The binding characteristics of isoniazid with copper–zinc superoxide dismutase and its effect on enzymatic activity
© Du et al.; licensee Chemistry Central Ltd. 2013
Received: 17 April 2013
Accepted: 30 May 2013
Published: 6 June 2013
Isoniazid (INH) is front-line anti-tuberculosis (TB) drugs, which are usually prescribed to TB patients for a total period of 6 months. Antituberculosis drug-induced hepatotoxicity (ATDH) is a serious adverse reaction of TB treatment. It is reported that INH-induced hepatotoxicity is associated with oxidative stress. Superoxide dismutase (SOD, EC 22.214.171.124) is the key enzyme for the protection of oxidative stress, which catalyzes the removal of superoxide radical anion, thereby raising the need to better understand the interaction between INH and SOD.
The experimental results showed that the fluorescence intensity of Cu/Zn-SOD regularly decreased owing to form a 1:1 INH-SOD complex. According to the corresponding association constants (KSV) between INH and SOD obtained from Stern–Volmer plot, it is shown that values of KA are 1.01 × 104, 5.31 × 103, 3.33 × 103, 2.20 × 103 L · mol−1 at four different temperatures, respectively. The binding constants, binding sites and the corresponding thermodynamic parameters (ΔH, ΔG and ΔS) were calculated. A value of 3.93 nm for the average distance between INH and chromophore of Cu/Zn-SOD was derived from Förster theory of non-radiation energy transfer. The conformational investigation showed that the presence of INH resulted in the microenvironment and conformational changes of Cu/Zn-SOD. In addition, Effects of INH on superoxide dismutase activity was examined.
The results show that the hydrogen bonding and van der Waals forces play major roles in stabilizing the 1:1 INH-SOD complex. After addition of INH during the range of the experiment, the conformation and microenvironment of Cu/Zn-SOD are changed, but the activity of Cu/Zn-SOD is not changed.
Tuberculosis (TB) is one of the leading causes of death due to a single disease, accounting for up to 2 million lives each year . Isoniazid (INH) is the foremost first-line antibiotic used to treat TB, which continues to be the cornerstone of all antituberculosis regimens and remains the only agent recommended for tuberculosis chemoprophylaxis for children .
Despite numerous and intensive studies, we have a limited knowledge of the action mechanism of INH [3–6]. The consensus opinion is that in the presence of a slow flux of H2O2 or superoxide, KatG converts INH into a radical species which is subsequently coupled to NADH to form an INH-NAD(P) adduct. The INH-NAD adduct is a potent inhibitor of Mtb InhA, an enoyl reductase required for the elongation steps in mycolic acid biosynthesis [7, 8]. It has been recently suggested that the superoxide reactivity affects antitubercular activity of INH to some extent, and thereby raises the needs to better understand the interaction between INH and superoxide .
Furthermore, with increasing occurrence of TB all over the world, a growing number of patients may be at risk for severe adverse drug reactions (ADRs) such as antituberculosis drug-induced hepatotoxicity (ATDH) when treated with antituberculosis chemotherapy [10, 11]. ATDH can be fatal if it is not recognised at an early stage, after which therapy should be interrupted timely. Moreover, ATDH has a negative impact on therapy adherence, decreases success rates of treatment and eventually increases treatment failure, relapse or the emergence of drug resistance. The occurrence of hepatotoxicity related to INH has been well-defined  and shown to increase as a result of drug-drug pharmacokinetic or pharmacodynamic interactions . It is generally acknowledged that INH-induced hepatotoxicity is associated with oxidative stress .
Superoxide dismutase (SOD, EC 126.96.36.199) is an antioxidant enzyme in animals, plants, fungi and bacteria, which catalyzes the removal of superoxide radical anion (O2−•) to hydrogen peroxide (H2O2) that can be subsequently converted to water by the enzyme catalase [15, 16]. Therefore, it is the key enzyme for the protection of oxidative stress and could be a suitable candidate drug to protect liver from ATDH [17–19]. To better understand the protective mechanism of SOD, the interaction between SOD and INH should be investigated.
Protein–drug interaction is a hot topic in fields of medicine, chemistry and biology. Also, due to the effects on the drug of pharmacokinetics when bounded to protein, there is an increasing interest in protein from clinical and pharmaceutical perspectives [20–22]. Furthermore, the binding of drugs to protein in vitro is considered as a model in protein chemistry to study the binding behavior of protein. Consequently, the investigation of the binding between drugs and SOD is of fundamental importance in pharmacology and pharmacodynamics. In addition, the analysis of the interaction between SOD and small drug molecules is interesting to some extent, because the binding of specific small molecules to SOD has only been characterized in detail for a few examples [23, 24].
In this paper, the interaction between INH and Cu/Zn-SOD has been investigated with fluorescence and ultraviolet spectroscopy. The binding mechanism, binding constants, binding sites and binding distance were obtained. The nature of the binding force was analyzed based on the thermodynamic parameters. Also, the effects of INH on the conformation of Cu/Zn-SOD were examined by synchronous fluorescence and three-dimensional fluorescence spectroscopy. What is more, the effects of INH on the activity of Cu/Zn-SOD were also explored. We hope that this work can provide useful information for pharmacology of INH.
Results and discussion
Effects of INH on Cu/Zn-SOD fluorescence
The quenching mechanism of Cu/Zn-SOD fluorescence by INH
where F0 and F are fluorescence intensities of Cu/Zn-SOD in the absence and presence of the quencher (INH), respectively, Kq is the quenching rate constant, KSV is the Stern–Volmer constant, τ0 is the average life time of the molecule without quencher and the fluorescence lifetime of the biopolymer is 10−8 s, [Q] is the quencher concentration.
Calculation of binding parameters
Binding parameters, number of binding sites and thermodynamic parameters of INH–SOD at four temperatures
KSV(L · mol-1)
KA(L · mol-1)
Δ G(kJ · mol-1)
Δ H(kJ · mol-1)
Δ S(J · mol-1 · K-1)
5.06 × 103
1.01 × 104
−22.38 (s = 1.69)
−105.70 (s = 9.54)
−284.66 (s = 18.50)
4.45 × 103
5.31 × 103
−21.04 (s = 1.73)
−286.31 (s = 15.67)
3.89 × 103
3.33 × 103
−20.16 (s = 1.54)
−285.42 (s = 19.92)
3.41 × 103
2.20 × 103
−19.36 (s = 1.26)
−284.29 (s = 19.31)
where A0 and A are the absorbance intensities of Cu/Zn-SOD in the absence and presence of the INH, respectively, KA and [Q] are the binding constant and INH concentration. As shown in the inset in Figure 3, plot of 1/(A0-A) against 1/[Q] gives a straight line, the correlation coefficient of which is 0.9986 and the binding constant (KA) is equal to 2.32 × 103 L · mol−1. The results further prove that only one molecule of INH binds Cu/Zn-SOD.
Thermodynamic analysis and intermolecular forces
where R is the gas constant, T is the absolute temperature and KA is the apparent binding constant at corresponding temperature T. If ΔS > 0 and ΔH > 0, the main force of interaction is the hydrophobic force, if ΔS > 0, ΔH < 0, the main force is electro static attraction, if ΔS < 0, ΔH < 0, the main force includes both van der Waals forces and hydrogen bonding. Table 1 shows the values of ΔH, ΔS and ΔG at four temperatures. The negative values of ΔG reveal that the interaction process is spontaneous. As ΔS < 0 and ΔH < 0, which indicates that the interaction between INH and Cu/Zn-SOD is mainly due to hydrogen bonding and van der Waals forces [34, 35].
Energy transfer between Cu/Zn-SOD and INH
Conformation investigation of Cu/Zn-SOD
Effects of INH on Cu/Zn-SOD activity
Cu/Zn-SOD was purchased from Doulai Biotechnology Company (Nanjing, China), INH was purchased from Taizhou Medical Corporation (Zhejiang, China). SOD was directly dissolved in redistilled water to prepare the stock solution (0.12 mM), and the stock solution was kept in the dark at 0–4°C. INH solution was obtained by dissolving it in redistilled water (1.2 mM). 0.05 M Tris-HCl buffer solution of pH 7.4 and 8.2 were prepared. Other reagents are local products of analytical grade. The water used was redistilled and ion-free.
Fluorescence measurements were carried out in 1.0 cm quartz cells on a FluoroMax-4 spectrophotometer (Horiba, Japan) equipped with a thermostat bath. UV–vis spectra were recorded on an 8000A spectrophotometer (Beijing Purkinje General Corporation, China). All pH measurements were made using a pHS-3 digital pH-meter (Chengdu Sanke Instrument Corporation, China) with a combined glass electrode.
A 3.0 mL Tris–HCl (pH 7.4, 0.05 M) buffer solution containing 4 μM SOD was titrated by successive additions of 1.2 mM INH solution and the concentration of INH varied from 0 to 40 μM. Titrations were done manually by using the microinjectors. After reaction for 10 min, fluorescence spectra were measured in the range of 290–450 nm at the excitation wavelength of 280 nm. The fluorescence spectra were performed at four temperatures (292, 295, 299 and 303 K). The excitation and emission slits were 5 nm.
The synchronous fluorescence spectra were recorded at 299 K from 260 nm to 350 nm (for tyrosine residues) at Δλ = 20 and from 280 to 400 at Δλ = 80 nm (for tryptophan residues). The excitation and emission slits were10 nm.
Three-dimensional fluorescence spectra of SOD were recorded at 299 K in the presence and absence of INH with an excitation wavelength in the range 230–300 nm and an emission wavelength in the range 260–500 nm. The excitation and emission slits were 5 nm.
A 3.0 mL Tris–HCl (pH 7.4, 0.05 M) buffer solution containing 4 μM SOD was titrated by successive additions of 1.2 mM INH solution and the concentration of INH varied from 20 to 200 μM. Titrations were done manually by using the microinjectors. After reaction for 10 min at 303 K, absorption spectra of SOD-INH were recorded in the range 190–400 nm.
The activity of Cu/Zn-SOD was assayed by using the pyrogallol autoxidation method. The assay mixture contained 5.0 ml Tris–HCl (pH 8.2, 0.05 M) buffer, 0.1 mM pyrogallol solution, 2.5 nM SOD solution and different concentrations of INH. The concentration of INH varied from 0 to 2.4 mM. Absorption at 325 nm at 303 K against time was recorded using a spectrophotometer. The SOD activity was determined as pyrogallol autoxidation rate.
In this paper, the interaction between INH and SOD was studied using fluorescence and ultraviolet spectroscopy at different temperatures under imitated physiological conditions. The results show that the quenching mechanism of fluorescence of SOD by INH is a static quenching process. The binding constants, binding sites and the corresponding thermodynamic parameters (ΔH, ΔG and ΔS) were determined, which indicates that hydrogen bonding and van der Waals forces play a major role in the binding process. According to Förster theory of nonradiation energy transfer, the binding distance between Cu/Zn-SOD and INH is 3.93 nm and the energy transfer occurs between SOD and INH with high probability. UV–vis, synchronous fluorescence and three-dimensional fluorescence studies indicates that the interaction leads to a change in the conformation and microenvironment of Cu/Zn-SOD. Moreover, determination of SOD activity in different concentration INH leads to the conclusion that the binding of INH with SOD does not affect the activity of Cu/Zn-SOD. This report has special significance in pharmacology and clinical medicine as well as methodology.
This work was financially supported by the International Sea Area Resources Survey and Development of the 12th Five-year Plan of China (DY125-15-E-01), the Nature Science Foundation of China (20971024) and the Higher Education Institutions Key Nature Science Foundation of Anhui (kj2009A127).
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