Electrochemical behaviour of an antibiotic ertapenem sodium and its interactions with a model protein

Transcription

1 Electrochemical behaviour of an antibiotic ertapenem sodium and its interactions with a model protein ABSTRACT Electrochemical behavior of an antibiotic drug, ertapenem sodium (ERS) was investigated employing cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Effect of different electrolytes, ph, scan rate and concentration on the peak current of ERS was studied. The results revealed the involvement of two-electrons and one proton in the electrode process. Irreversible oxidation of ERS was observed to be diffusion controlled electrode process. Linear relationship was observed between the oxidation peak current and concentration of ERS in the range of µm. The proposed electrochemical method was effectively applied for the determination of ERS in spiked urine samples. Interaction of ERS with BSA was also explored by DPV method. Decreased peak current of ERS with positive peak shift in the presence of increased concentrations of BSA indicated the formation of an electro-inactive ERS-BSA system. In addition, binding number (m) and the equilibrium constant (β) of ERS-BSA was evaluated. Further, the interaction process was investigated by different spectroscopic methods. Dynamic quenching mechanism between ERS and BSA was proposed based on steady state fluorescence and fluorescence lifetime results. ERS-BSA system was found to be stabilized through hydrogen bond and van der Waals forces. Conformational change in BSA upon its interaction with ERS was evident from FT-IR, 3D fluorescence and CD results. Karnatak University, Dharwad. Page 219

2 Ertapenem sodium (ERS) Chemical name (4R,5S,6S)-3-[(3S,5S)-5-[(3- carboxyphenyl)carbamoyl] pyrrolidin-3- yl]sulfanyl-6-(1-hydroxyethyl)-4-methyl-7- oxo-1-azabicyclo[3.2.0]hept-2-ene-2- carboxylic acid monosodium salt H 3 C OH H H CH 3 O Structure O N S NH 2 + N H O - O Na + COO - Molecular formula C 22 H 24 N 3 O 7 SNa Molecular mass Description White to off-white hygroscopic and weakly crystalline powder Solubility Soluble in water and 0.9 % sodium chloride slolution Category Carbapenem β-lactam antibiotic Karnatak University, Dharwad. Page 220

3 INTRODUCTION ERS is a group 1 carbapenem β-lactam antibiotic that possesses a broad antibacterial spectrum including common community-acquired Gram-positive and Gram-negative aerobic and anaerobic pathogens. However, low activity against some nosocomial pathogens such as Acinetobacter spp., Pseudomonas aeruginosa, methicillin-resistant staphylococci and enterococci [1-3]. ERS is structurally different from meropenem and imipenem. ERS contains β-methyl group at C4-position like in meropenem. This provides stability against human dehydropeptidase. The benzoate anionic side chain contributes to high proteinbinding (~ 94 %) and prolongs the t 0.5 thereby allowing daily dosage once [4]. Interaction between a drug and blood protein is directly related to absorption, transportation, distribution and metabolism of drugs [5, 6]. The binding properties of drugs towards plasma proteins are extremely important for understanding the pharmacokinetics and pharmacodynamic properties of drugs and are essential for drug s transportation and distribution [7]. BSA is an ideal model protein due to its availability, stability and inimitable binding property [8]. Capillary electrophoresis and HPLC methods have been reported for the determination of ERS [9-13]. Literature survey disclosed that attempts have not been made so far to study the electrochemical behavior of ERS and its interaction with BSA. This prompted us to investigate the electrochemical behavior of ERS and its binding characteristics with BSA using electrochemical and spectroscopic methods. Reagents EXPERIMENTAL ERS was obtained a gift sample from Cipla Pvt. Ltd., India. Stock solutions of ERS of 2.5 mm and 250 µm were prepared in water for voltammetric and spectroscopic methods, respectively. Karnatak University, Dharwad. Page 221

4 Apparatus Voltammetric studies were carried out on a CHI-1110a Electrochemical analyzer (CH Instruments Ltd. Co., USA, version 4.01) with a three electrode system consisting of GCE (3 mm diameter) as the working electrode, a platinum wire as the counter electrode and an Ag/AgCl (3M KCl) electrode as reference electrode. Details of other instruments employed in the present study are given on page 35. Procedures Electrochemical behavior of ERS Electrochemical behavior of ERS (125 µm) was investigated by CV in the potential range of V at a scan rate of 100 mv s -1. Quantitative determination of ERS was carried out by DPV method in the potential range of V with the following parameters: pulse amplitude V, pulse width -0.2 s and pulse period 0.5 s. All experiments were carried out at room temperature. Multi-sweep cyclic voltammetric studies Multi-sweep cyclic voltammetry at a scan rate of 0.1 V s -1 was applied in the potential range of V with 12 sweep segments in phosphate buffer of ph 3. The concentration of ERS was maintained constant at 125 µm. Determination of ERS in spiked human urine sample Spiked urine samples were prepared by spiking 9 ml urine with 1 ml ERS solution (25 mm) to obtain 2.5 mm ERS. Suitable amount of the sample solution was transferred to the voltammetric cell containing phosphate buffer of ph 3 and differential pulse voltammograms were recorded under optimized conditions. The content of the drug in urine sample was determined referring to the calibration graph or regression equation. Karnatak University, Dharwad. Page 222

5 Differential pulse voltammetric study of interaction of ERS with BSA Differential pulse voltammograms of ERS in the absence and presence of BSA in phosphate buffer of ph 3 were recorded in the range of V. Concentration of ERS was kept constant at 125 µm and that of BSA was varied from 0 to 6.25 µm. Fluorescence quenching studies Based on preliminary experiments, concentration of BSA was kept constant at 2.5 µm and that of ERS was varied from 2.5 to 22.5 µm. Fluorescence spectra of BSA and ERS-BSA were recorded in the range of nm upon excitation at 296 nm at 289, 294, 299 and 304 K. Both the excitation and emission slit widths were set at 5 nm. Fluorescence lifetime measurements Fluorescence lifetime measurement of BSA was performed in the absence and presence of ERS. Concentration of BSA was maintained at 30 µm while that of ERS was varied from 0 to 180 µm. Obtained decay curve was analyzed using Vinci2ver 2.1 software. The quality of fitting was evaluated by χ 2 values. Competitive binding of site markers Competitive binding experiments were performed using different site probes viz., WAR, IBU and DIGI for site I, II and III, respectively. Concentrations of the protein and site probe were kept constant at 2.5 µm and that of ERS was varied from 2.5 to 22.5 µm. Absorption studies Absorption spectra of ERS in the presence of BSA and vice-versa were performed in phosphate buffer of ph 3 and ph 7.4 and were recorded in the range of nm and nm, respectively. Absorbances of ERS in the absence and presence of BSA were recorded at ph 3 by keeping the concentration of ERS constant at µm and varying the concentrations of BSA (0, 0.625, 1.313, and 4 µm). Absorption measurements of BSA in Karnatak University, Dharwad. Page 223

6 the absence and presence of ERS were recorded at ph 7.4 by keeping the concentration of BSA fixed at 2.5 µm while that of the drug was changed from 2.5 to 22.5 µm. CD spectral analysis CD spectra of BSA in the range of nm in the absence and presence of ERS were recorded in the concentration ratio of BSA to ERS at 1:0, 1:4, 1:8, 1:12 and 1:16. FT-IR spectral studies The FT-IR spectra of BSA (2.5 µm) in the absence and presence of ERS (10 µm) were recorded in the range of cm -1. Three-dimensional excitation-emission matrix (3DEEM) fluorescence studies 3DEEM fluorescence spectra were recorded in the range of nm keeping the emission wavelength and excitation wavelength at 200 nm with an increment of 10 nm up to 350 nm. Other scanning parameters were identical to those maintained for steady state fluorescence studies. Concentration of BSA and ERS was fixed at 2.5 and 12.5 µm, respectively. Effects of some cations Fluorescence spectra of ERS-BSA were recorded in the absence and presence of various common ions viz., K +, Co 2+, Cu 2+, Ni 2+, Mn 2+ and Zn 2+ in the range of nm upon excitation at 296 nm. The overall concentration of BSA and cation was fixed at 2.5 µm. The concentration of ERS was changed from 2.5 to 22.5 µm. Energy transfer between BSA and ERS Absorption spectrum of ERS (2.5 µm) was recorded in the range of nm while emission spectrum of BSA (2.5 µm) was noted down in the range of nm. The overlap of absorption spectrum of ERS with fluorescence emission spectrum of BSA was used to calculate the energy transfer. Karnatak University, Dharwad. Page 224

7 RESULTS AND DISCUSSION Electrochemical behavior of ERS Electrochemical behavior of ERS at GCE was investigated by CV. Cyclic voltammogram of ERS (125 µm) in phosphate buffer of ph 3 at GCE in the potential range of V (Fig. 1) showed one oxidation peak at V. No peaks were observed in the reverse scan suggesting that the oxidation of ERS at GCE was irreversible. Electrochemical behavior of a compound is influenced by various parameters for quantitative and reproducible results. So, we have examined the effect of supporting electrolyte, ph and scan rate. Effect of supporting electrolyte The electrochemical behavior of ERS was examined in different electrolytes such as Britton-Robinson (BR) buffer and phosphate buffer. ERS showed better peak shape and peak current in phosphate buffer compared to that in BR buffer. Therefore, we have selected phosphate buffer as the supporting electrolyte for further studies. Effect of ph Effect of ph on the peak current and peak potential of ERS was investigated in the range of (Fig. 2). ERS showed a well defined oxidation peak at ph 3. Further, the oxidation peak became broader with a negative shift in potential with increase in ph (from V at ph 3 up to V at ph 10.6). The peak shift towards negative potential revealed the involvement of protons in the electrode process. Plot of peak potential (E p ) versus ph showed linear segments between ph 4.2 and 9 (Fig. 3). These intersection points of the curve were found to be close to pka values of ERS (3.37 and 9.03) [14]. The slope of the plot of E p versus ph (Fig. 3) was found to be 29.5 mv indicating that the electrode process involved transfer of twoelectrons and one proton. Karnatak University, Dharwad. Page 225

8 Effect of scan rate Scan rate is an important parameter to assess the electrochemical process. Cyclic voltammograms of ERS in the scan rate range of mv s -1 are shown in Fig. 4. The oxidation peak current increased with increase in scan rate with positive shift in peak potential indicating that the electrode process was irreversible. Linear increase in the peak current of ERS with square root of scan rate [Fig. 4 inset (A)] revealed that the electrode process was diffusion controlled [15]. This was further confirmed by a linear relationship observed between log I p and log υ [Fig. 4 inset (B)] with a slope value of This is close to the theoretical value of 0.5 for a diffusion controlled electrode process [15]. The corresponding regression equation is log I p = log υ ; r 2 = Multi-sweep cyclic voltammetric studies In order to study the adsorption of ERS on GCE, we have carried out multi-sweep cyclic voltammertic experiment. Multi-sweep cyclic voltammograms of ERS are shown in Fig. 5. The oxidation peak current decreased with increase in sweep segment revealing the strong adsorption of oxidation products of ERS on the electrode surface. Construction of calibration curve We have employed DPV for the determination of ERS since DPV is more sensitive compared to CV. For this, the DPV parameters are as follows: phosphate buffer of ph 3, amplitude 0.05 V, pulse width 0.2 s, sampling width s and pulse period 0.5 s. A linear relation between the peak current and concentration of ERS was observed in the range of µm (Fig. 6). The plot of I p versus [ERS] showed linearity for the said concentration with a correlation coefficient of Karnatak University, Dharwad. Page 226

9 Validation of the proposed method Validation of the optimized procedure for quantitative assay of ERS was examined by the evaluation of limit of detection (LOD), limit of quantification (LOQ), precision and recovery. Detection and quantification limits The values of LOD and LOQ were calculated based on the peak current using the following equations: LOD= 3s/m (4A.1) LOQ= 10s/m (4A.2) where s is the standard deviation of the intercept (n= 5) of calibration plot and m is the slope of the calibration curve. The values of LOD and LOQ were found to be 3.10 and µm, respectively. Low values of both LOD and LOQ confirmed the sensitivity of the proposed method. Precision Inter- and intra-day assay was carried out to check the reproducibility of the results. For this, we have recorded the voltammograms of 3 replicates each of 50, 150, 300 and 450 µm ERS on same and on different days. The RSD values for inter-day assay were found to be 1.02, 0.60, 1.04 and 0.88 %, while those of intra-day assay were 0.74, 0.42, 0.53 and 0.39 %, respectively. Low values of RSD revealed the good precision of the proposed method for the assay of ERS. Determination of ERS in spiked human urine samples DPV method was applied for the determination of ERS in spiked urine samples. Drug recovery from the human urine was examined by spiking drug free urine with known amounts of ERS. The results of analysis are listed in Table 1. Higher average recovery and lower RSD values suggested good accuracy and reproducibility of the proposed method, respectively. The Karnatak University, Dharwad. Page 227

10 proposed method is simple, easy to perform and sensitive enough for the assay of ERS in urine samples. Interaction of ERS with BSA studied by DPV Optimal reaction conditions Supporting electrolyte and ph Cyclic voltammetric studies revealed that ERS has maximum peak current in phosphate buffer of ph 3. So, this buffer was selected for the interaction of ERS with BSA by DPV method. Concentration of ERS In order to check the effect of ERS the concentration of BSA was kept constant at 5 µm and that of ERS was varied (0 to 175 µm). Peak current was observed to be increased with increase in the concentration of ERS and reached maximum value at 125 µm. With further increase in the concentration of ERS, the peak current was observed to be decreased. So, 125 µm ERS was selected for further studies. Interaction time Effect of interaction time on peak current responses of ERS was also studied. For this, the concentration of ERS and BSA was kept constant at 125 and 5 µm, respectively. Increased peak current was noticed with increase in interaction time and reached maximum at 4 min and then remained constant up to 2 h. These results indicated that the reaction system possesses ample time for routine applications. DPV of ERS and ERS-BSA Differential pulse voltammograms of ERS in the absence and presence of BSA in phosphate buffer of ph 3 are shown in Fig. 7. Peak current of ERS decreased with the addition of BSA with positive shift in peak potential. No new peaks were observed in the selected potential range. These results suggested the interaction between ERS and BSA. The peak of ERS did not Karnatak University, Dharwad. Page 228

11 disappear completely with increase in the concentration of BSA. The decrease in peak current suggested the formation of an electro-inactive ERS-BSA system. The formation of electro-inactive system resulted in the decreased concentration of free ERS in solution. Effects of scan rate in the interaction process The peak current of ERS in the presence of BSA was observed to be regularly increased with increase in the scan rate (Fig. 8) with a slight positive peak potential shift. These results demonstrated an irreversible electrode process for ERS in the presence of BSA [16-18]. Further, linearity was observed in the plot of log I p versus log υ (Fig. 8 inset) with a slope of This is close to the theoretical value of 0.5 indicating the diffusion controlled process for ERS in the presence of BSA. Stoichiometry of the ERS-BSA system Stoichiomerty of ERS-BSA system was determined using voltammetric data. It was assumed that ERS interacted with BSA and formed ERS-BSA complex. The binding number (m) and equilibrium constant (β) of the binding reaction can be deduced as follows: BSA+ mers BSA-mERS (4A.3) The equilibrium constant was deduced as follows: [BSA-mERS] β= (4A.4) [BSA] [ERS] m Since I max =k C BSA (4A.5) I= k [BSA-mERS] [BSA] + [BSA-mERS]= C BSA Threfore, I max I= k (C BSA [BSA-mERS])= k [BSA] Equations (4A.4), (4A.6) and (4A.8) give (4A.6) (4A.7) (4A.8) log [ I/ ( I max I)]= log β+ m log [ERS] (4A.9) Karnatak University, Dharwad. Page 229

12 where I is the difference between the peak current of sample and blank, I max is the maximum value of difference of peak current, [BSA-mERS] and [BSA] are the total bound and free concentration of BSA in the solution, respectively. The plot (Fig. 9) of log [ I/( I max I)] versus log [ERS] showed linearity with a correlation coefficient of The values of m and β were obtained from the slope and intercept of the plot and the corresponding values were ~ 1 and x 10 4, respectively. The value of m equal to unity indicated that a stable 1:1 ERS-BSA system was formed. Interaction between ERS and BSA by spectroscopic methods To investigate the interaction between ERS and BSA by spectroscopic methods, we have selected phosphate buffer of ph 7.4 as the working solution. Fluorescence quenching studies Fluorescence spectra of ERS and ERS-BSA are shown in Fig. 10. Fluorescence intensities of BSA decreased with the addition of increased concentrations of ERS. This revealed the interaction between ERS and BSA. The observed isobestic point at 393 nm indicated the equilibrium between the free ERS and ERS-BSA system. In order to evaluate the quenching mechanism between ERS and BSA, the fluorescence data was analysed using Stern-Volmer equation shown on page 38. The linear Stern-Volmer plot (Fig. 11) indicated the existence of only one type of quenching mechanism in the interaction process. The values of K sv are given in Table 2. These values were found to be increased with increase in temperature indicating the presence of dynamic type of quenching. Lifetime measurements To confirm the quenching mechanism between the drug and protein, we have carried out fluorescence lifetime measurements of BSA and ERS-BSA. Fluorescence decay curves of BSA in the absence and presence of ERS are shown in Fig. 12. Decay curve fit was found to be bi-exponential function. The average lifetimes were calculated using the equation shown on page 36. Karnatak University, Dharwad. Page 230

13 Fluorescence lifetimes, pre-exponential factors, average lifetime and χ 2 values are listed in Table 3. Fluorescence decay curves and average lifetimes of BSA were decreased in the presence of increasing concentrations of ERS thereby confirming the dynamic quenching mechanism between ERS and BSA [19]. Binding constant and number of ERS binding to BSA Binding constant (K) and the number of ERS molecules bound to BSA (n) was calculated using the equation shown on page 40. The plot of the values of log [(F o -F)/F] versus log [Q] is shown in Fig. 13. Corresponding results are listed in Table 2. Higher magnitude of K values indicated the strong binding between ERS and BSA. Decreased K values with increase in temperature revealed the reduction of stability of ERS-BSA system. The values of n approximately equal to unity indicated that one molecule of ERS bound to one molecule of BSA. Binding mode Thermodynamic parameters such as H, S and G were used to propose the binding forces between the drug and protein and these values were calculated using the equations shown on page 40 and 41. The plot of the values of log K versus 1/T is shown in Fig. 14 and the corresponding thermodynamic parameters are shown in Table 2. Negative G values revealed the spontaneity of binding process. The negative values of H and S revealed that the hydrogen bond and van der Waals forces played a major role in the interaction process [20]. Binding site of ERS in BSA To identify the binding site of ERS in BSA, displacement experiments were carried out using WAR, IBU and DIGI as site markers. Binding constant values of ERS-BSA without probe, with WAR, IBU and DIGI were evaluated to be (1.21±0.0135) x 10 4, (1.11±0.0156) x 10 4, (2.69±0.0320) x 10 4 and (1.67±0.0195) x 10 4 M -1, respectively. These results indicated that ERS bound to site I of BSA. Karnatak University, Dharwad. Page 231

14 FRET The overlap of emission spectrum of BSA with the absorption spectrum of ERS is shown in Fig. 15. The values of E, R o and J were calculated using equations shown on page 43. The calculated values were found to be: J= x cm 3 L mol -1, R o = 1.45 nm, E= and r= 1.98 nm. The value of r (less than 8 nm) indicated that the energy has been transferred from BSA to ERS [21]. Conformational investigations Absorption studies To confirm the interaction between ERS and BSA in phosphate buffer of ph 3, we have recorded the absorption spectra of ERS in the absence and presence of BSA [Fig. 16 (A)]. ERS showed two absorption peaks at 230 and 295 nm. Absorbance of ERS increased at 230 and 295 nm regularly upon the addition of BSA with a blue shift of 5 nm. The characteristic peak of BSA at 206 nm showed a significant red shift (215 nm) in the presence of different concentrations of ERS. This red shift revealed the interaction between ERS and BSA. Further, the shift in λ max indicated the change in polarity around the Trp residue and the change in peptide strand of BSA molecules [22]. The absorption spectra of BSA in the absence and presence of ERS in phosphate buffer ph 7.4 were recorded in the range of nm and are shown in Fig. 16 (B). Absorption of BSA increased with the addition of increased concentrations of ERS indicating the interaction between ERS and BSA. CD spectra CD spectra of BSA in the absence and presence of ERS are shown in Fig. 17. The α-helical contents of free and combined BSA were calculated using the equations shown on page 45. The α-helicity of BSA was decreased from % in free BSA to % in ERS-BSA system. These results revealed that the drug was bound through amino acid residue of the main Karnatak University, Dharwad. Page 232

15 polypeptide chain of the protein and altered the secondary structure of BSA upon interaction with ERS. FT-IR studies Additional evidence regarding the secondary structural changes in BSA upon interaction with ERS was obtained from FT-IR spectroscopy. FT-IR spectra of BSA and ERS-BSA were recorded (Fig. 18). It was noticed that the peak position of amide I was shifted from to cm -1 while amide II was shifted from to cm -1 when BSA interacted with ERS. These peak shifts indicated the conformational changes in BSA upon interaction with ERS. 3DEEM fluorescence of BSA and ERS-BSA 3D fluorescence contour maps of BSA and ERS-BSA are shown in Fig. 19. Fluorescence intensity of peak 1 and 2 was clearly quenched by ERS and the corresponding results are summarized in Table 4. These results indicated the slight changes in the polypeptide structure of BSA upon interaction with ERS. Effects of some cations Trace amounts of some metal ions are important to mammal. In blood plasma, there are some metal ions which can play structural role in many proteins. These can affect the interactions of drugs with serum albumins. Hence, we have studied the effects of common ions such as K +, Co 2+, Cu 2+, Ni 2+, Mn 2+ and Zn 2+ on the binding of ERS with BSA. The binding constants of ERS-BSA system were calculated in the presence of above common ions and the corresponding results are summarized in Table 5. Binding constants of ERS-BSA increased in the presence of Cu 2+, Mn 2+ and Zn 2+ thereby enhancing the storage time of ERS in blood plasma. This might increase the effectiveness of the drug in the body. Further, the values of K decreased in the presence of K +, Co 2+ and Ni 2+ signifying the reduction of the storage time of ERS in blood plasma. Thus, more amount of free drug would be available in plasma [23]. Karnatak University, Dharwad. Page 233

16 CONCLUSIONS In the present work, the electrochemical behavior of ERS and its interaction with BSA was investigated at the molecular level using voltammetric and spectroscopic techniques. The voltammetric results showed that the electrode processes of free ERS and ERS-BSA system were diffusion controlled. Voltammetric method was successfully applied for the determination of ERS in spiked urine samples and to characterize the binding mode of ERS-BSA system. ERS was bound to BSA to form an electro-inactive ERS-BSA system. The presence of dynamic quenching mechanism between ERS and BSA was proposed by steady state fluorescence and lifetime measurement studies. Hydrogen bond and van der Waals forces stabilized the binding between ERS and BSA. Changes in the secondary structure of the protein upon interaction with BSA were confirmed by FT-IR, CD and 3D fluorescence studies. Karnatak University, Dharwad. Page 234

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