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versión On-line ISSN 0717-9707
J. Chil. Chem. Soc. v.54 n.1 Concepción 2009
J. Chil. Chem. Soc, 54, N° 1 (2009); págs: 46-50
A VOLTAMMETRIC STUDY ON THE INTERACTION OF NOVOBIOCIN WITH CYSTEINE: pH EFFECT
ENDER BİÇER*, PAKİZE QETİNKAYA
Ondokuz Mayis University, Faculty of Arts and Sciences, Department of Chemistry, 55139 Kurupelit-Samsun, TURKEY *e-mail: eBiçer@omu.edu.tr
* Author to whom correspondence should be addressed.
The interaction of novobiocin (NOV), an aminocoumarin antibiotic, with cysteine was studied by square-wave voltammetry technique on the hanging mercury drop electrode in different pH values. After the addition of NOV into the cysteine solution, the peak current of mercurous cysteine thiolate decreased and its voltammetric peak potential shifted to more positive values. Voltammetric results showed that NOV binds with cysteine forming 1:1 nonelectroactive molecular complex by means of electrostatic and hydrogen-bonding interactions. The binding constants of NOV with cysteine at pHs 5, 7 and 10 were calculated to be 3.06x103, 1.54x104 and 1.06x105 M-1, respectively. Furthermore, apossible mechanism of such interaction was also discussed.
Key words: Voltammetry, Novobiocin, Cysteine, Interaction, pH effect.
NOV (Scheme 1) is an aminocoumarin antibiotic which is apotent inhibitor of bacterial DNA gyrase1 and is produced by Streptomyces spheroids2. Although classified as a non-essential amino acid, in rare cases, cysteine (Scheme 1) may be essential for infants, the elderly, and individuáis with certain metabolic disease or who suffer from malabsorption syndromes3. Cysteine can usually be synthesized by the human body under normal physiological conditions if a sufficient quantity of methionine is available3. Cysteine is potentially toxic and is catabolized in the gastrointestinal tract and blood plasma3. The cysteine thiol group is nucleophilic. The reactivity is enhanced when the thiol ionised, and cysteine residues in proteins have pKa values close to neutrality, so are often in their reactive thiolate form in the cell4. Because of its high reactivity, the thiol group of cysteine has numerous biological functions4. Cysteine has gained a special interest among the amino acids for voltammetric investigations due to its electrochemical activity5.
Almost all drugs exert their pharmacologic effect by interactions with some kind of protein in the body and are eliminated either by combining with several transport systems or by drug-metabolizing enzymes6. The equilibrium constants and the stoichiometry of the drug-protein complexes play fundamental roles in determining the free drug concentration in plasma: this, in turn, influences the pharmacological and toxicological activities of the drugs7. In addition, the binding of drugs with non-receptor sites, such as serum proteins, has important pharmacological implications because such interactions frequently determine the rates at which drugs are adsorbed from the gastrointestinal tract, transported to various tissues and eliminated from the body6.
Immunoassay is widely used in many áreas based on the specific interaction of antibody and antigen, and the principies of electrochemical immunosensors are now well established8,9. Antibodies are commonly used in the development of biosensors for the binding affinity with small molecules8,10. Nowadays, the interactions of small molecules such as organic dyes, drugs, and toxic substances with bio-molecules have aroused great interest among chemists and biologists11,12. Studies on this type of the interactions are very useful for understanding their structure and functions11,12. Electrochemical methods are useful techniques for the study of the interaction of small molecules with biomolecules11,12. Compared with a spectroscopic method, electrochemical assay is simple, reliable and practical with low detection limit and wide dynamic range11,12. Because, electrochemical reaction occurs on the electrode/ liquid surface, it is especially suitable for small amounts of sample11,12.
No voltammetric references on the interaction of NOV with cysteine could be traced in the literature although the interactions of cysteine with folates1314, some monosaccharides15, saccharin16, pentoxifylline17 and oxacilline18 were reported. It was therefore considered important to report the use of square-wave voltammetry technique to monitor the interaction of NOV with cysteine in different pH values.
Novobiocin sodium salt and cysteine were purchased from Fluka and Merck, respectively. All chemicals were of analytical grade. The solutions of novobiocin sodium salt and cysteine were prepared by directly dissolving them in triply distilled and deionized water and used immediately. Britton-Robinson (B-R) buffer solution was used as supporting electrolyte.
A three-electrode potentiostatic control system (EG&G PARC 303A SMDE) with a hanging mercury drop electrode (HMDE), a Ag | AgCl | KClsat reference electrode and a platinum auxiliary electrode has been used in all experiments. The potential sean was generated by means of an EG&G PAR 384B Polarographic Analyzer. The recording of current-potential curves was obtained by means of a Houston Instrument DMP-40 plotter connected to the polarograph. The pH values of the buffer solution were measured with a Jenway 3010 pH meter. A UNICAM UV2-100 UV-VISIBLE spectrometer was used to record the UV-Visible absorption spectra.
Before voltammetric experiments, the solution within the electrochemical cell was deareted by purging with pure nitrogen gas for 5 min, and during measurements a stream of nitrogen gas was passed over the solution. The voltammograms were obtained by using equilibrium time of 5 s; sean rate of 200 mVs-1; sean increment of 2 mV; drop size, medium (unless stated otherwise). The interaction between cysteine and NOV was studied by using the amperometric titration. The reduction peak current of mercurous cysteine thiolate (Hg2(RS)2) was followed when the NOV concentration was increased. Also, the electronic spectra of NOV in the presence and absence of cysteine were recorded to support the interaction between them. All experiments were carried out at room temperature.
RESULTS AND DISCUSSION
The square-wave voltammograms of novobiocin and its mixture with cysteine
The reduction process of NOV was previously studied in Britton-Robinson (B-R) buffer 5.0-12.019. Typical square-wave voltammograms obtained for 9.90x10-6 M NOV in Britton-Robinson B-R buffer at pH 7.0 and 10.0 are shown in Fig. 1. As can be seen in Fig. 1, only one well-defined reduction peak was obtained. In addition, its potential depends on the nature and/or pH of supporting electrolytes, and the concentration of NOV. With the increase of buffer pH, its peak potential shifts to positive values. This peak has previously been attributed to the reduction in the coumarin group of the molecule19. This reduction has been probably carried out by means of the attack of hydroxyl ion to the coumarin moiety and then the consumption of two electrons19.
The square-wave voltammograms of 3.85x10-5 M cysteine (RSH) in Britton-Robinson B-R buffer at various pHs are shown in Figs. 2, 3 and 4. In general, cysteine exhibits two peaks which are attributed to the reduction of mercuric cysteine thiolate (Hg(RS)2) to mercurous cysteine thiolate (Hg2(RS)2) and the reduction of Hg2(RS)2 to metallic mercury and free thiolate (RS-) ions16,20,21. However, the reduction of Hg(RS)2 (1U) to Hg2(RS)2 generally appeared at the high cysteine concentrations. On the other hand, the reduction peak of Hg2(RS)2 (2U) was often seen and the main peak on the voltammograms of cysteine. As a result, the currents and potentials of these peaks (1U and 2U) also depend on the experimental conditions. The formation of mercury complexes of cysteine proceeds according to the following equations20,21:
Square-wave voltammetry were previously used to study the interaction between the molecules16,22-24. The interactions of NOV with cysteine in aqueous solutions at different pHs were monitored with square-wave voltammetry (SWV). Considering the sensitivity, the cathodic peak of Hg2(RS)2 was chosen as a probé to study the interaction of NOV with cysteine. Figs. 2, 3 and 4 show square-wave voltammograms with and without adding NOV into cysteine solution. With the addition of NOV to cysteine solution, the peak current of Hg2(RS)2 (2U) decreased and its peak potential shifted to more positive values and no new reductive peaks appeared. For the decrease of the peak current of Hg2(RS)2, the different causes can be thought: (1) the competitive adsorption between NOV and cysteine on the mercury electrode surface; (2) the formation of an electrochemically active complex; (3) the formation of electro-inactive complex11,12. An added adsorbable substance acts usually as an inhibitor of a reversible electrode reaction and shifts reduction processes to negative and oxidation processes to positive potentials25. So, the decrease in the current can not be sourced from the competitive adsorption between NOV and cysteine on the electrode surface. This current decrease showed that there were interactions between NOV and cysteine. On the other hand, the formation of an electrochemically active complex is not also said because of the factthat new reductive peaks are not seen in the same potential sean. According to the experimental data, it can be said that the interaction of NOV with cysteine forms an electro-inactive molecular complex, which can not be reduced on the mercury electrode. On the other hand, the reduction peak potential of NOV in the presence of cysteine (3U in Figs. 2, 3 and 4) lies at more positive potentials compared to that of the free NOV (Fig. 1) under the same conditions.
The voltammetric results, observed between cysteine and NOV are similar to those of folates with thiols13,14. Bard and co-workers26 reported that positive shifts in the peak potential of intercalators were observed in the binding form via hydrophobic interactions (intercalation) while electrostatic interactions led to negative shifts. However, Heyrovský and Prokopová13 reported that the effect of a chemical reaction following a reversible electron transfer is a facilitation of the redox process, i.e., a shift of electroreduction towards positive and of electrooxidation towards negative potentials. This has been established both experimentally27-29 and theoretically30-34.
Hence, for the clarification of the intercalation phenomenon or a follow-up chemical reaction, the stoichiometry of NOV-cysteine molecular complex was determined (see next section).
Measurement of stoichiometry of NOV-cysteine molecular complex and effect of pH on the equilibrium constant
According to the reported method35, the current titration equation on the binding of cysteine (RSH) with NOV is expressed as:
where, [NOV] is the concentration of NOV, K is the apparent formation constant, i and i0 and are the peak current of Hg2(RS)2 without and with NOV. A is the proportional constant. The condition of using this equation is that a 1:1 association complex is formed.Fig. 5 shows the plots of 1/ [NOV] versus 1/ (1 -i/i0) at different pH values. From the linear regression equations (Table 1), K values for pH 5, 7 and 10 are calculatedto be 3.06x103, 1.54x104and 1.06x105 M-1, respectively. These linear relationships also revealed that the interaction of NOV with cysteine was a 1:1 association complex. According to the stoichiometry of molecular complex, apart from a typical intercalation interaction, the formation of 1:1 association complex between cysteine (RSH) and NOV was observed as the result of a chemical reaction following a reversible electron transfer.
As can be seen in Table 1, with increasing pH value , the Kf value increases. This result indicated that the pH of the buffer solution greatly influences the strength of binding between them. In aqueous solutions, the ionization of cysteine depends on pH and can be described as sheme 236:
Kf value of the interaction of cysteine (RSH) with NOV at pH 10 is more close than those of other pHs 5 and 7 to the association constant (Ka= 1.8x105 M-1) that is reported by Garrido et al.37 for the binding between the peptide model, built using the natural fragment 131 -146 of DNA gyrase B (GyrB) protein and novobiocin in standard buffer (10 mM Tris-HCl, pH 7.2 / 20mM NaCl /5 mM MgCl2) at 37 °C using the relative peptide fluorescenee. The hydrogen bonds, and not ionic interactions, are suggested as the main determinants of peptid-drug binding37,38. As can be seen in this study, however, the ionization of the biomolecule or the pH of medium is effective in the cysteine-novobiocin interaction. With increasing pH, the increase in Kf value is probably due to the increasing number of negative charge centers in the cysteine molecule, forms hydrogen bonds.
On the other hand, UV/Vis absorption studies were carried out to provide an evidence for possible binding between NOV and cysteine in solution medium.
Fig. 6 shows the UV-Vis. absorption spectra of NOV in the absence and presence of different concentrations of cysteine in pH 10.0. In B-R buffer solution and in the sean range of 200 - 800 nm, NOV showed three maximum absorption peaks at 214, 238 and 303 nm, respectively (curve a). It is well known that cysteine has UV absorption at 231 nm39. When NOV was mixed with cysteine, the absorbances of NOV at 214, 238 and 303 nm decreased without the movement of maximum absorption wavelength (curves b, c). As the amount of cysteine was increased, the absorbance of NOV gradually decreased. According to these experimental results, it can be said that a binding phenomenon between cysteine and NOV is taken place and thus a new molecular complex is formed. In the literature, the similar results were observed for interactions of acid chrome blue K with protein11 and of amaranth with albumin12.
The above reported results showed that the intermolecular interactions between cysteine and NOV were present not only on the mercury electrode surface but also in solution phase in spite of the fact that a reaction between them was not.
Brief mechanism for the intermolecular interaction
It was demonstrated that NOV interaets with cysteine by means of intermolecular attraction forces. At voltammetric studies, this case is a fast chemical interaction inactivating the product of a reversible electroreduction. According to the voltammetric data, the interaction of NOV with the electrochemically generating RSH on the mercury electrode plays an important role to facilitate the electron transfer from the electrode to Hg2(RS)2. The possible mechanism about the binding of NOV to cysteine on mercury electrode surface was briefly deduced as follows:
Furthermore, UV-Vis. results showed that this interaction was also determined in solution medium. As similar to folate-thiol14 and NOV-synthetic peptide fragments of gyrase B protein37 interactions, the nature of the interaction between NOV and cysteine may be perhaps descríbed as the bonding with intermolecular forces, including hydrogen bonds.
Inthis work, the easy electron transfer of Hg2(RS)2in the presence ofNOV was first presented. The voltammetric results suggested that 1:1 molecular complex between NOV and cysteine forms with electrostatic interactions. This study also showed that voltammetric techniques could provide an effective way to characteríze both the binding mode and the interaction mechanism of NOV binding to cysteine.
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(Received 24 March 2008 - Accepted 14 July 2008)