Journal of Biomolecular Structure and Dynamics, 2015 http://dx.doi.org/10.1080/07391102.2015.1009946
Study of the interaction between DNP and DIDS with human hemoglobin as binary and ternary systems: spectroscopic and molecular modeling investigation Shamim Rashidipour, Samane Naeeminejad* and Jamshidkhan Chamani* Faculty of Sciences, Department of Biochemistry and Biophysics, Mashhad Branch, Islamic Azad University, Mashhad, Iran Communicated by Ramaswamy H. Sarma
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(Received 14 November 2014; accepted 16 January 2015) The combination of several drugs is necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease in the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. Here, the interaction between 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and 2,4-Dinitrophenol (DNP) with Hemoglobin (Hb) was investigated by different spectroscopic and molecular modeling techniques. Fluorescence analysis was used to estimate the effect of the DIDS and DNP on Hb as well as to define the binding properties of binary and ternary complexes. The distance r between donor and acceptor was obtained by the FRET and found to be 2.25 and 2.13 nm for DIDS and DNP in binary and 2.08 and 2.07 nm for (Hb–DNP) DIDS and (Hb–DIDS) DNP complexes in ternary systems, respectively. Time-resolved fluorescence spectroscopy confirmed static quenching for Hb in the presence of DIDS and DNP in both systems. Furthermore, an increase in ellipticity values of Hb upon interaction with DIDS and DNP showed secondary structural changes of protein that determine to disrupt of hydrogen bonds and electrostatic interactions. Our results showed that the Hb destabilize in the presence of DIDS and DNP. Molecular modeling of the possible binding sites of DIDS and DNP in binary and ternary systems in Hb confirmed the experimental results. Keywords: Hemoglobin; DNP; DIDS; fluorescence quenching; time-resolved fluorescence; circular dichroism
1. Introduction Drug–protein complex is one of the essential factors in analyzing pharmacokinetics and pharmacodynamics of a drug because it can affect the distribution, excretion, metabolism, and interaction with the target tissues. In this study, we investigated the interactions of 2,4-Dinitrophenol (DNP) and 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) with Hemoglobin (Hb) and evaluate the competition between the binding of one drug in the presence and absence of the other. Hb, the major protein component in erythrocytes, contains of two α-chains and two β-chains. The subunit structure of Hb is α2 β2 . The α-chains and β-chains contain 141 amino acids and 146 amino acids, respectively (Lei, Wollenberger, Bistolas, Guiseppi-Elie, & Scheller, 2002; Cui et al., 2004). Each α-chain is in contact with β-chain. (Boys & Konermann, 2007) Each (α,β) dimer contains three Trp residues, namely, α-14 Trp, β-15 Trp, and β-37 Trp, giving a total of six Trp residues in the tetramer. Among the three above-mentioned Trp moieties, only β-37 Trp is situated at the dimer–dimer interface, which corresponds to the region where structural differences among the quaternary states are the most pronounced (Chen, Lalezari, Nagel, & Hirsch,
2005; Jang, Liu, Chen, & Zou, 2009; Xiao et al., 2007). This region has also been designated the primary source of fluorescence emission, though it may also contain some contribution from the surface Trp residues, i.e. α-14 and β-15 Trps (Yuan, Liu, Kang, Lv, & Zou, 2008). Hb also contains five Tyr residues in each dimer: α-24, 42, 140 Tyr, β-34, and 144 Tyr, giving a total of 10 Tyr residues in the tetramer. Hb has a molecular weight of 64,500 kDa. There are four oxygen-binding sites on the Hb molecule. Hb is a carrier of oxygen, it removes hydrogen ions in the capillaries and carries them to the lungs (Bao, Zhu, Li, & Chen, 2001). In addition, it is involved in many clinical diseases such as leukemia, anemia, heart disease, and excessive loss of blood. (Zhang, Wang, & Zhou, 2009). The concentration of Hb in plasma is about (140 g L−1), which is higher than that of serum albumin (40 g L−1), another important carrier of biomolecules. Hb can thus accumulate certain biomolecules (Wang, Zhang, Zhou, & Xu, 2008). Several reports have been published on the interactions of heteropolyacids, drugs, artemisinins, herbicides, surfactants, hematoporphyrins, flavonoids, and insecticides with Hb. Use of a combination of several drugs is often necessary, especially during long-term therapy. But using
*Corresponding authors. Email:
[email protected] (S. Naeeminejad);
[email protected] (J. Chamani) © 2015 Taylor & Francis
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several drugs simultaneously can cause a decrease in the amount of a particular drug bound to Hb, which results in an increase of the free biologically active fraction of the drug. DNP, C6H4N2O5 (Molecular structure, Scheme 1), is an inhibitor of efficient energy (ATP) production in cells with mitochondria. It uncouples oxidative phosphorylation by carrying protons across the mitochondrial membrane, leading to a rapid consumption of energy without generation of ATP. In addition, DNP, which has been shown to increase permeability of mitochondrial and chloroplast membranes to H+. These effects of DNP may not be due entirely to changes in ATP–ADP levels caused by its uncoupling action, but may be the direct result of its ability to increase permeability of membranes to H+. DIDS, C16H10N2O6S4 (Molecular structure, Scheme 2), inhibits anion channels by reversible and nonreversible mechanisms (Kawasaki & Kasai, 1989). The nonreversible affects presumably the result of covalent bonds formed between isothiocyanate groups of DIDS and a variety of amino acid residues of the channel. DIDS has pronounced effects on several channel type: it irreversibly activates SR Ca release channels, while it blocks chloride channels of various origin by causing either a reversible flicker block or an irreversible conductance decrease (Hals & Palade, 1990; Kawasaki & Kasai, 1989). The two negatively charged sulfonate groups render DIDS membrane impermanent, and are probably responsible for the flicker block and covalent amino group modification by the two isothiocyanate groups of DIDS was proposed to cause the irreversible effects. DIDS and DNP have been used as simultaneously for treatment. Binding of various drugs to Hb can results in a decrease or increase of their affinity. The aim of this work was to determine the interaction of DIDS and DNP
Scheme 1.
Molecular structure of DNP.
Scheme 2.
Molecular structure of DIDS.
with Hb, as well as to evaluate the competition between the binding of one drug in the presence and absence of the other. Also the influence of DIDS and DNP on the conformation of Hb and protein fluorescence was investigated. The general affinity of Hb toward a particular drug as well as the changes of free and bound fraction concentrations are important from a pharmacological point of view, and the mechanism of competition between various drugs provides vital information concerning drug design. 2. Materials and methods 2.1. Materials and solutions Hb, DNP, and DIDS were purchased from the Sigma Chemical Corporation. The Hb solution was prepared with a concentration of 4.6 × 10−3 mM and its volume in the cell was 2 mL. The concentrations of DNP and DIDS solutions were [DNP] = 10−5 mM and [DIDS] = 10−8 mM. The interaction between DNP and DIDS has been done by spectroscopic methods as a standard experiment. The spectrum changes induced with interaction between two drugs corrected by inner filter effects. In ternary systems, the first drug concentration is constant and the second drug is injected to form the complex. The Hb, DNP, and DIDS solutions were freshly prepared for each experiment. All of these mixtures were dissolved in 50 mM potassium phosphate buffer (pH 7.4) and kept in a refrigerator. All experiments were performed at room temperature. All pH measurements were performed with a Metrohm digital pH meter (Metrohm, Germany). 2.2. Apparatuses UV–vis spectra were collected at room temperature on a double-beam UV-630 spectrophotometer (Hitachi Japan) in 1.0 cm quartz cells. The slit width was set to 5 nm, and the wavelength range was 200–600 nm. All fluorescence measurements were recorded on a Hitatchi model F-2500 spectrofluorometer (Japan) equipped with a 1.0 cm quartz cell and Xenon pulse lamp and a thermostate bath. Instrument settings, i.e. the slit widths (excitation at 10.0 nm, emission at 10.0 nm), scan speed (1500 nm min−1), and response time (.08 s), were kept constant for all experiments. All the experiment were repeated at least three times and performed at room temperature. Fluorescence spectra of Hb were obtained with excitation at 280 and 295 nm, and the excitation and emission slit widths were set to 5 nm. Synchronous fluorescence spectra were obtained by simultaneously scanning the excitation and emission monochromators. Synchronous fluorescence spectra are characteristic of the Tyr and Trp residues of Hb when the wavelength interval (Δλ) is 15 and 60 nm, respectively.
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Study of the interaction between DNP and DIDS In all titration experiments, the dilution factor of the ligand titration was corrected. Time-resolved fluorescence spectra were executed in a time-correlated single photon counting system from ARCUS fluorescence (LKB, Turku, Finland) with excitation wavelength at 295 nm. The data are fitted to biexponential function by an iterative reconvolution approach by the DAS6 decay analysis software utilizing reduced the error in the calculated fluorescence (χ2) and weighted residuals as parameters for goodness of fit. The conductance values of Hb–DIDS and Hb–DNP complexes as binary and ternary systems were measured with a conductometer (Metrohm, Germany) at 25.0 ± .1 °C, which was calibrated by .01 mol/L KCl solutions before measurement. The error limitation for conductivity values was within ±.5%. Zeta potential experiments were carried out on a Malvern Instruments Zetasizer, model nano-zs (UK). The effective charges on a protein particle can be affected by pH, ionic strength, and the accumulation of ligands or surfactants at the interface. As the zeta potential is pH dependent, the pH was kept constant at 7.4 for all samples. CD spectra were recorded with a Jasco J-815 spectropolarimeter (Jasco, Tokyo, Japan) in 1.0 cm cells at room temperature. Dry nitrogen gas was utilized to purge the testing environment before and during the course of the measurements. The bandwidth was 1 nm and the scanning speed was 200 nm min−1. The samples for CD analysis were prepared with a fixed concentration of Hb (.03%) and varying drug concentrations, resulting in equal volumes. The instrument was calibrated with ammonium d-10-camphor sulfuric acid. The induced ellipticity, expressed in degrees, was obtained for the drug–Hb mixtures by subtracting the ellipticity of the drug at the same wavelength. The results were expressed as the mean residue ellipticity [θ], defined as [θ] = 100 × θobsd/(LC), where θobsd is the observed ellipticity in degrees, C is the concentration in the residue in mol cm−3, and L is the length of the light path in the cell. The secondary structure contents of Hb in the presence of DIDS and DNP in binary and ternary systems were analyzed by SILCON III software. The crystal structure of Hb was retrieved from RCSB Protein Data Bank (PDB: 1GZX). MOE 2008.10 was used as the docking software in docking study. In docking procedure, we assumed drug as flexible molecules and let the docking software to rotate all rotatable bonds of drugs to catch the best and optimized conformer of the drugs in Hb. To study the ternary systems of (Hb–DNP) DIDS and (Hb–DIDS) DNP, one drug docked with Hb and followed by minimizing the energy, Hb–drug complex merged with the second drug in MOE. For add surface, Viewerlite software was used. Partial atomic charge for each atom was added and the
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rigid root and rotable bonds for each ligand were calculated automatically. 3. Results and discussion 3.1. UV–vis spectroscopy measurements UV–vis absorption measurement is a very simple method and applicable to explore the structural changes and to investigated protein–ligand complex formation. Hence, absorption spectra of Hb in presence and absence of DIDS and DNP in binary and ternary systems were recorded (Bi et al., 2005). Figure 1(A) shows the absorption spectral changes of Hb and Hb–DNP complex in
Figure 1. (A) Absorption spectra of Hb, DIDS, (Hb–DIDS), and (Hb–DNP) DIDS systems. (a) the absorption spectrum of Hb only; (b) the difference absorption between Hb–DIDS; (c) the difference absorption spectrum Hb–DNP in the presence of DIDS; (d) the absorption spectrum of DIDS only, Hb = .03 mM, DIDS = 10−8 mM. (B) Absorption spectra of Hb, DNP, (Hb–DNP), and (Hb–DIDS) DNP systems. (a) the absorption spectrum of DNP only; (b) the absorption spectrum of Hb only; (c) the absorption spectrum Hb–DNP; (d) the difference absorption spectrum of Hb–DIDS in the presence of DNP, Hb = .03 mM, DNP = 10−5 mM.
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the presence of DIDS as binary and ternary systems at wavelength limitation of 200–600 nm. UV–vis absorption spectrum of Hb in Figure 1(A) shows a band in the near-UV region with a maximum at 275 nm, which appears due to phenyl group of Trp and Tyr residues. The peak at 410 nm correspond the Soret-band of Hb. The absorption maximum of Soret-band is decreased after DIDS treatment in binary system and after interaction DIDS with Hb–DNP complex in ternary system, while the maximum absorption wavelengths remain unchanged. This means that the heme is not exposed from the crevices at the exterior of the subunit and DIDS is easily integrated into the hydrophobic pocket of Hb in binary and ternary systems (Moreno & GonzálezJiménez, 1999). This indicated that there exists interaction between DIDS and Hb and ground-state complex has formed. Figure 1(B) shows the absorption spectra changes of Hb in the presence of DNP in binary system and Hb–DIDS complex in the presence of DNP as ternary system in the wavelength limitation of 200–600 nm. There are two absorption peaks like 275 and 410 nm in Figure 1(A) with increasing DNP concentration to Hb and Hb–DIDS complex, the absorbance of the Soretband increases with the increment of the band situated at 275 nm (Wu, Wu, Guan, Su, & Cai, 2007). The results indicate that the interaction between DNP with Hb and Hb–DIDS complex leads to the mild unfolding of the protein skeleton and decreases the hydrophobicity of the micro-environment of the aromatic amino acid residue (Zhou et al., 2007). With increasing DNP concentration, the intensity of the peak at 410 nm increased, suggesting that DNP can also affect the structure of the heme group but there is no direct interaction between DNP and the heme groups of Hb. 3.2. Fluorescence quenching of Hb by DIDS and DNP in binary and ternary systems For macromolecules, the fluorescence measurements can give some information of the binding of small molecule substances to protein on the molecular level, such as the binding mechanism, binding mode, binding constant, and intermolecular distances. (Silva, Cortez, & Cunha-Bastos, 2004; Tang, Chen, Chen, Xie, & Li, 2008). Fluorescence quenching is the decrease in the quantum yield of fluorescence from a fluorophore induced by variety molecular interactions with quencher molecule, such as excited-state reaction, molecules rearrangement, energy transfer, ground-state complex formation, and collision quenching (Chen & Tianqing, 2008). On the other hand, the fluorescence behavior can provide information about the molecular micro-environment in the vicinity of chromophore groups (Mandal, Bardhan, & Ganguly, 2010). The fluorescence of Hb originates from Trp, Tyr, and
Phe residues. But in fact, the main contribution to the intrinsic fluorescence of Hb is solely from the Trp residues. A valuable feature of intrinsic fluorescence of Hb is the high sensitivity of Trp to its local environment (Ravasi, Masserini, Vecchio, Li, & Li, 2002). Changes in emission spectra of Trp are common in response to protein conformational transitions, subunit association, substrate binding, or denaturation (Zhang, Que, Pan, & Guo, 2008). Thus, the intrinsic fluorescence of proteins can provide considerable information about their structure and dynamics, and it often considered on the study of protein folding and association reactions. Here, the fluorescence of Hb excited at 280 and 295 nm in the presence of different concentrations of the DIDS and DNP as binary systems was shown in Figure 2. As can be seen from Figure 2(A), fluorescence intensity of Hb decreased regularly with enhancement of DIDS, which indicated that DIDS interacted with Hb and a complex is formed between DIDS and Hb (Anbazhagan & Renganathan, 2008; Chen, Huang, Xu, Zheng, & Wang, 1990; He & Carter, 1992). It can also be noticed from the spectra that the interaction of Hb with DIDS led to a slight blue shift at the maximum wavelength of Hb fluorescence emission, especially at a high drug concentrations, which indicated that the chromophore of protein has been brought to a more hydrophobic environment and the conformation of the protein has been changed. The quenching takes place when the quencher is sufficiently close to the Trp or/and Tyr residues. Then the energy transfer between a ligand and fluorophore is possible. The inset of Figure 2(A) illustrates the excitation at 295 nm. As can be seen from Figure 2(A), the maximum wavelength of Hb emission spectra did not change; thus, the micro-environment of Tyr residues plays an important role in conformational changes of Hb. Figure 2(B) demonstrates the effect of DNP on the Hb fluorescence intensity at 280 and 295 nm. When the Hb solution was titrated with increasing amounts of DNP, its fluorescence intensities at 280 nm were found to be significantly decreased and a slight blue shift was occurred. The strong quenching of the Hb fluorescence suggests that the chromophore of Hb was positioned in a more hydrophobic environment blue shift when the concentration of DNP is increased. These results indicated that the interaction between DNP and Hb can cause with changing the electronic states’ distances of protein. In order to confirm the quenching mechanism, the fluorescence quenching data are analyzed by the Stern– Volmer equation (Lakowicz, 1999): F0 =F ¼ 1 þ kq s0 ½Q ¼ 1 þ Ksv ½Q
(1)
where F0 and F are the relative fluorescence intensities of Hb in absence and presence of quenchers (DNP and
Study of the interaction between DNP and DIDS
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Figure 2. (A); Fluorescence emission spectra of Hb in the presence of various concentrations of DIDS. The Hb concentration was 4.6 × 10−3 mM and the DIDS concentration was increased from (0 to 1.67)×10−9 mM. T = 298 K, pH 7.4, λex = 280 nm. Inset (A) quenching curves of Hb in the presence of DIDS. T = 298 K, pH 7.4, λex = 295. (B) Fluorescence emission spectra of Hb in the presence of various concentrations of DNP. The Hb concentration was 4.6 × 10−3 mM and the DNP concentration was increased from (0 to 1.67) × 10−6 mM. T = 298 K, pH 7.4, λex = 280 nm. Inset (B) quenching curves of Hb in the presence of DNP. T = 298 K, pH 7.4, λex = 295.
DIDS). kq is the quenching rate constant of the biomolecule; τ0 is the average lifetime of the biomolecule without quencher; [Q] is quencher concentration; and Ksv is the Stern–Volmer dynamic quenching constant that can be interpreted as the extent of the quenching process
(Lakowicz (second ed.), 1999). This extent and the degree of accessibility of the fluorophores to the quencher show a dependence on its size and change obviously: Ksv ¼ kq s0
(2)
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Some previous studies have determined that the lifetime of Trp in the Hb system is 5.0 ns (He & Carter, 1992; Lakowicz (second ed.), 1999). As a rule, the maximum scatter collision quenching constant, kq of various quenching with the biopolymer was 2.0 × 1010 L mol−1 s−1. The fluorescence quenching mechanism can be analyzed with the modified Stern–Volmer equation as follows: F0 =DF ¼ 1=ðfa KQ ½QÞ þ 1=fa
(3)
Fa ¼ F0a =ðF0a þ F0b Þ
(4)
Here, F0, F, and [Q] are the same as in Equation (1), ΔF = F0−F is the decrease in fluorescence intensity due to the concentration of quencher drug, fa is the fraction of the initial fluorescence that is accessible to the quencher, and KQ is the effective quenching constant for the accessible fluorophores. The dependence of F0/ΔF on the reciprocal of the quencher concentration [Q]−1 is linear and equal to (faKQ)−1(see Figure 3) (Mohammed-Sultan, Rao, Nadimpalli, & Swamy, 2006). The value of fa−1 is fixed by the ordinate. KQ is the effective quenching constant for the accessible fluorophores. The dependence of F0/ΔF on the reciprocal value of the quencher concentration [Q]−1 is linear with the intercept and equal to the value of fa−1.29 In this case, F0a is the fluorescence of the fluorophore moieties that can complex with the quencher and F0b is the fluorescence of the inaccessible fluorophore moieties. For the static quenching interaction, if there are similar and independent binding sites in the biomolecule, the association constant and the number of binding sites can be obtained from the double logarithmic equation (Sarzehi & Chamani, 2010): log½ðF0 FÞ=F ¼ log Ka þ n log½Q
(5)
where Ka is the association constant of DIDS and DNP with Hb and n is the number of binding sites. The latter can be determined by the slope and the intercept of the double logarithm regression curves of log[(F0−F)/F] vs. log[Q]. The values of n are listed in Table 1. Analysis of the modified Stern–Volmer equation [Equation (1)] allowed the determination of the quenching constants KQ and fa for binary and ternary systems. The constant KQ is a mean value of the quenching constants characterizing all binding sites of the Hb (MaciazelJurczyk, Sulkowsla, Bojko, Rownicka, & Sulkowski, 2009). In order to compare the effect of DIDS and DNP on Hb, the experiment data were analyzed by the modified Stern–Volmer equation and recorded in Table 1. In order to decrease the inner filter effect, the fluorescence intensities used in this study were all corrected for absorption of the exciting light and reabsorption of emitted light using the formula: Fcor ¼ Fobs eðAex þAem Þ=2
where Fcor and Fobs are the fluorescence intensities corrected and observed, Aex and Aem are the absorption of the system at excitation and emission wavelength, respectively (Lakowicz, 2006). The result displayed in Figure 3(A) and (B) indicates that DIDS and DNP have two and one set of binding sites in Hb as binary systems, respectively. Moreover, the type of binding site did not change with the presence of a second drug. Figure 3(A) indicated that for the first type of binding site, kq1 and kq2 values increased with enhancement of DIDS concentration. Moreover, it can be concluded that the interaction between DIDS and Hb decreases in the presence of DNP (Maciazel-Jurczyk et al., 2009). In other words, the transfer of energy from excited protein fluorophores to DIDS is more difficult in the presence of DNP, and it is also harder for DIDS to access the Hb. Figure 3(B) demonstrated that DNP had two independent binding sites in Hb–DIDS complex in comparison with Hb. It indicated that after enhancement of DNP to Hb–DIDS complex, another binding site was revealed. The comparison between kq in Hb–DNP and (Hb–DIDS) DNP complexes as binary and ternary systems suggests that for the first type of binding site kq1 and kq2 decrease and increase, respectively. It could be concluded that the interaction between DNP and Hb in the presence of the first set of binding site is lower than in the second. The comparison between kq values in binary and ternary systems indicates that the kq values of Hb, during quenching by DIDS in binary and ternary systems were greater than the corresponding value of another complex. This indicates that the binding of DIDS to Hb was quite strong and that the quenching process involved a static quenching mechanism. The kq values of interaction in Table 1 show that kq values of interaction are more than 2 × 1012 M−1 s−1, therefore, the quenching behavior is static mechanism. On the other hand, the results of Table 1 pointed to the fact that the number of binding site was about one for all systems except for the (Hb–DIDS) DNP complex, suggesting that the interaction between Hb and both mentioned drugs as binary and ternary systems is 1:1. 3.3. Synchronous fluorescence measurements Usually, each protein has only one emission band in its normal fluorescence spectrum. This is due to the emission by the Trp and Tyr residues in the protein since their emission peaks cannot be separated in normal fluorescence spectroscopy. In order to the separation of emission peaks of Trp and Tyr residues, one can employ synchronous fluorescence spectroscopy to the protein. This technique is useful when studying the molecular micro-environment in the vicinity of fluorophore functional groups by measuring the shift in position of
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Study of the interaction between DNP and DIDS
Figure 3. (A) Modified Stern–Volmer plot adapted to estimate kq for one (dashed line) and two (solid line) binding sites of: the Hb–DIDS complex and insert is about (Hb–DNP) DIDS at λex = 280 nm, and the Hb–DIDS complex and insert about (Hb–DNP) DIDS at λex = 295 nm. T = 298 K, pH 7.4. (B) Modified Stern–Volmer plot adapted to estimate kq for one (dashed line) and two (solid line) binding site of: the Hb–DNP complex and insert is about (Hb–DIDS) DNP at λex = 280 nm, and the Hb–DNP complex and insert about (Hb–DIDS) DNP at λex = 295 nm. T = 298 K, pH 7.4.
the emission maximum corresponding to changes of the micro-structure around the chromophore molecule (Wang, Tang, Zhang, Zhou, & Zhang, 2009; Zhou et al., 2009). The method has several advantages, such as
sensitivity, spectral simplification, spectral bandwidth reduction, and the possibility of avoiding a variety of perturbing effects (Divya & Ashok, 2008). In this case, the synchronous fluorescence spectra only show the
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Table 1.
Stern–Volmer quenching constants for the interaction of Hb with DIDS and DNP in binary and ternary systems.
System
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Hb–DIDS (Hb–DNP) DIDS Hb–DNP (Hb–DIDS) DNP
KSV1/M−1
KSV2/M−1
11
11
8.1 × 10 4.2 × 1011 4.5 × 108 2.6 × 108
32.9 × 10 4.5 × 1011 – 95.5 × 108
kq1/M−1 S−1 19
8.1 × 10 4.2 × 1019 4.5 × 1016 2.6 × 1016
kq2/M−1 S−1 19
32.9 × 10 4.5 × 1019 – 95.5 × 1016
n1
n2
.75 1.37 .93 .57
.80 1.5 – .92
System
f1
f2
R1
R2
Hb–DIDS (Hb–DNP) DIDS Hb–DNP (Hb–DIDS) DNP
.30 .39 .54 .37
.15 .02 – .026
.98 .98 .99 .99
.99 .97 – .99
presence of the Tyr and the Trp residues of Hb when the wavelength interval (Δλ) is 60 and 15 nm, respectively. Because the β-37 Trp, α-42 Tyr, α-140 Tyr, and β-144 Tyr residues are located at the interface of Hb, an X-ray study pointed out that α-42 Tyr and β-37 Trp are part of the switch region and flexible joint region, respectively, which are responsible for the stabilization of Hb’s quaternary structure (Divya & Ashok, 2008; Jang, Liu, Chen, & Zou, 2009). Changes in these regions imply a mild destabilization of the protein’s quaternary structure. Figure 4(A) and (B) shows the synchronous fluorescence spectrum of Hb in the presence of various concentration of DIDS and DNP at Δλ = 60 nm and Δλ = 15 nm, respectively. It was apparent from Figure 4(A) that the emission maximum of Trp residues did a slight blue shift which indicated that the conformation of Hb was changed, the polarity around the Trp residues was decreased and the hydrophobicity was increased. This may be due to the changes of residue micro-environment with the insertion of DIDS (see Figure 4(A)). Furthermore, Figure 4(B) shows the synchronous fluorescence spectrum of Hb in the presence of DNP at Δλ = 60 nm and Δλ = 15 nm. After the addition of DNP, a significant blue shift is observed for the emission maximum of Trp residues. The inset of Figure 4(B) shows that the emission peaks of Tyr residues has a blue shift (Wang, Zhang, & Zhou, 2009). For aromatic Tyr and Trp residues, the fluorescence emission peak is sensitive to the polarity of their micro-environment; the blue shift signifies an enhancement of hydrophobicity. Therefore, the binding of DIDS and DNP with Hb probably induces the tertiary structure changes of the adsorbed Hb and produces perturbation of micro-environments around aromatic amino acid residues. Figure 4(C) and (D) showed that a decrease in fluorescence emission of Hb occurred with increasing concentration of the drugs in binary and ternary systems. As can be shown from Figure 4(C), the slope of F/F0 vs. [Q] for (Hb–DNP) DIDS complex is more than Hb–DIDS that determine the presence of DNP, interaction behavior between DIDS and Hb has been changed. The conformational changes of the Hb–DIDS in the
presence of DNP as ternary system caused to this behavior changes. The inset of Figure 4(C) shows that the initial part of the quenching curves of Hb–DIDS and (Hb–DNP) DIDS practically overlap, then the quenching of Hb–DIDS is higher than the (Hb–DNP) DIDS complex. Figure 4(D) shows that the quenching of (Hb–DIDS) DNP is more than Hb–DNP. The comparison between Figure 4(C) and (D) determined that the Hb–DIDS complex quenching is more than Hb–DNP systems. These results suggest that both the Trp and Tyr residues play important role in formation of complex (Cui et al., 2008; Chi, Liu, Yang, & Zhang, 2010) that the Trp plays an important role during fluorescence quenching of Hb. This means that it approached the Trp more than Tyr residues. 3.4. Thermodynamic parameters and binding force The thermodynamic parameters, i.e. the enthalpy (ΔH°) and entropy (ΔS°), of the binding reaction are important for confirming binding modes. Therefore, the thermodynamic parameters were analyzed as a function of temperature in order to depict the type of force acting between the protein and drugs. These forces acting between the drugs and the biomacromolecule may include hydrogen bonds, van der Waals forces, electrostatic forces, and hydrophobic interactions. For this purpose, the temperature dependence of the quenching constant was measured at three temperatures, i.e. 298, 302, and 306 K (Table 2). In order to elucidate the interaction of DIDS and DNP with Hb, the thermodynamic parameters were calculated from van’t Hoff plots (spectra are not shown) and are presented in Table 2. For a constant ΔH° value over the investigated temperature range, the values of ΔH° and ΔS° can be determined from the following Equation (6): ln K ¼ DH =RT þ DS =R
(6)
here K is analogous to the Stern–Volmer quenching constant Ksv at the corresponding temperature (i.e. 298, 302, and 306) and R is the gas constant. The standard enthalpy changes (ΔH°), the standard entropy changes
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Study of the interaction between DNP and DIDS
Figure 4. (A) Synchronous fluorescence spectra of Hb in the presence of DIDS at Δλ = 60 nm. Inset is synchronous fluorescence spectra of Hb in the presence of DIDS at Δλ = 15 nm. (B) Synchronous fluorescence spectra of Hb in the presence of DIDS at Δλ = 60 nm. Inset is synchronous fluorescence spectra of Hb in the presence of DIDS at Δλ = 15 nm. (C) Synchronous fluorescence spectra of the quenching of Hb by DIDS in the absence and presence of DNP, T = 298 K; pH 7.4. Hb–DIDS (ο), (Hb–DNP) DIDS (●) at Δλ = 60 nm. Inset: Hb–DIDS (ο), (Hb–DNP) DIDS (●) at Δλ = 15 nm. (D) Hb–DNP (♢), (Hb–DIDS) DNP (♦) at Δλ = 60 nm. Inset: Hb–DNP (♢), (Hb–DIDS) DNP (♦) at Δλ = 15 nm.
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Figure 4.
(Continued).
(ΔS°), and the standard Gibbs energy changes (ΔG°) of the reaction are important parameters for confirming the binding mode. The value of ΔH° and ΔS° was obtained from Equation (6), the slope and intercept of the fitted curve of Ksv against 1/T, while the change in Gibbs
energy (ΔG°) was estimated from the following relationship (Tang, Luan, & Chen, 2006): DG ¼ DH T DS
(7)
Study of the interaction between DNP and DIDS Table 2.
Thermodynamic parameters for the drugs when bound to Hb in binary and ternary systems at 298, 302, 306 K (pH 7.4).
System Hb–DIDS (Hb–DNP) DIDS Hb–DNP (Hb–DIDS) DNP
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DH1 kJ mol−1
DH2 kJ mol−1
DS1 J mol−1 K−1
DS2 J mol−1 K−1
−72.9
−76.8
−11.2
−14.9
−70.7
−74.5
−9.7
−10.3
−9.1
–
−10.6
−13.7
−54.4 −60.3
−53.9
The negative sign for ΔG° signifies that the binding of the drugs with Hb is spontaneous and facile (Zhang et al., 2010). On the other hand, the ΔG° values decrease as the temperature is raised, suggesting that hydration of Hb occurs at higher temperature. Also, ΔG° of (Hb–DIDS) DNP is lower than for Hb–DIDS, indicated that formation an Hb–DIDS complex in the presence DNP is difficult. Nonetheless, ΔG° of the binary Hb–DNP system is higher than the corresponding value of the ternary system. From a thermodynamic standpoint, ΔH° > 0 and ΔS° > 0 imply hydrophobic interactions, ΔH° < 0 and ΔS° < 0 reflect van der Waals forces or hydrogen bonds, whereas ΔH° ≈ 0 and ΔS° > 0 suggest an electrostatic force. Negative ΔH° and ΔS° values indicate that the binding process is enthalpy driven since the entropy factor is not favorable. Because negative ΔH° and ΔS° values are generally taken as evidence of van der Waals forces and hydrogen binding in low-dielectric media, the interaction of drugs with Hb may also be determined from these two parameters. The negative ΔH° and ΔS° values for (Hb–DIDS) DNP are larger than those of Hb–DNP, implying that the former system demonstrates more significant van der Waals forces and hydrogen bonding (Zhang et al., 2010). Moreover, the values of standard changes in entropy (ΔS°) and enthalpy (ΔH°) of the binding between DIDS and DNP with Hb were found to be negative, suggesting that the van der Waals forces and hydrogen bond interactions played an important role in the binding of DIDS and DNP to Hb. 3.5. Fluorescence resonance energy transfer (FRET) measurements Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the various electronic excited states of small molecules in which the excitation energy is transferred from one molecule (donor) to another (acceptor) without emission of photon from the former molecular system (Wang et al., 2010).
DG1 kJ mol−1
DG2 kJ mol−1
−69.1 −69.5 −69.7 −67.4 −67.7 −67.8 −51.1 −51.6 −51.7 −56.9 −57.1 −57.7
−71.7 −72.3 −72.7 −67.7 −68.3 −68.4 – – – −49.9 −49.8 −49.6
FRET is an important technique for investigating the structure, conformation spatial distribution, and assembly of complex proteins (Laib & Seeger, 2004; Wang et al., 2007). Energy transfer is likely to take place if the following conditions are: (1) the donor can produce fluorescent light, (2) the appropriate orientation of a transition dipole between donor and acceptor, (3) fluorescence emission spectra of the donor and UV–vis absorption spectra of the acceptor overlap, and (4) the distance between the donor and acceptor is less than 7 nm. The efficiency of energy transfer between the donor and the acceptor (E) is described by the following equation (8).40 Where r is the distance from the ligand to the fluorophore residues of the protein, and R0 is the Forster critical distance, at which 50% of the excitation energy is transferred to the acceptor. It can be calculated from donor emission and acceptor absorption spectra using the Forster formula Equation (8): E ¼ 1 F=F0 ¼ R60 =R60 þ r6
(8)
R60 ¼ 8:79 1025 K 2 N 4 J
(9)
2
In Equation (6) (Wang et al., 2007), K is the orientation factor related to the geometry of the donor and acceptor of the dipole, K2 = 2/3 for a random orientation as occurs in a fluid solution, N is the average refractive index of the medium in the wavelength range where spectral overlap is significant, and is the fluorescence quantum yield of the donor. J is the spectrum overlap integral of the donor fluorescence emission spectrum and acceptor absorption spectrum, and is given by: J ¼ ðFðkÞeðkÞk4 DkÞ=FðkÞDk
(10)
where F(λ) is the fluorescence intensity of the donor at wavelength, and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. The overlap of fluorescence emission spectrum of Hb and the absorption spectrum of the DIDS and DNP is shown in Figure 5(A) and (B).
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Obviously, the values of R0 and r for binary and ternary systems at different drug concentrations are found to be lower than the maximal critical distance for R0 (i.e. 5–10 nm) and the maximum critical distance between donor and acceptor for r (i.e. 7–10 nm), which indicates that energy transfer from the drugs to Hb occurred with a high probability (Mark, Hink-Nina, Borst, Hoek, & Visser, 2003; Sklar, Hudson, & Simoni, 1977). Furthermore, these results again indicated the presence of a static quenching mechanism for the interaction between the drugs and Hb. It also suggested the association between DIDS and DNP with Hb can make the behaviors and micro-structure of Hb changes and the fluorescence quenching. 3.6. Time-resolved fluorescence measurements Fluorescence lifetime decay measurements supply one of the best parameters that help us to distinguish between static and dynamic process. In order to further substantiate the quenching mechanism of DIDS and DNP to Hb, fluorescence lifetime of Hb was ascertained in the absence and presence of DIDS and DNP as binary and ternary systems. Average fluorescence lifetime (τ) for biexponential iterative fittings was calculated from the decay times and the relative amplitude (α) using the following equation: s ¼ s 1 a1 þ s 2 a2 Figure 5. (A) Overlap of the fluorescence emission of Hb (a) with the absorption spectra of DIDS (b). T = 298 K, pH 7.4. (B) Overlap of the fluorescence emission of Hb (a) with the absorption spectra of DNP (b). T = 298 K, pH 7.4.
According to the theory, value of the overlap integral J was calculated to be equal 2.91 × 10−14 and 1.53 × 10−14 cm3 dm3 mol−1 for (Hb–-DNP) DIDS and (Hb–DIDS) DNP systems, respectively. The critical distance R0 and the distance r between donor and acceptor were calculated 1.92, 2.25 and 1.83 nm, 2.13 nm for Hb–DIDS and Hb–DNP complexes as binary systems, respectively. In ternary systems for (Hb–DNP) DIDS and (Hb–DIDS) DNP complexes, R0 and r values have been obtained 1.86, 2.08 and 1.75 nm, 2.07 nm, respectively.
(11)
Time-resolved fluorescence decay of Hb in the absence and presence of DIDS and DNP is summarized in Table 3. With the addition of drugs, the lifetime changed and a much faster decay of the Trp fluorescence was observed in the presence of the second drug in the ternary systems. The average fluorescence lifetime of Hb reduces, without and with the DIDS and DNP for binary and ternary systems. Such a reduction in the average fluorescence lifetime in all tested systems, suggested the formation of complexes and a significant energy transfer between the drugs and Hb. It also attested to the fluorescence quenching being essentially a static mechanism due to the ground-state complex formation (Ding, Liu, Zhang, & Sun, 2010; Pan et al., 2010; Wang et al., 2008). Hence, both steady-state and time-resolved
Table 3. Time-resolved fluorescence data of Hb and Hb/drug complexes in all interacting systems (λem = 295 λem = 345 nm, pH 7.4, T = 298 K). System
τ1/ns
α1
τ2/ns
α2
τ/ns
χ2
Hb Hb–DIDS (Hb–DNP) DIDS Hb–DNP (Hb–DIDS) DNP
1.962 1.883 1.819 1.824 1.804
.6281 .6629 .6679 .6471 .6519
5.929 5.875 5.481 5.538 5.377
.4711 .4155 .3526 .4375 .4085
4.02 3.69 3.15 3.60 3.37
.9633 .9573 .9519 .9605 .9701
Study of the interaction between DNP and DIDS measurements hint to the occurrence of static-type fluorescence quenching caused by specific interaction, mainly by ground-state complex formation.
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3.7. Conductometry measurements Electrolytic conductance was used as a common value of the electrical conductivity of principally aqueous electrolyte solution and conductivity has been developed into an electrochemical analytical method based on measuring the conductance in electrolyte solution. Figure 6(A) shows the plot of the conductance of Hb–DIDS and (Hb–DNP) DIDS complexes. We can recognize two regions in curve, that each of them exerts different conductance behavior. The first region which has a larger slope and the second region is the area with a smaller slope. The region with the smaller slope results from the formation of micelles (Peterlin, 2010). With changing of conductometry slope, interaction behavior of DIDS with Hb has been changed at binary and ternary systems. On the other hand, Figure 6(B) shows the plot of the conductance of Hb–DNP and (Hb–DIDS) DNP complexes. In Figure 6(B), observed two CCIAC points in
13
curve that demonstrate Hb can increase the CCIAC of DNP, which indicate that there exists an interaction between Hb and DNP in binary and ternary systems. The values of CCIAC for Hb–DIDS and (Hb–DNP) DIDS systems were determined to be 8.6 × 10−11 mM. In addition, for Hb–DNP and (Hb–DIDS) DNP systems, two CCIAC points were evaluated to be 1.5 × 10−7 and 12.7 × 10−7 mM. Comparing between obtained results from zeta potential and conductometry measurements clearly express that the presence of free ions in the environment shows remarkable effect on interaction between Hb with DIDS (CCIAC zeta-potential = .26 × 10−11), and −7 with DNP (CCIAC and zeta-potential = 2.4 × 10 −7 8.8 × 10 ). Whereas, the presence of free ions for DIDS has more effect than DNP and also amplify DIDS effects upon Hb structural changes. On the other hand, the inflection points of plots showed, there were two different slopes of curves that determined two different interaction behaviors between Hb and both mentioned drugs as binary and ternary systems. 3.8. Circular dichroism (CD) analysis Circular dichroism (CD) is one of the most sensitive physical techniques for determining structures and for monitoring structural changes of biomolecules (Donskova, Stasovskaya, & Repko, 1975). It can directly interpret the changes of a protein’s secondary structure even though the method is empirical. The Far-UV CD spectra of proteins are extremely sensitive toward protein structure. To further verify the possible influence of DIDS and DNP (binary and ternary systems) on the secondary structure of Hb, CD studies were performed with DIDS and DNP. The α-helical content of Hb in the absence and presence of DIDS and DNP was calculated from Equations (12) and (13): MRE ¼ Observed CD ðmedgeÞ=Cp nl 10
(12)
where Cp is the molar concentration of the protein, n is the number of amino acid residues, and l is the path length of the cell. a-Helix ð%Þ ¼ MRE208 4000=33000 4000 100 (13)
Figure 6. (A) Electric conductivity of Hb–DIDS (ο) and (Hb–DNP) DIDS (●) complexes. (B) Electric conductivity of Hb–DNP (♢) and (Hb–DIDS) DNP (♦) complexes.
where MRE208 is the observed MRE value at 280 nm, 4000 is the MRE of the β-form and random coil conformation cross at 208 nm, and 33,000 is the MRE value of a pure α-helix at 208 nm. The CD spectra of Hb in the absence and presence of DIDS are displayed in Figure 7(A) (Xiao et al., 2007). These spectra exhibited two negative bands in the UV region at 208 and 222 nm, both contributed by n → π* transfer for the peptide bond of the α-helix. As can be seen in Table 4, Hb is comprised of 33.14% regular α-helix, 10.7% regular β-sheet and 13.47% turn.
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S. Rashidipour et al. & Agnishwar, 2006). The decrease in α-helix content indicates that DIDS combines with amino acid residues of the main polypeptide chain of the protein and destroyed their hydrogen bond networks (Shen, Liou, Ye, Liang, & Wang, 2007). The binding of DIDS to Hb induces some conformational changes in Hb which may affect Hb functions. Figure 7(B) shows two negative bands in UV region at 208 and 222 nm, which are characteristics of a high α-helical content. According to Table 3, regular α-helix content decreased from 33.14% in free Hb to 31.28% in Hb–DNP and 29.36% in (Hb–DIDS) DNP complex. An increase unordered coil and an increase in β-sheet structures were observed with the absorbed Hb. As mentioned above, DIDS and DNP can bind to Hb central cavity to form Hb–DIDS and Hb–DNP complexes, leading to the mild denaturation of the protein skeleton, increasing the hydrophobicity of the micro-environment of Tyr and Trp residues, and changing of the structure of the heme groups. The α-helix content of Hb decreased due to the binding DIDS and DNP.
Figure 7. (A) Far-UV CD spectra of Hb in the absence and presence of DIDS. T = 298 K, pH 7.4. [Hb] = 4.6 × 10−3 mM. (B) Far-UV CD spectra of Hb in the absence and presence of DNP. T = 298 K, pH 7.4. [Hb] = 4.6 × 10−3 mM.
The binding of drugs to Hb caused a dramatic reduction in the band intensity in its CD spectrum, and a decrease of the ellipticity was also noted. According to Table 3, it was shown that with addition of DIDS to Hb, the regular α-helix decreased from 33.14% in free Hb to 30.28% in Hb–DIDS and 29.22% in (Hb–DNP) DIDS complex, regular and disordered β-sheet and unordered coil increased with increasing DIDS concentration in binary and ternary systems (Lu, Jin, Sun, & Zhou, 2007; Swati
3.9. Zeta potential measurements The first layer around a suspended particle, close to the surface of the particle, consists of strongly bound ions, and is called the Stern layer. Ions further away from the surface of the particle are more loosely bound and the layer of these loosely attached ions is called a diffuse layer. Inside this layer, there is an imaginary boundary called the slipping plane. When a suspended particle surrounded by attached ions moves in the liquid, all ions within the slipping plane boundary move with the particle whereas the ions outside this boundary do not (Pinho Melo, Aires-Barros, Costa, & Cabral, 1997). There is a potential between the particle and the liquid, and at the slipping plane, this potential is called the zeta potential. The zeta potential is a function of the surface charge of the particle and the nature and composition of the surrounding medium in which the particle is dispersed. It can provide a measure of the net surface charge on the particle and potential distribution at the interface (Zhang, Hassanali, Shin, Knight, & Singer, 2011). Moreover, the zeta potential serves as an important parameter in characterizing the electrostatic interaction between particles in
Table 4. CD data showing drug-derived alterations in the secondary structure of Hb. H(r) regular alpha helix; H(d) disordered alpha helix; S(r) regular beta sheet; S(d) disordered beta sheet, T(r) Turn; Un unordered structure. System
H(r)%
H(d)%
S(r)%
S(d)%
T(r)%
Un%
Hb Hb–DIDS (Hb–DNP) DIDS Hb–DNP (Hb–DIDS) DNP
33.14 30.28 29.22 31.28 29.36
20.83 19.42 19.03 19.11 17.67
10.27 11.37 11.14 11.72 11.04
8.04 9.82 9.07 9.37 9.13
13.47 13.45 13.41 11.38 13.19
14.25 15.66 18.13 15.14 19.61
Study of the interaction between DNP and DIDS dispersed systems and the properties of the dispersion as affected by this electrical phenomenon. Although the zeta potential is a physical property that cannot the measured directly, it can be calculated from its electrophoretic mobility by applying the Henry equation. The Henry equation describes the relationship between the zeta potential and the electrophoretic mobility, and is expressed as:
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UE ¼ 2ezf ðKa Þ=3g
(14)
where z is the zeta potential, UE is the electrophoretic mobility, ε is the dielectric constant, η is the viscosity, and f (Ka) is Henry function. Ka is the ratio of the particle radius to the double layer thickness (Debye length) (Kerec, Bogataj, Mugerle, Gaperlin, & Mrhar, 2002). Two values are usually used as approximations of f (Ka), namely, 1.5 or 1.0. A zeta potential is a very usual index of the magnitude of the interaction between colloidal particles and measurements of zeta potential are commonly used to assess the stability of colloidal systems. Low zeta potentials results in agglomeration and greater measured particle sizes. It was important to note that if the particles have low zeta potential values, there will be no force to prevent the particles coming together to form aggregates. The adsorption of drugs over Hb may influence the kinetics and particle aggregation (Shcharbin et al., 2007; Shen et al., 2007). By bringing the zeta potential close to zero, the attractive forces become dominant and coagulation occurs. To explore the electrostatic effects of the drug/protein interactions, the zeta potential values of the systems were measured at various concentrations of drugs at pH 7.4 and room temperature. Figure 8(A) and (B) shows the zeta potential plots of DIDS and DNP upon interaction with Hb as a function of the concentration of the drugs is the two- and threecomponent systems. As we can see from low values of zeta potential, interaction occurred between the drugs and the protein. The adsorption of both drugs over the Hb surface increased the signal of the zeta potential and enhancement of drug concentrations, the zeta potential values dropped in consequence. The positive values of the zeta potential suggested that hydrophobic interactions had an essential role in the interaction between Hb and drugs. On the other hand, in all interacting systems, with increasing drug concentration, the amino acid residues on the Hb surface declined; therefore, the zeta potential values of the systems decreased. In other words, due to the addition of negatively charged carboxylic groups over the Hb, the zeta potential values became more negative. We should also note that the zeta potential values in all interacting systems fluctuated between 0 and −12, and that they were generally low enough for aggregates to form in solution. The inflection points of the zeta potential curves of Hb–DIDS and Hb–DNP were 2.8 × 10−10 mM
15
and 2.3 × 10−7 mM and 8.8 × 10−7 mM, respectively. Consequently, the critical aggregation point of the drug was induced by changes in the hydrophobic interactions due to the presence of other drugs. This phenomenon revealed that there occurred changes over the Hb surface upon the interaction with the drugs. The inflection points of the zeta potential curves for the binary and ternary Hb–drug systems confirmed the obtained RLS results. As can be shown from inset of Figure 8(A) and (B), the RLS intensity of the Hb–DIDS complex was higher than for the (Hb–DNP) DIDS system. It was found that the enhancement of the RLS intensity differed for various concentration of the Hb–drug solution. In the RLS spectrum, there was a nonlinear relationship between the enhanced intensity and the concentration of the drug. When DIDS and DNP concentrations were too low, the RLS intensity of the drug–Hb system hardly changed, the RLS intensity of the systems gradually increased, and precipitation occurred in the solutions that contained high concentrations of drugs. For comparison of the ability of the drugs to form complexes and aggregations on the Hb surface, we studied the critical-induced aggregation concentration (CCIAC) values of the interacting systems (Kabiri, Amiri-Tehranizadeh, Baratian, Saberi, & Chamani, 2012). Under identical experimental conditions, a smaller CCIAC value signified a smaller concentration of drug-induced protein aggregation. Due to a smaller interaction between the drug and Hb in the presence of the other drug, the CCIAC values for both ternary systems were much greater than for the binary ones. Consequently, in the presence of DNP in the [Hb–DNP] DIDS system and DIDS in the [Hb–DIDS] DNP system, the affinity of DIDS and DNP to form aggregates on the Hb surface decreased and the aggregated form generated at higher concentrations. 3.10. Molecular modeling To obtain more insight into the interaction of DIDS and DNP with Hb, molecular modeling simulations were applied to improve the understanding of the interaction between Hb and both anticancer drugs at the active binding of Hb in binary and ternary systems. This study should offer an explanation on the molecular level of the participation of specific chemical groups and their interactions in complex stabilization. The experimental observations were supported with docking studies where DNP and DIDS were docked to Hb. All calculations were performed with the MOE program. The crystal structure of Hb was downloaded from protein data bank, while the DNP and DIDS compound was prepared in the Molegro molecular viewer. The best energy-ranked results of the interaction between drugs and protein in all runs of docking procedure are shown in Figure 9(A) and (B), respectively. It is important to
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Figure 8. (A) Zeta potential spectra at varying concentrations of DIDS in the absence and presence of DNP. [Hb] = 4.6 × 10−3 mM. (B) Zeta potential spectra at varying concentrations of DNP in the absence and presence of DIDS. [Hb] = 4.6 × 10−3 mM.
note that the six Trp (Trp14, Trp158, Trp180, Trp414, Trp558, Trp580) located in various subunit are averagely 3.1 nm away from DIDS and 2.9 nm away from DNP in binary system which is within the distance (2–7 nm) suitable for efficient energy transfer from this residue to
DIDS and DNP located to Hb, to cause quenching (Figure 9(A) and (B)). Amino acids located near DIDS were obtained Leu 171,174,249 Val 210 and His 235 and amino acids located near DNP is Leu 174,248,249, and Val 252. The most of these amino acids were
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Study of the interaction between DNP and DIDS
Figure 9. (A) Docking interaction of Hb–DIDS. DIDS was shown as CPK and the amino acid around DIDS in Hb was shown in inset A. (B) docking interaction of Hb–DNP. DNP was shown as CPK and the amino acid around DNP in Hb was shown in inset B. (C) docking interaction of Hb–DNP in the presence of DIDS in ternary system and distance of DIDS and DNP with six Trp was determined. DIDS and DNP are shown as CPK and the amino acids around DIDS in Hb–DNP complex was shown in inset C. (D) docking interaction of Hb–DIDS in the presence of DNP in ternary system and distance of DIDS and DNP with six Trp was determined. DIDS and DNP are shown as CPK and the amino acids around DNP in Hb–DIDS complex was shown in inset D.
S. Rashidipour et al.
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Figure 9.
(Continued).
Study of the interaction between DNP and DIDS aliphatic and have hydrophyl chain and situated on the inside. In ternary system, averagely distance of Hb Trps and DIDS was determined 2.8 nm and for DNP, the averagely distance was measured 2.4 nm (Figure 9(C) and (D)). This result indicates that when DIDS or DNP interacted with Hb, the distance between second drug in ternary system decreased, suggesting that DNP and DIDS can also effect the structure of the Hb.
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4. Conclusions This work explored the interaction and binding of DNP and DIDS to Hb by different spectroscopic and molecular modeling techniques. This is done through an opening of its structural state, which is of vital importance in case of structure-specific binders, e.g. Hb-oxygen binding. DIDS and DNP are two uncoupler mitochondria drugs and they both can affect the structure of Hb. The central focus of this research was to investigate the influence of these substances on the structure and function of Hb. The binding of DIDS and DNP to Hb results in either a decrease or increase in affinity of the second drug to Hb. The obtained results indicated that a complex was formed between the drugs and Hb, as evidenced by the fact that the fluorescence quenching mechanism for Hb by the drugs is static and that the distance between drugs and Hb is lower than 7 nm. On the basis of binding and quenching constants determined from Stern–Volmer equations, it can be stated that the formation of Hb–DIDS and Hb–DNP complexes decreased in the presence of DNP and DIDS, respectively. Synchronous fluorescence spectroscopy showed that the micro-environment changes of the Trp and Tyr residues altered their stability in the presence of the drugs, but only Trp played a role in the fluorescence quenching. The CD data revealed that the presence of DIDS and DNP decreased the α-helical content of Hb that caused destabilization of protein. The Time-resolved fluorescence spectroscopy demonstrated a reduction in the average fluorescence lifetime in all tested systems that showed a significant energy transfer and the formation of complexes occurred between the drugs and Hb. The interaction of DIDS and DNP with Hb resulted in an enhancement of the RLS and zeta potential intensity in binary and ternary systems. It was possible to determine the critical-induced aggregation concentration (CCIAC) of DIDS and DNP on Hb. The results obtained can be used in pharmacokinetic, drug delivery, and determination of usage drug doses. Acknowledgments The financial support of the Research Council of the Mashhad Branch, Islamic Azad University is gratefully acknowledged. The authors thank Dr Ljungberg for English editing.
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