, it was registered for use in food animals in the United States since 1995 and it is still widely applied in poultry production and fish farming worldwide today [1,2]. The extensive use of fluoroquinolones has led to their widespread presence in environment [3–5]. The potential hazards of fluoroquinolone in environment including animal origin foods, waters, soils and marsh sediments to human being and ecosystem have aroused wide attention [6–11]. Superoxide dismutases (SODs, EC 1.15.1.1) are ubiquitous antioxidant enzymes which catalyze the dismutation of superoxide radicals (O 2 ) to oxygen (O2) and hydrogen peroxide (H2O2). Superoxide radicals are byproducts of normal aerobic metablism. About two percent of the oxygen involved in aerobic metabolism turns into superoxide radicals. The excessive accumulation of these highly reactive species can attack biological macromolecules, strip their electrons, destroy their function, and thereby lead to cellular oxidative damage [12]. Superoxide dismutases, serving as the first-line oxidant defense in biological system, play major role in controlling cellular redox state and maintaining proper cellular function through efficient removal of superoxide radicals [13,14]. Changes in the SODs structures and activities are related to cell biology, aging and a number of diseases such as psychiatric diseases, cardiovascular diseases, diabetes and cancers [15–20]. Therefore, the investigations on the effects of contaminants on the structures and activities of SODs are of great importance for assessing their toxicity. It was reported, fluoroquinolones are associated with oxidative stress and the change in SOD activity [11,21–23]. Orally intake of ciprofloxacin, levofloxacin and gatifloxacin induced from substantial to slight degrees depletion in plasma SOD levels of urinary tract infection patients [21]. Administration of enrofloxacin without withdrawal period led to significant decrease in SOD activities in broiler liver [11]. The injection of danofloxacin enhanced the SOD activities in Balb/C mice liver while the ofloxacin feeding lowered the SOD activities in liver, blood and renal tissues of Mus musculus mice [22,23]. However, investigation focus on the impact of sarafloxacin to SODs has not been reported. Protein structures are closely related to their functions [24,25]. Given this relation, study on the conformational change of protein is very important for understanding their molecular basis functions. Fluoroquinolones tend to directly interact with biomacromolecules and change their structure [26–28]. Previous studies showed that sarafloxacin can bind to HAS and DNA [29,30]. Our investigation also demonstrated that sarafloxacin can bind to the a-helix area of catalase (another primary antioxidant enzyme) and induce alteration in its peripheral substituents of the porphyrin ring of

heme and lead to inhibition in its activity [31]. In this work, the interaction mechanism of sarafloxacin with Cu/ZnSOD and the relation between the change in structure and activity of Cu/ZnSOD were investigated using multi-spectroscopic methods, isothermal titration microcalorimetry (ITC) and molecular modeling method. The experiment results indicated that sarafloxacin binds to Cu/ ZnSOD mainly through hydrogen and hydrophobic interactions. The binding results in the structure change of Cu/ZnSOD but does not affect its activity. Molecular docking supports the experiment results. This study proved in molecular level that the toxicity of sarafloxacin to Cu/ZnSOD is limited.

Materials and methods Materials and reagents Sarafloxacin was provided by the Institute of Veterinary Medicine of China. Cu/ZnSOD from porcine erythrocyte was provided by Beijing BioDee BioTech Corporation Ltd. NaH2PO42H2O and Na2HPO412H2O (analytical reagent grade) were obtained from Tianjin Damao Chemical Reagent Factory. Cu/ZnSOD activity assay kit was provided by Nanjing Jiancheng Bioengineering Institute. Tap water filtered by Milli-Q system (Millipore, Watford, U.K.) was used throughout the experiment. 0.2 mol L1 phosphate buffer (PBS, mixture of NaH2PO42H2O and Na2HPO412H2O) was used to control the pH. Cu/ZnSOD stock solution was prepared by directly dissolving Cu/ZnSOD in water. Sarafloxacin stock solution (5.0  103 mol L1) was prepared by dissolving sarafloxacin in water with 0.1 mol L1 NaOH solution as hydrotropic agent. All stock solutions were preserved at 0–4 °C. Absorption measurements Absorption measurements were performed on U-4100 spectrophotometers (Hitachi, Japan) equipped with 10 mm quartz cell. The slit width and scan speed were set as 5.0 mm and 120 nm/ min, respectively. The absorption spectra of Cu/ZnSOD with different concentrations of sarafloxacin were collected in the range of 202–240 nm and 270–315 nm with corresponding concentrations sarafloxacin as reference while the absorption spectra of sarafloxacin in the absence and in the presence of Cu/ZnSOD were collected in the range of 250–300 nm with corresponding concentrations of Cu/ZnSOD as reference. Synchronous fluorescence measurements Synchronous fluorescence spectra were recorded on F-4600 fluorescence spectraphotometer (Hitachi, Japan) with kex = 230– 320 nm and Dk = 15 nm. The excitation and emission slit widths were set at 5.0 nm. Multiplier Tube (PMT) voltage was 750 V. Time-resolved fluorescence Measurements

Scheme 1. Chemical structure of sarafloxacin.

Fluorescence lifetimes of Cu/ZnSOD in the absence and in the presence of sarafloxacin were measured on FLS920 fluorometer (Edinburgh, U.K.) with nanosecond flash lamp as excitation light source and time correlated single photon counting (TCSPC) as probing method. All spectra were collected at room temperature in a 0.5 mm quartz cell with kex/em = 278/324 nm. Both excitation and emission slit widths were fixed at 14 nm. The fluorescence decay curves were fitted to a double-exponential decay function using F900 software. In order to better explain the alteration in structural dynamics of Cu/ZnSOD upon sarafloxacin binding the average decay time s and the standard deviations r were calculated using the following equations [29]

603

Z. Cao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 601–606

s ¼ s1 f 1 þ s2 f 2 h

ð1Þ i1=2

r ¼ ððs  s1 f 1 Þ2 þ ðs  s2 f 2 Þ2 Þ=2

ð2Þ

where s1 and s2 represent the lifetimes of the decay components while f1 and f2 represent the corresponding fractional intensity. Circular dichroism measurements Circular dichroism (CD) spectra were collected on Jasco-810 spectrophotometer (Jasco, Japan) equipped with a 0.5 mm quartz cell. The measurements were performed in the range of 200– 260 nm with scanning speed of 100 nm/min. Each spectrum is the average of three measurements. All CD spectra were analyzed by Jasco secondary structure manager software with Yang.jwr as reference [32]. ITC measurements Calorimetric titrations were performed on a MicroCal ITC200 microcalorimeter (Microcal Inc., Northampton, MA) at 298 K. All solutions were prepared using 0.04 M PBS (pH 7.4). Saraflxacin solution (1.1 mM) was filled into the 200 lL sample cell and Cu/ZnSOD solution (0.1 mM) was loaded into the 40 lL injection syringe. The titration process was computer-controlled by injecting 3 lL sarafloxacin solution into the isothermal sample cell each time (the first injection was 0.4 lL). There are 14 consecutive injections with a 150 s interval in each experiment. The solution in the sample cell was stirred at 1000 rpm to ensure thorough mixing. Control experiment was performed under the identical operation conditions by titrating sarafloxacin solution into 0.04 M PBS. The experiment results were analyzed using the calorimeter software.

Fig. 1a and b, Cu/ZnSOD exhibites two absorption bands. The strong absorption band with maximum absorption wavelength at 206 nm is corresponding to the p ? p⁄ electronic transitions of C@O in protein polypeptide backbone while the weak absorption band with maximum absorption wavelength at 275 nm is associated with the conjugated system in aromatic amino acids. The gradual addition of sarafloxacin lead to dramatic decreases in the absorption at 275 nm and moderate decreases in the absorption at 206 nm accompanied by obvious red shifts. This phenomenon suggests that sarafloxacin binds to Cu/ZnSOD and the binding makes aromatic amino acid and peptide strands of Cu/ ZnSOD expose to a more hydrophilic microenvironment. The binding of sarafloxacin to Cu/ZnSOD was also proved by the absorption spectra of sarafloxacin (Supporting information, Fig. 1). The absorption band in the range of 250–300 nm with maximum at 272 nm is typical for the p ? p⁄ electronic transition in sarafloxacin aromatic ring. Upon increasing Cu/ZnSOD concentration, significant decrease in absorption intensity and slight blue shift at 272 nm were observed, indicating sarafloxacin aromatic ring moves into a hydrophobic cavity of Cu/ZnSOD or (and) the side chain atoms of sarafloxacin bind to Cu/ZnSOD amino acid residues which causes inhibition of p ? p⁄ electronic transition and shift in absorption band. Besides, both Cu/ZnSOD and sarafloxacin absorption spectra are gradual changing with the increasing concentration of sarafloxacin and Cu/ZnSOD, suggesting that the binding process should be reversible. Especially for Cu/ZnSOD, the decreases in the intensities of its two absorption bands almost simultaneously stopped as the molar ratio of sarafloxacin to Cu/ZnSOD reaches 4. This may be because as the reversible binding process achieves equilibrium at sarafloxacin concentration is 4 times of Cu/ZnSOD the binding tends to be saturated. Furthermore, the uniformity in the alteration trend of the two absorption bands of Cu/ZnSOD upon sarafloxacin

0.5

The activities of CuZnSOD in the absence and presence of sarafloxacin were detected using a commercial bioassay kit. Superoxide radicals generated from the incubation of xanthine and xanthine oxidase system can react with the included chromagen to produce a water-soluble dye which causes absorbance at 550 nm. The presence of CuZnSOD catalyzes the dismutation of superoxide radicals and therefore reduces the colorimetric signal. The absorption values were measured on U-4100 spectrophotometers (Hitachi, Japan) in 10 mm quartz cuvet. The CuZnSOD activity is calculated by the following formula:

0.4

SOD activity ð%Þ ¼ ðODblank  ODsample Þ=ODblank  100

Absorbance

SOD activity determination

(a)

a b c d e f g

0.3 0.2 0.1 0.0 210

220

230

240

Wavelength (nm)

ð3Þ 0.09

Molecular docking was performed on a bovine superoxide dismutase crystal model (PDB code: 1CBJ) using molecular operating environment software (MOE) (Version 2009, Chemical Computing Group Inc, Canada).The 3D structure of sarafloxacin was generated and optimized using the MOE Builder module and the Energy Minimize module, respectively. Results and discussion

Absorbance

Molecular modeling investigation

(b)

a b c d e f g

0.06

0.03

0.00 270

285

300

315

Wavelength (nm) Studies on Cu/ZnSOD conformation changes UV–vis absorption studies UV–vis absorption spectroscopy is an efficient technique for exploring conformational changes of molecules. As shown in

Fig. 1. UV–vis spectra of Cu/ZnSOD in absence and presence of sarafloxacin. Experimental conditions: pH = 7.4; At room temperature. (a) CCu/ZnSOD: 1.25  106 mol L1; Csarafloxacin/(106 mol L1): a. 0; b. 1.25; c. 2.5; d. 3.75; e. 5.0; f. 6.25; g. 7.5. (b) CCu/ZnSOD: 1.25  105 mol L1; Csarafloxacin/(105 mol L1): a. 0; b. 1.25; c. 2.5; d. 3.75; e. 5.0; f. 6.25; g. 7.5.

604

Z. Cao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 601–606

Fluorescence intensity

5000

a

4000 3000

g 2000 1000 0

260

280

300

320

Wavelength (nm) Fig. 2. Synchronous fluorescence spectra of Cu/ZnSOD. Experimental conditions: pH = 7.4; Dk = 15 nm; At room temperature; CCu/ZnSOD: 1.0  105 mol L1; Csarafloxacin/(105 mol L1): a. 0; b. 1.0; c. 2.0; d. 3.0; e. 4.0; f. 5.0; g. 6.0.

binding indicates that the conformational change in polypeptide backbone should basically be induced by the change in the microenvironment around aromatic amino acid residues. Thus one can speculate that sarafloxacin may exclusively bind to the vicinity of aromatic amino acid residues.

Synchronous fluorescence investigations Porcine erythrocyte Cu/ZnSOD is a single tyrosine protein [33]. In order to obtain the characteristic information of the microenvironment around tyrosine, synchronous fluorescence scans were performed by setting the excitation and emission wavelengths interval as 15 nm (Fig. 2). The increasing concentration of sarafloxacin induces a gradual red shift in the maximum emission wavelength of Cu/ZnSOD synchronous spectra demonstrating that sarafloxacin binds to Cu/ZnSOD and the binding moves Tyr residue to a more hydrophilic microenvironment. It is noteworthy, the bathochromic effects of Cu/ZnSOD synchronous spectra become much weak while the molar ratio of sarafloxacin to Cu/ZnSOD is greater than 4. These are in good agreement with the absorption spectroscopy results of Cu/ZnSOD at 275 nm, suggesting the binding site of sarafloxacin should be in the vicinity of tyrosin and as the concentration of sarafloxacin reaches 4 times of Cu/ZnSOD the binding of sarafloxacin to Tyr area basically reaches saturation.

Time-resolved fluorescence studies Fluorescence decay of porcine erythrocyte Cu/ZnSOD reflects the structural dynamics of the protein matrix surrounding Tyr residues [34]. Cu/ZnSOD exhibits heterogeneous fluorescence decay with two lifetimes (Fig. 3) which has been attributed to the existence of the conformational substates in Cu/ZnSOD and the interconversion between them [31,34,35]. In order to better illustrate the effect of srafloxacin on Cu/ZnSOD, average fluorescence lifetimes s and standard deviations r were calculated (Table 1). Increasing concentration of sarafloxacin promotes gradual decrease in the average fluorescence lifetime and standard deviation of Cu/ZnSOD, which reflects the change in the conformational dynamics of the protein. The decrease in the s value can be interpreted as the microenvironment change around Tyr residues and the energy transfer from Tyr residues to the bound sarafloxacin. The reduction in r value is associated with the interconversion between the conformational substates in Cu/ZnSOD [34,35]. The binding of sarafloxacin may decrease the height of the energy barriers between the substates and thus lead to an increase in substate interconversion rates and then to a lower r value. Moreover, the variations in s and r values become insignificant as the molar ratio of sarafloxacin to Cu/ZnSOD changes from 4 to 6, which lends support to the binding saturation conclusion once more. Circular dichroism studies In order to characterize the secondary structure change of Cu/ ZnSOD upon sarafloxacin binding, circular dichroism (CD) measurements were performed. The addition of sarafloxacin to Cu/ZnSOD induced a slight increase in the intensity of Cu/ZnSOD CD signal which corresponds to a slight secondary structural alteration (Table 2 and Supporting information Fig. 2). As the concentration of sarafloxacin is 4 times of Cu/ZnSOD, the contents of b-pleated sheet decrease from 57.1% to 55.1% while the contents of b-turn and Random coil increase from 2.2% to 3.7% and from 26.3% to 27.0%, respectively. As the concentration of sarafloxacin to Cu/ZnSOD increased from 4 to 8, only mild alterations in the contents of b-turn and Random coil were observed (both a-helix and b-pleated sheet contents almost keep same), indicating the affect of sarafloxacin to the Cu/ZnSOD secondary structure become very little. These supported the binding saturation conclusion further. Besides, the relatively stable a-helix contents suggest that the binding site of sarafloxacin should be far away from a-helix area.

Fig. 3. Time-resolved fluorescence decay of Cu/ZnSOD in the presence of different concentrations of sarafloxacin. Experimental conditions: pH = 7.4; At room temperature; CCu/ZnSOD: 25 lM; Csarafloxacin (lM): a. 0; b. 50; c. 100; d. 150.

Z. Cao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 601–606

605

Table 1 Average fluorescence lifetime and standard deviation of Cu/ZnSOD at different sarafloxacin concentrations. Csarafloxacin (lM)

0

50

100

150

s (ns) r (ns)

2.8726 1.7435

2.6564 1.6216

2.5358 1.5535

2.5051 1.5249

Experimental conditions: Cu/ZnSOD concentration is 25 lM, kex/em = 278/324 nm.

Table 2 Changes in the secondary structure of Cu/ZnSOD in the absence and presence of sarafloxacin. Molar ratio of sarafloxacin to SOD

Secondary structural elements in Cu/ZnSOD

a-helix (%)

b-sheet (%)

b-turn (%)

Random coil (%)

0:1 4:1 8:1

15.4 15.1 15.3

58.3 54.9 55.1

1.1 2.9 3.8

25.2 27.1 25.8

Experimental conditions: the concentration of Cu/ZnSOD is 25 lM.

Cu/ZnSOD activity

100 80 60 40 20 0

0

5

10

Sarafloxacin concentration (x 10-5) Fig. 4. Effect of sarafloxacin on the activity of Cu/ZnSOD. Experimental conditions: T = 310 K; CCu/ZnSOD: 5.0  106 mol L1; Csarafloxacin/(105 mol L1): a. 0; b. 1.0; c. 2.0; d. 3.0; e. 5.0; f. 10.0. Error bars are the standard deviations of three.

Thermodynamic properties of SOD-sarafloxacin binding Isothermal titration calorimetry (ITC) is a physical technique used to directly measure the heat changes of biomolecular reactions. Raw ITC data of the titration of sarafloxacin (1.11  103 M, pH 7.4, 298 K) into Cu/ZnSOD (1.0  104 M, pH 7.4, 298 K) are obtained as a plot of heat flow spikes against time (Supporting information, Fig. 3, the upper panel). These heat flow spikes are integrated and normalized to obtain the plot of enthalpy change of per mole injectant (DH/mole) against molar ratio. The heats of the blank experiment are subtracted from the titration experiment of sarafloxacin into Cu/ZnSOD to correct the dilution effect (Supporting information, Fig. 3, the bottom panel). The binding of sarafloxacin to Cu/ZnSOD is a weak exothermic process, indicating that the binding is mainly driven by entropy change (DS). The hydrogen interaction and hydrophobic force should play major role in the formation of sarafloxacin-SOD complex [36].

Effect of sarafloxacin on Cu/ZnSOD activity In order to understand the impacts of sarafloxacin on Cu/ZnSOD function, the activities of Cu/ZnSOD with different concentrations of sarafloxacin were measured. As shown in Fig. 4, the increasing concentration of sarafloxacin does not alter the Cu/ZnSOD activity,

Fig. 5. (a) The binding site of sarafloxacin to Cu/ZnSOD. (b) The hydrogen bond interactions between sarafloxacin and the amino acid residues around it. (c) Sarafloxacin binds into a hydrophobic area of Cu/ZnSOD. The green-colored part represents hydrophobic area; The pink-colored part represents hydrogen bond interaction area; The blue-colored part represents mild polar area. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

even as the concentration of sarafloxacin reaches 20 times of Cu/ ZnSOD. This proves that the toxicity of sarafloxacin to Cu/ZnSOD is limited. Molecular docking studies To obtain further insight into the binding modes of sarafloxacin with Cu/ZnSOD and the relation between the conformational change induced by sarafloxacin binding and Cu/ZnSOD activity, molecular docking was performed using MOE. Because porcin erythrocyte Cu/ZnSOD is a homodimer with high homology sequence to that of the bovine erythrocyte Cu/ZnSOD [34], one

606

Z. Cao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 601–606

subunit of bovine erythrocyte superoxide dismutase crystal model (PDB code: 1CBJ) was used in this docking. Sarafloxacin binds to a hydrophobic area and tends to form hydrogen bonds with Tyr 108, Asp 25, Pro 100 and Ser 103 residues of Cu/ZnSOD (Fig. 5c, b and Supporting information, Table 1), indicating that hydrophobic force and hydrogen bond force play key role in the binding process, which is in agreement with the thermodynamic study result. The bound sarafloxacin locates on the surface of b-barrel close to Tyr 108 (Fig. 5a and b) which makes it easy to explain why the binding of sarafloxacin can induce significant change in the microenvironment around Tyr and produces obvious change in the contents of b-sheet and b-turn. More important, the binding site of sarafloxacin is in the proximity of the GK2 loop and V-loop and far away from the active site and the active site channels (b4/b5 loop and b7/b8 loop) of Cu/ZnSOD (Fig. 5a) [20], demonstrates that the structural alteration in Cu/ZnSOD induced by the binding of sarafloxacin should not significantly affect the microenvironment of the active site and the active site channels which explains why the binding of sarafloxacin does not affect the activity of Cu/ZnSOD. Conclusions In this paper, the interaction of sarafloxacin with Cu/ZnSOD and the activity alteration of Cu/ZnSOD induced by sarafloxacin binding were studied. Sarafloxacin binds to the surface of b-barrel mainly through hydrophobic and hydrogen bond forces. The binding results in structure change around Tyr residue of Cu/ZnSOD but does not affect the activity of the enzyme, which should attribute to the binding site being far away from the enzyme active sites and active site channels and thus their microenvironment does not be affected. This study provided the detailed data for evaluating the toxicity of sarafloxacin to Cu/ZnSOD in molecular level and proved that the direct toxicity of sarafloxacin to Cu/ZnSOD is very weak. Acknowledgements This work is supported by NSFC (21277081), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education’’, Ministry of Education of China (708058, 20130131110016), and Independent innovation program of Jinan (201202083) are also acknowledged. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.09.073. References [1] R.N. Jones, M.E. Erwin, In vitro susceptibility testing and quality control parameters for sarafloxacin (A-56620): a fluoroquinolone used for treatment and control of colibacillosis in poultry, Diagn. Microbiol. Infect. Dis. 32 (1) (1998) 55–64. [2] A. Tyrpenou, E. Iossifidou, I. Psomas, G. Fotis, Tissue distribution and depletion of sarafloxacin hydrochloride after in feed administration in gilthead seabream (Sparus aurata L.), Aquaculture 215 (2003) 291–300. [3] V. Andreu, C. Blasco, Y. Picó, Analytical strategies to determine quinolone residues in food and the environment, TrAC, Trends Anal. Chem. 26 (2007) 534–556. [4] Y. Picó, V. Andreu, Fluoroquinolones in soil–risks and challenges, Anal. Bioanal. Chem. 387 (2007) 1287–1299. [5] P. Sukul, M. Spiteller, Fluoroquinolone antibiotics in the environment, Rev. Environ. Contam. Toxicol. 191 (2007) 131–162. [6] J.H. Choi, T.W. Na, M. Rouf Mamun, Bufferized solvent extraction and HPLC fluorometric detection method for sarafloxacin in pig and chicken muscles, Biomed. Chromatogr. 25 (2011) 405–411.

[7] B. Roudaut, J.-C. Yorke, High-performance liquid chromatographic method with fluorescence detection for the screening and quantification of oxolinic acid, flumequine and sarafloxacin in fish, J. Chromatogr. B 780 (2002) 481– 485. [8] L. Aristilde, A. Melis, G. Sposito, Inhibition of photosynthesis by a fluoroquinolone antibiotic, Environ. Sci. Technol. 44 (2010) 1444–1450. [9] S. Kusari, D. Prabhakaran, M. Lamshöft, M. Spiteller, In vitro residual antibacterial activity of difloxacin, sarafloxacin and their photoproducts after photolysis in water, Environ. Pollut. 157 (2009) 2722–2730. [10] H.-C. Holten Lützhøft, B. Halling-Sørensen, S.E. Jørgensen, Algal toxicity of antibacterial agents applied in Danish fish farming, Arch. Environ. Contam. Toxicol. 36 (1999) 1–6. [11] I. Carreras, M. Castellari, J.G. Regueiro, L. Guerrero, E. Esteve-Garcia, C. Sarraga, Influence of enrofloxacin administration and a-tocopheryl acetate supplemented diets on oxidative stability of broiler tissues, Poult. Sci. 83 (2004) 796–802. [12] I.A. Abreu, D.E. Cabelli, Superoxide dismutases–a review of the metalassociated mechanistic variations, Biochim. Biophys. Acta 2010 (1804) 263– 274. [13] J.V. Bannister, W.H. Bannister, R.C. Bray, E.M. Fielden, The superoxide dismutase activity of human erythrocuprein, FEBS Lett. 32 (2) (1973) 303–306. [14] I. Fridovich, Superoxide anion radical (O2), superoxide dismutases, and related matters, J. Biol. Chem. 272 (1997) 18515–18517. [15] R.P. Singh, S. Sharad, S. Kapur, Free radicals and oxidative stress in neurodegenerative diseases: relevance of dietary antioxidants, Indian Acad. Clin. Med. 5 (3) (2004) 218–225. [16] J.J.P. Perry, L. Fan, J.A. Tainer, Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair, Neuroscience 145 (2007) 1280–1299. [17] T. Fukai, R.J. Folz, U. Landmesser, D.G. Harrison, Extracellular superoxide dismutase and cardiovascular disease, Cardiovasc. Res. 55 (2002) 239–249. [18] R.M. Cardoso, M.M. Thayer, M. DiDonato, T.P. Lo, Insights into Lou Gehrig’s disease from the structure and instability of the A4V mutant of human Cu, Zn superoxide dismutase, J. Mol. Biol. 324 (2002) 247–256. [19] M.E. Gurney, R.G. Liu, J.S. Althaus, E.D. Hall, Mutant Cu, Zn superoxide dismutase in motor neuron disease, Age 21 (2) (1998) 85–90. [20] J. Perry, D. Shin, E. Getzoff, J. Tainer, The structural biochemistry of the superoxide dismutases, Biochimica et Biophysica Acta (BBA)-Proteins Proteomics 1804 (2) (2010) 245–262. [21] P. Veerareddy, Oxidative stress induced by fluoroquinolones on treatment for complicated urinary tract infections in Indian patients, J. Young Pharmacists 3 (2011) 304–309. [22] E. Yazar, B. Tras, Effects of fluoroquinolone antibiotics on hepatic superoxide dismutase and glutathione peroxidase activities in healthy and experimentally induced peritonitis mice, Revue de Med. Vet. 152 (2001) 235–238. [23] I. Thomson, A. Soni, M. Chaudhary, A. Tamta, R. Sehgal, Therapeutic efficacy of ofloxacin and ornidazole vs mebatic: toxicity profile and antioxidant defense study, Res. J. Parasitol. 4 (2009) 79–86. [24] H. Frauenfelder, E. Gratton, Protein dynamics and hydration, Methods Enzymol. 127 (1986) 207–216. [25] H. Frauenfelder, F. Parak, R.D. Young, Conformational substates in proteins, Ann. Rev. Biophys. Biophys. Chem. 17 (1988) 451–479. [26] P.F. Qin, R.T. Liu, Oxidative stress response of two fluoroquinolones with catalase and erythrocytes: a combined molecular and cellular study, J. Hazard. Mater. 252–253 (2013) 321–329. [27] J. Chamani, N. Tafrishi, M. Momen-Heravi, Characterization of the interaction between human lactoferrin and lomefloxacin at physiological condition: multi-spectroscopic and modeling description, J. Lumin. 130 (2010) 1160– 1168. [28] Y. Lu, G.K. Wang, X.M. Lu, Molecular mechanism of interaction between norfloxacin and trypsin studied by molecular spectroscopy and modeling, Spectrochim. Acta 75 (2010) 261–266. [29] L.M. Cao, H. Lin, V.M. Mirsky, Detection of antibiotics in food: extraction of fluoroquinolones by DNA, Anal. Bioanal. Chem. 388 (1) (2007) 253–258. [30] L.W. Zhang, K. Wang, X.X. Zhang, Study of the interactions between fluoroquinolones and human serum albumin by affinity capillary electrophoresis and fluorescence method, Anal. Chim. Acta 603 (2007) 101– 110. [31] Z.Z. Cao, R.T. Liu, B.J. Yang, Potential toxicity of sarafloxacin to catalase: spectroscopic, ITC and molecular docking descriptions, Spectrochim. Acta 115 (2013) 457–463. [32] J.T. Yang, C.S. Wu, H.M. Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol. 130 (1986) 208–269. [33] M.E. Schininà, D. Barra, M. Simmaco, F. Bossa, Primary structure of porcine Cu– Zn superoxide dismutase, FEBS Lett. 186 (2) (1985) 267–270. [34] S.T. Ferreira, L. Stella, E. Gratton, Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue, Biophys. J. 66 (1994) 1185–1196. [35] J.R. Alcala, E. Gratton, F. Prendergast, Fluorescence lifetime distributions in proteins, Biophys. J. 51 (1987) 597–604. [36] Y.-L. Zhang, X. Zhang, X.-C. Fei, S.-L. Wang, H.-W. Gao, Binding of bisphenol A and acrylamide to BSA and DNA: insights into the comparative interactions of harmful chemicals with functional biomacromolecules, J. Hazard. Mater. 182 (2010) 877–885.

ZnSOD structure and activity.

The effect of sarafloxacin to Cu/ZnSOD was evaluated via investigating the change in Cu/ZnSOD structure and the structure basis activity upon saraflox...
983KB Sizes 0 Downloads 5 Views