Cell Biochem Biophys DOI 10.1007/s12013-014-9863-1
ORIGINAL PAPER
Analysis of Binding Interaction Between Antibacterial Ciprofloxacin and Human Serum Albumin by Spectroscopic Techniques Ankita Varshney • Yunus Ansari • Nida Zaidi Ejaz Ahmad • Gamal Badr • Parvez Alam • Rizwan Hasan Khan
•
Ó Springer Science+Business Media New York 2014
Abstract The binding of ciprofloxacin (CFX) to human serum albumin (HSA) has been investigated by fluorescence displacement and induced circular dichroism (ICD) measurements. Displacement measurements were performed with CFX in the absence and presence of marker ligands (hemin for domain I, bilirubin for interspace of domain IA and IIA, chloroform for domain II, and diazepam for domain III) to establish CFX binding site in one of the three major domains of HSA. The primary binding site of CFX is located in site I of HSA (domain IIA) in close vicinity to the site where chloroform (CHCl3) binds. It is depicted from the decrease in quenching constant of HSA–CHCl3 system (0.02 ± 0.06) 9 10-3 L mol-1 compared to HSA–CFX– CHCl3 system (0.01 ± 0.06) 9 10-3 L mol-1 as obtained by the fluorescence displacement spectroscopy. Furthermore, far-UV CD results show that the binding of CFX leads to change in the helicity of HSA. The ICD results indicated that the CFX binds to the domain IIA of HSA which is in agreement with the fluorescence displacement results. Keywords Ciprofloxacin (CFX) Binding parameters Circular dichroism Human serum albumin
A. Varshney Y. Ansari N. Zaidi E. Ahmad P. Alam R. H. Khan (&) Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, U.P., India e-mail: [email protected]; [email protected] G. Badr Laboratory of Immunology & Molecular Biology, Zoology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt
Introduction Human serum albumin (HSA) is most abundant serum protein in humans. It binds and transport a large variety of ligands including hormones, fatty acids, drugs, etc. [1–5]. It is a globular, multifunctional protein composed of three structurally similar domains each containing two subdomains and having molecular weight of 67 kD stabilized by 17 disulfide bonds [6–8]. Apart from ligands binding and transport, it involves in maintaining pH and osmotic pressure, preventing photodegradation of folic acid, and is also a marker of inflammatory state [9, 10]. The group of fluoroquinolones is one of the most successful classes of antibacterial drugs. These compounds are of exceeding interest because their clinical role has greatly expanded since they were introduced in the 1980s. One of these quinolones, ciprofloxacin (CFX), has in vitro activity against a wide range of Gram-negative and Gram-positive microorganisms. The mechanism of action of quinolones, including CFX, is different from that of other antimicrobial agents such as beta-lactams, macrolides, tetracyclines, and aminoglycosides; therefore, organisms resistant to these drugs may be susceptible to CFX. There is no known crossresistance between CFX and other classes of antimicrobials. Notably, the drug has 100 times higher affinity for bacterial DNA gyrase than for mammalian. This fluoroquinolone has been applied in the empirical treatment of a variety of infections, particularly those of genitourinary, gastrointestinal, and respiratory tracts [11]. Chemically, CFX is a 1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-quinolone3-carboxylic acid (Fig. 1). Since it has an extended aromatic part and functional groups suitable for hydrogen bonding, it can be expected that this phenolic type molecule is able to interact strongly with biomacromolecules and that these noncovalent interactions may play a decisive role in its
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Cell Biochem Biophys
was purchased from Hemedia. The number in the parenthesis corresponds to the purity of the compounds. All of the other reagents were of analytical grade. Methods Protein Concentration Determination
Fig. 1 Chemical structure of ciprofloxacin
mechanism of action. Because of its pharmacological activity, the investigation of the interactions between this compound and serum albumins is very important [12]. Furthermore, binding of drugs to albumin alters the pattern and volume of distribution, lowers the rate of clearance, and increases the plasma half-life of the drug [12–15]. Since CFX is practically insoluble in water (*1.1 mg L-1) at neutral pH and rapidly decomposes in alkaline solution, its binding to serum albumin is very important to exert the beneficial therapeutic activities. Protein binding has long been considered one of the most important physicochemical characteristics of drugs, playing a potential role in distribution, excretion, and therapeutic effectiveness [16]. The multiplicity of binding sites on HSA for endogenous and exogenous small molecules makes it difficult to assess interactions, whether competitive or cooperative, between different ligands bound to the protein. The flexible structural organization allows the protein structure to adapt to a variety of ligands. However, it is important to address this issue in order to obtain a fuller description of the ligand-binding properties of HSA [8, 17, 18]. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA a multimeric protein. In this study to explore the binding of CFX to HSA, quenching of tryptophan fluorescence was carried out. Furthermore far-UV CD spectroscopy was employed to confirm the secondary structural changes upon CFX binding to HSA. The probable binding site of CFX on HSA is also predicted from marker displacement experiment.
Materials and Methods
Protein concentration was determined spectrophotometrically using E1cm of 5.30 at 280 nm [19] on a Hitachi spectrophotometer, model U-1500 or alternately by the method of Lowry et al. [20]. Sample Preparation HSA and drug solutions were prepared in 20 mM sodium phosphate buffer (pH 7.4). HSA was passed through Sephacryl-S200 gel filtration column and dialyzed. Site markers for HSA were also prepared in 20 mM phosphate buffer and ethanol [ethanol concentration did not exceed 5 % (v/v)] and their concentration was calculated appropriately. All the solutions were prepared by weight/volume (w/v). Binding Displacement Measurement Using Site Markers Different site markers, Hem for site in subdomain IA [21], DIA for site II (subdomain III A) [22, 23], chloroform (CHCl3) for site in subdomain 1IA [24], and BR for site in interspace of subdomain IA and IIA [25], were used for performing displacement experiments. The titration of CFX was carried out to the solution having protein and site marker in the ratio of 1:1. The fluorescence emission spectra were recorded in the 300–400 nm range after exciting at 295 nm. The binding constant values of drug–protein–marker were evaluated using Stern–Volmer equation. Fluorescence Quenching Measurement of HSA Fluorescence measurements were performed on a Shimadzu spectrofluorimeter, model RF-5301 PC. The fluorescence spectra were measured at 25 ± 0.1 °C with a 1 cm path length cell. Both excitation and emission slits were set at 3 nm. Intrinsic fluorescence was measured by exciting the protein solution at 295 nm and emission spectra were recorded in the range of 300–400 nm.
Materials HSA (A1887; [96 %), CFX (17850; [98.0 %), warfarin (A2250; [98 %), hemin (Hem) (H5533; [80 %), diazepam (DIA) (D0406; [98 %), and chloroform (C2432; >99.5 %) purchased from Sigma-Aldrich. Bilirubin (BR)
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Calculations/Data Analysis The quenching equation is presented by F0 =F ¼ 1 þ kq s0 ½Q ¼ 1 þ Ksv ½Q;
ð1Þ
Cell Biochem Biophys
where F and F0 are the fluorescence intensities with and without quencher, respectively, kq is the quenching rate constant of the biomolecule, Ksv is the Stern–Volmer quenching constant, s0 is the average lifetime of the biomolecule without ligand, and [Q] is the concentration of the quencher used. Fluorescence quenching data of HSA complexed with markers in the absence and presence of CFX were analyzed to obtain various binding parameters. The binding constant (Kb) and binding affinity were calculated according to the given equation
CFX. On the other hand, stability of HSA–Hem complex (Kb = 3.01 9 108 M-1, DG0 = -48.30 kJ mol-1) decreases compared to the HSA–CFX–Hem complex (Kb = 29.37 9 108 M-1, DG0 = -53.93 kJ mol-1). These results suggest that the presence of CFX did not affect the binding of Hem to domain IB.
Log ½ðF0 FÞ=F ¼ log Kb þ n log ½Q;
The binding of BR, a toxic metabolite of heme, to HSA [18, 26] has been studied extensively for many years. A number of studies that measured the affinity of proteolytic fragments of HSA for BR showed that the high-affinity BR-binding site was located near subdomain IIA. There is, therefore, great clinical interest in understanding the binding of BR to albumin and the effects of drugs and other competitors on this binding. Aliquots of BR to the protein solution were added in the absence and presence of CFX, and decrease in protein fluorescence were measured after each addition of BR. The fluorescence intensity of HSA decreased regularly, and slight blue shift was observed for the emission wavelength with increasing BR concentration up to BR/albumin molar ratio of 0–10, indicating that the presence of BR could quench the fluorescence of HSA– CFX complex. Furthermore, determining the various binding parameters (Fig. 3a, b; Tables 1, 2) depicts clearly that there was no competitive binding between CFX and BR since we observed very less change in the binding pattern when two ligands were allowed to bind. We observed decreased stability of HSA–BR complex (Kb = 0.1088 9 108 M-1, DG0 = -40.08 kJ mol-1) when compared to HSA–CFX–BR complex (Kb = 20.73 9 108 M-1, DG0 = -53.07 kJ mol-1).
ð2Þ
where F0 and F are the fluorescence intensities with and without the ligand, respectively. A plot of log [(Fo - F)/F] versus log [Q] gave a straight line using least-squares analysis whose slope was equal to n (binding affinity) and the intercept on Y-axis to logK (K = binding constant). The binding constant (K) thus obtained was used to calculate the standard free energy change DG0binding of the ligand binding to HSA from the relationship DG0binding ¼ 2:303 RT ln Kb :
ð3Þ
Circular Dichroism Spectroscopy Circular dichroism (CD) was performed on a Jasco J-715 spectropolarimeter at 25 ± 0.2 °C, in a rectangular cell with 1.0 cm path length equipped with magnetic stirring. Each spectrum was signal-averaged at least three times with a bandwidth of 1.0 nm and a resolution of 0.5 nm at a scan speed of 100 nm min-1. Induced CD (ICD) spectra resulting from the interaction of the drug with HSA were obtained by subtracting the CD spectrum of the protein from that of the complex.
Results and Discussions Binding of Hemin in the Absence and Presence of Ciprofloxacin X-ray crystal structure of HSA–Hem complex has shown a single binding site for Hem on domain I [18, 19]. Hem is a large planar molecule and can be used as a probe for monitoring the effect of drug on the binding properties of domain I. The fluorescence quenching spectra of HSA at various concentrations of Hem in the absence and presence of CFX are shown in Fig. 2a, b and the data are summarized in Tables 1 and 2. Equilibration of HSA with Hem caused concentration-dependent quenching in the intrinsic fluorescence intensity, which suggests the binding of Hem to HSA. A little decrease in association constant and almost no change in the binding sites n were observed in the presence of
Binding of Bilirubin in the Absence and Presence of Ciprofloxacin
Binding of Chloroform in the Absence and Presence of Ciprofloxacin The site of action of the volatile general anesthetics remains controversial, but evidence in favor of its binding to subdomain IIA of HSA is accumulating. In this study, in the absence and presence of CFX, binding to chloroform to HSA is monitored by fluorescence quenching measurements. Chloroform causes a decrease in the fluorescence emission quantum yield as shown in Fig. 4a, b. A slight blue shift of 2 nm in the emission wavelength maximum was observed, suggesting that the binding of chloroform is associated with the changes in the dielectric environment of the indole ring in HSA, because electron transfer from the excited indole ring to chloroform might be responsible for the observed fluorescence quenching. Figure 4 shows the Stern–Volmer plot from the slope of which
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Cell Biochem Biophys Fig. 2 a Fluorescence spectra of hemin quenching in the absence of CFX. b Fluorescence spectra of hemin quenching in the presence of CFX. c Stern– Volmer plots of hemin quenching in the absence (open circle) and presence (filled circle) of CFX. d Log [(F0 F)/F] versus Log [Q] plots of hemin quenching in the absence (open circle) and presence (filled circle) of CFX for determining the binding constant and binding sites
250
A
Fluorescence Intensity
Fluorescence Intensity
250 200 150 100 50 0 300
350
B 200 150 100 50 0 300
400
350
1.2
8
C
D
0.8
Log [(Fo-F)/F]
6
Fo/F
400
Wavelength (nm)
Wavelength (nm)
4
2
0.4 0 -0.4 -0.8
0
-1.2 0
1
2
Hemin [ µ M]
3
4
-7
-6.5
-6
-5.5
-5
Log [Hemin]
Table 1 Effects of domain-specific ligands on binding constants of HSA and HSA–CFX systems Ligands
Systems
Ksv 9 106 [L mol-1]a
Kq 9 1014 [L mol-1 s-1]a
R2
Hemin
HSA
1.77
1.77
0.9917
HSA–CFX
1.40
1.40
0.9895
HSA
0.4
0.4
0.9902
HSA–CFX
0.2
0.2
0.9572
HSA
0.02 9 10-3
0.02 9 10-3
0.9992
-3
0.01 9 10-3
0.9951
Bilirubin Chloroform Diazepam a
HSA–CFX
0.01 9 10
HSA
0.09
0.092
0.9962
HSA–CFX
0.06
0.06
0.9969
The mean value of 4 individual experiments with standard deviation ±0.06–±0.11 %
Table 2 Thermodynamic and binding parameters of HSA and HSA– CFX system Ligand
Systems
Kb [M-1]a
n
Hemin
HSA
3.01 9 108
1.40
-48.30
HSA–CFX
29.37 9 108
1.60
-53.93
HSA
0.1088 9 108
1.24
-40.08
Bilirubin Chloroform Diazepam a
8
DG0 [kJ mol-1]
HSA–CFX
20.73 9 10
1.68
-53.07
HSA
18.13
1.03
-7.16
HSA–CFX
11.98
1.08
-6.14
HSA
3.48 9 104
0.93
-25.87
HSA–CFX
3.09 9 104
0.94
-25.57
The mean value of 4 individual experiments with standard deviation ±0.06–±0.11 %
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Ksv = 0.02 ± 0.08 M-1 was calculated. It is observed that Ksv = 0.02 ± 0.08 M-1 was reduced to almost half (0.01 ± 0.08 M-1) in the presence of CFX which is suggestive of static interaction between fluorophore and CFX. Furthermore a little increase in the binding site of HSA n in the absence and presence of CFX depict that an allosteric binding exists on domain II for both the CFX and chloroform (Fig. 4; Tables 1, 2). The approach used allows direct monitoring of anesthetic binding to the protein and, in addition, provides information about the location of the anesthetic in the protein matrix. The results indicate that chloroform occupies the same binding site on this model mammalian protein, HSA, as that occupied by CFX.
Cell Biochem Biophys 320
250
A 240
Fluorescence Intensity
Fluorescence Intensity
Fig. 3 a Fluorescence spectra of bilirubin quenching in the absence of CFX. b Fluorescence spectra of bilirubin quenching in the presence of CFX. c Stern– Volmer plots of bilirubin quenching in the absence (open circle) and presence (filled circle) of CFX. d Log [(F0 F)/F] versus Log [Q] plots of bilirubin quenching in the absence (open circle) and presence (filled circle) of CFX for determining the binding constant and binding sites
a
160
j
80
0 300
350
B a
200 150
j 100 50 0 300
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Wavelength [nm]
Wavelength [nm] 2 0
C
D -0.4
Fo/F
Log [(Fo-F)/F]
1.6
1.2
-0.8 -1.2 -1.6
0.8
0
0.5
1
1.5
2
-2 -6.6
2.5
-6.4
Bilirubin [ µ M]
100
-5.8
B
80
Fluorescence Intensity
Fluorescence Intensity
-6
-5.6
100
A a
60 j
40 20 0 300
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60
j
40 20 0 300
400
a
80
350
Wavelength [nm]
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Wavelength [nm] 0.4
2.4
C
D
0 Log [(Fo-F)/F]
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Fo/F
Fig. 4 a Fluorescence spectra of chloroform quenching in the absence of CFX. b Fluorescence spectra of chloroform quenching in the presence of CFX. c Stern–Volmer plots of chloroform quenching in the absence (open circle) and presence (filled circle) of CFX. d Log [(F0 - F)/F] versus Log [Q] plots of chloroform quenching in the absence (open circle) and presence (filled circle) of CFX for determining the binding constant and binding sites
-6.2
Log [Bilirubin]
1.6
1.2
-0.4 -0.8 -1.2
0.8 0
20
40 60 CHCl3 [mM]
80
100
-1.6 -2.2
-1.8 -1.4 Log [CHCl3]
-1
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Cell Biochem Biophys 250 250
A 200
B
a Fluorescence Intensity
Fluorescence Intensity
Fig. 5 a Fluorescence spectra of diazepam quenching in the absence of CFX. b Fluorescence spectra of diazepam quenching in the presence of CFX. c Stern– Volmer plots of diazepam quenching in the absence (open circle) and presence (filled circle) of CFX. d Log [(F0 F)/F] versus Log [Q] plots of diazepam quenching in the absence (open circle) and presence (filled circle) of CFX for determining the binding constant and binding sites
150 j 100 50 0 300
350
200
100 50
350
Wavelength [nm] 0
C
D
2.1 Log [(Fo-F)/F]
-0.3
Fo/F
1.8 1.5
-0.6
-0.9
1.2 0.9
400
Wavelength [nm]
2.4
0
3
6 9 Diazepam [µM]
Binding of Diazepam in the Absence and Presence of Ciprofloxacin Accumulating evidence suggests that the primary diazepam binding site was located in HSA domain III [11]. Thus to trace the binding site of CFX, DIA binding to domain III of HSA in the absence and presence of CFX was examined. Figure 5a, b represents the fluorescence intensity spectra while Fig. 5c, d represents Stern–Volmer plot and modified Stern–Volmer plot, respectively. The values of binding constant and change in binding energy was obtained using Eqs. (1)–(3) and are listed in Tables 1 and 2. Almost 1.5 times decrease in the binding constant of HSA in the absence of drug compared to HSA–CFX complex was observed while no significant change in binding site is being noticed. This indicates increased stability of HSA–diazepam complex (Ksv = 0.09 9 106 M-1, DG0 = -25.87 kJ mol-1) compared to HSA–CFX–diazepam complex (Ksv = 0.06 9 106 M-1, DG0 = -25.57 kJ mol-1). These results suggest that the binding of diazepam at domain III is not affected by the presence and absence of drug.
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j
150
0 300
400
a
12
15
-1.2 -6.3
-5.8 -5.3 Log [Diazepam]
-4.8
Conformation Investigation To evaluate the structural change of HSA by addition of ligands, the far-UV CD spectra measurements were taken which have been widely used for studying the conformation and conformational change of proteins and polypeptides in solution [27]. Figure 6a, b shows the far-UV CD spectra of the HSA–marker and HSA–marker–CFX complexes obtained at pH 7.4 at room temperature, respectively. As expected for a protein that is predominantly ahelical, the CD spectrum of HSA shows a strong negative ellipticity at 208 and 222 nm (Fig. 6a, curve 3). The reasonable explanation is that the negative peaks at 208 and 222 nm are both contributed as a result of n ? K* transition for the peptide bond of a-helix [22]. As shown in Fig. 6b curve 3, binding of CFX to HSA decreases its helical content. The binding of chloroform to HSA–CFX complex induces increase in negative ellipticity as shown in Fig. 6b that might be due to shielding of the peptide strand due to the increase in hydrophobicity on binding. This conclusion agrees with the result of fluorescence quenching experiment.
Cell Biochem Biophys 20
characteristic CD of BR bound to HSA undergoes a remarkable sign inversion on the addition of CFX. This sign inversion reflects a pronounced conformational change of the bound ligand; probably a complete inversion of chirality. The observation suggests that association of ligand with proteins can markedly alter the internal topography of receptor sites and potentially influence the stereoselectivity of ligand binding.
A
CD [mdeg]
0
1 2 3 4 5
-50
-90
200
210
220
230
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250
Wavelength [nm]
Probing the Binding Site of Ciprofloxacin on HSA by Ligand Displacement Experiments
20
B CD [mdeg]
0
-50
1 2 3 4 5
-90 200
210
220
230
240
250
Wavelength [nm] 1 2 3 4 5
HSA: Hemin= 1:1 HSA:Bilirubin=1:1 Free HSA / HSA-CFX=1:1 (b& d) HSA:Diazepam=1:1 HSA:Chloroform=1:1
Fig. 6 Far-UV CD signals of HSA–marker complex (1:1) a in the absence and b in presence of equimolar CFX
Chiroptical Properties of the Ciprofloxacin–HSA Complexes at pH 7.4 The method of ICD is based on the observation that an optical activity arises from asymmetry in the ligand induced by its binding to the protein, since the free ligand has either no asymmetric center or, therefore, gives no signal in solution. Chiral conformation of the ligand due to conformational adaptation to its binding site, or interaction between ligand molecules held in chiral arrangement relative to each other by the protein sites, results in one or more ICD bands with different shapes, signs and intensities. These extrinsic Cotton effects present in lightabsorbing region of the optically active or inactive ligand molecules give qualitative and quantitative information of the binding process. Figure 7a–d shows the ICD spectra for marker complexed with protein in the absence and presence of CFX. A molar ratio of 0.1 and 1 was selected for both the conditions to ensure that all of the ligand is bound to the primary binding site, avoiding interactions with secondary, weaker binding sites. In Fig. 7a, the
The finding that the CFX–HSA complex exhibits induced a CD couplet in the visible spectral region provides a sensitive tool for studying the binding location of CFX on HSA. In the presence of a compound having the same binding site as CFX, amplitudes of the induced secondary structure should decrease due to competition. Therefore, CD displacement experiments were performed using domain-specific ligands. Primary binding location of Hem on HSA is domain IB with Ka = 1.1 9 108 M-1, BR binds at the interface of domain I and II with Ka = 9.5 9 107 M-1 [25], diazepam (Ka = 3.8 9 105 M-1) binds at subdomain IIIA, and high-affinity binding site of chloroform (Ka = 3.8 9 105 M-1) is on domain IIA of BSA.
Conclusion Ciprofloxacin binds to site I of HSA; in addition it is also a promising molecular probe to study biologically important, induced conformational polymorphisms of serum albumins. Pharmacological and pharmacodynamic properties of biologically active natural and synthetic compounds are crucially determined via their binding to proteins of the human serum. This paper is aimed to survey competitive binding of drug in the presence of domain-specific ligands and the results investigate the influence of domain-specific ligands in the presence of CFX binding on HSA and how the drug molecules can influence or compete with other ligand molecules bound to the protein. The experimental results revealed that CFX has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. All these experimental results and other data clarified that CFX could bind to HSA and be effectively transported and eliminated in body, which could be a useful guideline for further drug design. On the basis of the above-discussed results, a pictorial model depicting the binding of various domain specific ligands have been formulated (Fig. 8) which also predicts that the binding site of CFX exists on domain IIA as reported earlier by our group [28] in the close vicinity of
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Cell Biochem Biophys 4
40
A
B
2
CD [mdeg]
20
CD [mdeg]
Fig. 7 ICD (expressed in mdeg) of marker ligands: hemin (a), bilirubin (b), diazepam (c), and chloroform (d) bound to HSA and HSA–CFX at a molar ratio of 0.1 and 1. Line symbols are shown in inset. Protein concentration was 10 lM in 0.06 M sodium phosphate buffer, pH 7.4
0 -2 HE: HSA=0.1:1 HE: CFX: HSA=0.1:1:1 HE: HSA=1:1 HE: CFX: HSA=0.1:1:1
-4 -6 350
400
450
0
BR: HSA=0.1:1 BR: CFX: HSA=0.1:1:1 BR: HSA=1:1 BR: CFX: HSA=1:1:1
-20
-40 350
500
Wavelength [nm] 12
400
450
500
550
Wavelength [nm] 12
C
D CD [mdeg]
CD [mdeg]
9
0 DI: HSA=0.1:1 DI: CFX: HSA=0.1:1:1 DI: HSA=1:1 DI: CFX: HSA=1:1:1
280
300
Wavelength [nm]
3 0
CHCl3: HSA=0.1:1 CHCl3: CFX: HSA= 0.1:1:1 CHCl3: HSA= 1:1 CHCl3: CFX: HSA= 1:1:1
-3
-10 -13 250 260
6
320
-6 300
350
400
450
500
Wavelength [nm]
Fig. 8 A pictorial model indicating the binding of domain-specific ligands and predicting the binding site of ciprofloxacin on human serum albumin
the site where chloroform binds. Our work not only provides the multiplicity of binding sites on HSA and demonstrates the conformational plasticity of HSA on drug binding, but it may also provide structural information for
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the modulation of HSA–drug interaction by various spectroscopic approaches based on HSA–drug interaction. The binding of small molecules to proteins and protein– protein interactions are key processes in cell biochemistry.
Cell Biochem Biophys
The usual paradigm is that ligand binding induces a change in the conformation of the target protein which, in turn, produces a given response and fundamental importance. Using the binding of various domain-specific ligands to HSA, we have concluded that CFX interacts with domain II of HSA in the close vicinity to chloroform. Recent advances of gene cloning, together with complete understanding of albumin structure and function, provide for a greater abundance of future applications. Accordingly, our information on the relative stability of HSA and its domains should provide a basis for drug design, as they possess binding sites for a variety of exogenous and endogenous ligands. Acknowledgments Facilities provided by A.M.U are gratefully acknowledged. Ankita Varshney, Ejaz Ahmad, Nida Zaidi, and Parvez Alam thank the Council of Scientific and Industrial Research, New Delhi and Yunus Ansari thanks the department of Biotechnology, Govt. of India for financial assistance.
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11. Lode, H. (1999). Potential interactions of the extended spectrum fluoroquinolones with the CNS. Drug Safety, 2, 123–135. 12. Benet, L. Z., & Hoener, B. A. (2002). Changes in plasma protein binding have little clinical relevance. Clinical Pharmacology Therapeutics, 71, 115–121. 13. Herve, F., Urien, S., Albengres, E., Duche, J. C., & Tillement, J. P. (1994). Drug binding in plasma. A summary of recent trends in the study of drug and hormone binding. Clinical Pharmacokinetics, 26, 44–58. 14. Levine, R. R. (2000). Pharmacology: Drug actions and reactions (6th ed.). New York: The Parthenon Publishing Group. 15. Pratt, W. B., & Taylor, P. (1990). Principles of drug action: The basis of pharmacology (3rd ed.). New York: Churchill Livingstone Inc. 16. Khan, M. Y. (1986). Direct evidence for the involvement of domain III in the N-F transition of bovine serum albumin. Biochemical Journal, 236, 307–310. 17. Curry, S., Mandelkow, H., Brick, P., & Franks, N. (1998). Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites. Nature Structural Biology, 5, 827–835. 18. He, X. M., & Carter, D. C. (1992). Atomic structure and chemistry of human serum albumin. Nature, 358, 209–215. 19. Wallevik, K. (1973). Reversible denaturation of human serum albumin by pH, temperature, and guanidine hydrochloride followed by optical rotation. Journal of Biological Chemistry, 248, 2650–2655. 20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275. 21. Zunszain, P. A., Ghuman, J., Komatsu, T., Tsuchida, E., & Curry, S. (2003). Crystal structural analysis of human serum albumin complexed with hemin and fatty acid. BMC Structural Biology, 3, 1–9. 22. Kratochwil, N. A., Huber, W., Muller, F., Kansy, M., & Gerber, P. R. (2002). Predicting plasma protein binding of drugs: a new approach. Biochemical Pharmacology, 64, 1355–1374. 23. Kang, J., Liu, Y., Xie, M. X., Li, S., Jiang, M., & Wang, Y. D. (2004). Interactions of human serum albumin with chlorogenic acid and ferulic acid. Biochimica Biophysica Acta, 1674, 205–214. 24. Johansson, J. S. (1997). Binding of the volatile anesthetic chloroform to albumin demonstrated using tryptophan fluorescence quenching. Journal of Biological Chemistry, 272, 17961–17965. 25. Kragh-Hansen, U. (1988). Evidence for a large and flexible region of human serum albumin possessing high affinity binding sites for salicylate, warfarin, and other ligands. Molecular Pharmacology, 34, 160–171. 26. Silva, D., Cortez, C. M., Cunha-Bastos, J., & Louro, S. R. W. (2004). Methyl parathion interaction with human and bovine serum albumin. Toxicology Letters, 147, 53–61. 27. Kelly, S. M., & Price, N. N. (2000). The use of circular dichroism in the investigation of protein structure and function. Current Protein and Peptide Science, 1, 349–384. 28. Ahmad, B., Parveen, S., & Khan, R. H. (2006). Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site. Biomacromolecules, 7, 1350–1356.
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ORIGINAL PAPER
Analysis of Binding Interaction Between Antibacterial Ciprofloxacin and Human Serum Albumin by Spectroscopic Techniques Ankita Varshney • Yunus Ansari • Nida Zaidi Ejaz Ahmad • Gamal Badr • Parvez Alam • Rizwan Hasan Khan
•
Ó Springer Science+Business Media New York 2014
Abstract The binding of ciprofloxacin (CFX) to human serum albumin (HSA) has been investigated by fluorescence displacement and induced circular dichroism (ICD) measurements. Displacement measurements were performed with CFX in the absence and presence of marker ligands (hemin for domain I, bilirubin for interspace of domain IA and IIA, chloroform for domain II, and diazepam for domain III) to establish CFX binding site in one of the three major domains of HSA. The primary binding site of CFX is located in site I of HSA (domain IIA) in close vicinity to the site where chloroform (CHCl3) binds. It is depicted from the decrease in quenching constant of HSA–CHCl3 system (0.02 ± 0.06) 9 10-3 L mol-1 compared to HSA–CFX– CHCl3 system (0.01 ± 0.06) 9 10-3 L mol-1 as obtained by the fluorescence displacement spectroscopy. Furthermore, far-UV CD results show that the binding of CFX leads to change in the helicity of HSA. The ICD results indicated that the CFX binds to the domain IIA of HSA which is in agreement with the fluorescence displacement results. Keywords Ciprofloxacin (CFX) Binding parameters Circular dichroism Human serum albumin
A. Varshney Y. Ansari N. Zaidi E. Ahmad P. Alam R. H. Khan (&) Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, U.P., India e-mail: [email protected]; [email protected] G. Badr Laboratory of Immunology & Molecular Biology, Zoology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt
Introduction Human serum albumin (HSA) is most abundant serum protein in humans. It binds and transport a large variety of ligands including hormones, fatty acids, drugs, etc. [1–5]. It is a globular, multifunctional protein composed of three structurally similar domains each containing two subdomains and having molecular weight of 67 kD stabilized by 17 disulfide bonds [6–8]. Apart from ligands binding and transport, it involves in maintaining pH and osmotic pressure, preventing photodegradation of folic acid, and is also a marker of inflammatory state [9, 10]. The group of fluoroquinolones is one of the most successful classes of antibacterial drugs. These compounds are of exceeding interest because their clinical role has greatly expanded since they were introduced in the 1980s. One of these quinolones, ciprofloxacin (CFX), has in vitro activity against a wide range of Gram-negative and Gram-positive microorganisms. The mechanism of action of quinolones, including CFX, is different from that of other antimicrobial agents such as beta-lactams, macrolides, tetracyclines, and aminoglycosides; therefore, organisms resistant to these drugs may be susceptible to CFX. There is no known crossresistance between CFX and other classes of antimicrobials. Notably, the drug has 100 times higher affinity for bacterial DNA gyrase than for mammalian. This fluoroquinolone has been applied in the empirical treatment of a variety of infections, particularly those of genitourinary, gastrointestinal, and respiratory tracts [11]. Chemically, CFX is a 1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-quinolone3-carboxylic acid (Fig. 1). Since it has an extended aromatic part and functional groups suitable for hydrogen bonding, it can be expected that this phenolic type molecule is able to interact strongly with biomacromolecules and that these noncovalent interactions may play a decisive role in its
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was purchased from Hemedia. The number in the parenthesis corresponds to the purity of the compounds. All of the other reagents were of analytical grade. Methods Protein Concentration Determination
Fig. 1 Chemical structure of ciprofloxacin
mechanism of action. Because of its pharmacological activity, the investigation of the interactions between this compound and serum albumins is very important [12]. Furthermore, binding of drugs to albumin alters the pattern and volume of distribution, lowers the rate of clearance, and increases the plasma half-life of the drug [12–15]. Since CFX is practically insoluble in water (*1.1 mg L-1) at neutral pH and rapidly decomposes in alkaline solution, its binding to serum albumin is very important to exert the beneficial therapeutic activities. Protein binding has long been considered one of the most important physicochemical characteristics of drugs, playing a potential role in distribution, excretion, and therapeutic effectiveness [16]. The multiplicity of binding sites on HSA for endogenous and exogenous small molecules makes it difficult to assess interactions, whether competitive or cooperative, between different ligands bound to the protein. The flexible structural organization allows the protein structure to adapt to a variety of ligands. However, it is important to address this issue in order to obtain a fuller description of the ligand-binding properties of HSA [8, 17, 18]. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA a multimeric protein. In this study to explore the binding of CFX to HSA, quenching of tryptophan fluorescence was carried out. Furthermore far-UV CD spectroscopy was employed to confirm the secondary structural changes upon CFX binding to HSA. The probable binding site of CFX on HSA is also predicted from marker displacement experiment.
Materials and Methods
Protein concentration was determined spectrophotometrically using E1cm of 5.30 at 280 nm [19] on a Hitachi spectrophotometer, model U-1500 or alternately by the method of Lowry et al. [20]. Sample Preparation HSA and drug solutions were prepared in 20 mM sodium phosphate buffer (pH 7.4). HSA was passed through Sephacryl-S200 gel filtration column and dialyzed. Site markers for HSA were also prepared in 20 mM phosphate buffer and ethanol [ethanol concentration did not exceed 5 % (v/v)] and their concentration was calculated appropriately. All the solutions were prepared by weight/volume (w/v). Binding Displacement Measurement Using Site Markers Different site markers, Hem for site in subdomain IA [21], DIA for site II (subdomain III A) [22, 23], chloroform (CHCl3) for site in subdomain 1IA [24], and BR for site in interspace of subdomain IA and IIA [25], were used for performing displacement experiments. The titration of CFX was carried out to the solution having protein and site marker in the ratio of 1:1. The fluorescence emission spectra were recorded in the 300–400 nm range after exciting at 295 nm. The binding constant values of drug–protein–marker were evaluated using Stern–Volmer equation. Fluorescence Quenching Measurement of HSA Fluorescence measurements were performed on a Shimadzu spectrofluorimeter, model RF-5301 PC. The fluorescence spectra were measured at 25 ± 0.1 °C with a 1 cm path length cell. Both excitation and emission slits were set at 3 nm. Intrinsic fluorescence was measured by exciting the protein solution at 295 nm and emission spectra were recorded in the range of 300–400 nm.
Materials HSA (A1887; [96 %), CFX (17850; [98.0 %), warfarin (A2250; [98 %), hemin (Hem) (H5533; [80 %), diazepam (DIA) (D0406; [98 %), and chloroform (C2432; >99.5 %) purchased from Sigma-Aldrich. Bilirubin (BR)
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Calculations/Data Analysis The quenching equation is presented by F0 =F ¼ 1 þ kq s0 ½Q ¼ 1 þ Ksv ½Q;
ð1Þ
Cell Biochem Biophys
where F and F0 are the fluorescence intensities with and without quencher, respectively, kq is the quenching rate constant of the biomolecule, Ksv is the Stern–Volmer quenching constant, s0 is the average lifetime of the biomolecule without ligand, and [Q] is the concentration of the quencher used. Fluorescence quenching data of HSA complexed with markers in the absence and presence of CFX were analyzed to obtain various binding parameters. The binding constant (Kb) and binding affinity were calculated according to the given equation
CFX. On the other hand, stability of HSA–Hem complex (Kb = 3.01 9 108 M-1, DG0 = -48.30 kJ mol-1) decreases compared to the HSA–CFX–Hem complex (Kb = 29.37 9 108 M-1, DG0 = -53.93 kJ mol-1). These results suggest that the presence of CFX did not affect the binding of Hem to domain IB.
Log ½ðF0 FÞ=F ¼ log Kb þ n log ½Q;
The binding of BR, a toxic metabolite of heme, to HSA [18, 26] has been studied extensively for many years. A number of studies that measured the affinity of proteolytic fragments of HSA for BR showed that the high-affinity BR-binding site was located near subdomain IIA. There is, therefore, great clinical interest in understanding the binding of BR to albumin and the effects of drugs and other competitors on this binding. Aliquots of BR to the protein solution were added in the absence and presence of CFX, and decrease in protein fluorescence were measured after each addition of BR. The fluorescence intensity of HSA decreased regularly, and slight blue shift was observed for the emission wavelength with increasing BR concentration up to BR/albumin molar ratio of 0–10, indicating that the presence of BR could quench the fluorescence of HSA– CFX complex. Furthermore, determining the various binding parameters (Fig. 3a, b; Tables 1, 2) depicts clearly that there was no competitive binding between CFX and BR since we observed very less change in the binding pattern when two ligands were allowed to bind. We observed decreased stability of HSA–BR complex (Kb = 0.1088 9 108 M-1, DG0 = -40.08 kJ mol-1) when compared to HSA–CFX–BR complex (Kb = 20.73 9 108 M-1, DG0 = -53.07 kJ mol-1).
ð2Þ
where F0 and F are the fluorescence intensities with and without the ligand, respectively. A plot of log [(Fo - F)/F] versus log [Q] gave a straight line using least-squares analysis whose slope was equal to n (binding affinity) and the intercept on Y-axis to logK (K = binding constant). The binding constant (K) thus obtained was used to calculate the standard free energy change DG0binding of the ligand binding to HSA from the relationship DG0binding ¼ 2:303 RT ln Kb :
ð3Þ
Circular Dichroism Spectroscopy Circular dichroism (CD) was performed on a Jasco J-715 spectropolarimeter at 25 ± 0.2 °C, in a rectangular cell with 1.0 cm path length equipped with magnetic stirring. Each spectrum was signal-averaged at least three times with a bandwidth of 1.0 nm and a resolution of 0.5 nm at a scan speed of 100 nm min-1. Induced CD (ICD) spectra resulting from the interaction of the drug with HSA were obtained by subtracting the CD spectrum of the protein from that of the complex.
Results and Discussions Binding of Hemin in the Absence and Presence of Ciprofloxacin X-ray crystal structure of HSA–Hem complex has shown a single binding site for Hem on domain I [18, 19]. Hem is a large planar molecule and can be used as a probe for monitoring the effect of drug on the binding properties of domain I. The fluorescence quenching spectra of HSA at various concentrations of Hem in the absence and presence of CFX are shown in Fig. 2a, b and the data are summarized in Tables 1 and 2. Equilibration of HSA with Hem caused concentration-dependent quenching in the intrinsic fluorescence intensity, which suggests the binding of Hem to HSA. A little decrease in association constant and almost no change in the binding sites n were observed in the presence of
Binding of Bilirubin in the Absence and Presence of Ciprofloxacin
Binding of Chloroform in the Absence and Presence of Ciprofloxacin The site of action of the volatile general anesthetics remains controversial, but evidence in favor of its binding to subdomain IIA of HSA is accumulating. In this study, in the absence and presence of CFX, binding to chloroform to HSA is monitored by fluorescence quenching measurements. Chloroform causes a decrease in the fluorescence emission quantum yield as shown in Fig. 4a, b. A slight blue shift of 2 nm in the emission wavelength maximum was observed, suggesting that the binding of chloroform is associated with the changes in the dielectric environment of the indole ring in HSA, because electron transfer from the excited indole ring to chloroform might be responsible for the observed fluorescence quenching. Figure 4 shows the Stern–Volmer plot from the slope of which
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Cell Biochem Biophys Fig. 2 a Fluorescence spectra of hemin quenching in the absence of CFX. b Fluorescence spectra of hemin quenching in the presence of CFX. c Stern– Volmer plots of hemin quenching in the absence (open circle) and presence (filled circle) of CFX. d Log [(F0 F)/F] versus Log [Q] plots of hemin quenching in the absence (open circle) and presence (filled circle) of CFX for determining the binding constant and binding sites
250
A
Fluorescence Intensity
Fluorescence Intensity
250 200 150 100 50 0 300
350
B 200 150 100 50 0 300
400
350
1.2
8
C
D
0.8
Log [(Fo-F)/F]
6
Fo/F
400
Wavelength (nm)
Wavelength (nm)
4
2
0.4 0 -0.4 -0.8
0
-1.2 0
1
2
Hemin [ µ M]
3
4
-7
-6.5
-6
-5.5
-5
Log [Hemin]
Table 1 Effects of domain-specific ligands on binding constants of HSA and HSA–CFX systems Ligands
Systems
Ksv 9 106 [L mol-1]a
Kq 9 1014 [L mol-1 s-1]a
R2
Hemin
HSA
1.77
1.77
0.9917
HSA–CFX
1.40
1.40
0.9895
HSA
0.4
0.4
0.9902
HSA–CFX
0.2
0.2
0.9572
HSA
0.02 9 10-3
0.02 9 10-3
0.9992
-3
0.01 9 10-3
0.9951
Bilirubin Chloroform Diazepam a
HSA–CFX
0.01 9 10
HSA
0.09
0.092
0.9962
HSA–CFX
0.06
0.06
0.9969
The mean value of 4 individual experiments with standard deviation ±0.06–±0.11 %
Table 2 Thermodynamic and binding parameters of HSA and HSA– CFX system Ligand
Systems
Kb [M-1]a
n
Hemin
HSA
3.01 9 108
1.40
-48.30
HSA–CFX
29.37 9 108
1.60
-53.93
HSA
0.1088 9 108
1.24
-40.08
Bilirubin Chloroform Diazepam a
8
DG0 [kJ mol-1]
HSA–CFX
20.73 9 10
1.68
-53.07
HSA
18.13
1.03
-7.16
HSA–CFX
11.98
1.08
-6.14
HSA
3.48 9 104
0.93
-25.87
HSA–CFX
3.09 9 104
0.94
-25.57
The mean value of 4 individual experiments with standard deviation ±0.06–±0.11 %
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Ksv = 0.02 ± 0.08 M-1 was calculated. It is observed that Ksv = 0.02 ± 0.08 M-1 was reduced to almost half (0.01 ± 0.08 M-1) in the presence of CFX which is suggestive of static interaction between fluorophore and CFX. Furthermore a little increase in the binding site of HSA n in the absence and presence of CFX depict that an allosteric binding exists on domain II for both the CFX and chloroform (Fig. 4; Tables 1, 2). The approach used allows direct monitoring of anesthetic binding to the protein and, in addition, provides information about the location of the anesthetic in the protein matrix. The results indicate that chloroform occupies the same binding site on this model mammalian protein, HSA, as that occupied by CFX.
Cell Biochem Biophys 320
250
A 240
Fluorescence Intensity
Fluorescence Intensity
Fig. 3 a Fluorescence spectra of bilirubin quenching in the absence of CFX. b Fluorescence spectra of bilirubin quenching in the presence of CFX. c Stern– Volmer plots of bilirubin quenching in the absence (open circle) and presence (filled circle) of CFX. d Log [(F0 F)/F] versus Log [Q] plots of bilirubin quenching in the absence (open circle) and presence (filled circle) of CFX for determining the binding constant and binding sites
a
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j
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B a
200 150
j 100 50 0 300
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Wavelength [nm]
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Fo/F
Log [(Fo-F)/F]
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2
-2 -6.6
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Bilirubin [ µ M]
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0 Log [(Fo-F)/F]
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Fig. 4 a Fluorescence spectra of chloroform quenching in the absence of CFX. b Fluorescence spectra of chloroform quenching in the presence of CFX. c Stern–Volmer plots of chloroform quenching in the absence (open circle) and presence (filled circle) of CFX. d Log [(F0 - F)/F] versus Log [Q] plots of chloroform quenching in the absence (open circle) and presence (filled circle) of CFX for determining the binding constant and binding sites
-6.2
Log [Bilirubin]
1.6
1.2
-0.4 -0.8 -1.2
0.8 0
20
40 60 CHCl3 [mM]
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-1.6 -2.2
-1.8 -1.4 Log [CHCl3]
-1
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Cell Biochem Biophys 250 250
A 200
B
a Fluorescence Intensity
Fluorescence Intensity
Fig. 5 a Fluorescence spectra of diazepam quenching in the absence of CFX. b Fluorescence spectra of diazepam quenching in the presence of CFX. c Stern– Volmer plots of diazepam quenching in the absence (open circle) and presence (filled circle) of CFX. d Log [(F0 F)/F] versus Log [Q] plots of diazepam quenching in the absence (open circle) and presence (filled circle) of CFX for determining the binding constant and binding sites
150 j 100 50 0 300
350
200
100 50
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Wavelength [nm] 0
C
D
2.1 Log [(Fo-F)/F]
-0.3
Fo/F
1.8 1.5
-0.6
-0.9
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Wavelength [nm]
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0
3
6 9 Diazepam [µM]
Binding of Diazepam in the Absence and Presence of Ciprofloxacin Accumulating evidence suggests that the primary diazepam binding site was located in HSA domain III [11]. Thus to trace the binding site of CFX, DIA binding to domain III of HSA in the absence and presence of CFX was examined. Figure 5a, b represents the fluorescence intensity spectra while Fig. 5c, d represents Stern–Volmer plot and modified Stern–Volmer plot, respectively. The values of binding constant and change in binding energy was obtained using Eqs. (1)–(3) and are listed in Tables 1 and 2. Almost 1.5 times decrease in the binding constant of HSA in the absence of drug compared to HSA–CFX complex was observed while no significant change in binding site is being noticed. This indicates increased stability of HSA–diazepam complex (Ksv = 0.09 9 106 M-1, DG0 = -25.87 kJ mol-1) compared to HSA–CFX–diazepam complex (Ksv = 0.06 9 106 M-1, DG0 = -25.57 kJ mol-1). These results suggest that the binding of diazepam at domain III is not affected by the presence and absence of drug.
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j
150
0 300
400
a
12
15
-1.2 -6.3
-5.8 -5.3 Log [Diazepam]
-4.8
Conformation Investigation To evaluate the structural change of HSA by addition of ligands, the far-UV CD spectra measurements were taken which have been widely used for studying the conformation and conformational change of proteins and polypeptides in solution [27]. Figure 6a, b shows the far-UV CD spectra of the HSA–marker and HSA–marker–CFX complexes obtained at pH 7.4 at room temperature, respectively. As expected for a protein that is predominantly ahelical, the CD spectrum of HSA shows a strong negative ellipticity at 208 and 222 nm (Fig. 6a, curve 3). The reasonable explanation is that the negative peaks at 208 and 222 nm are both contributed as a result of n ? K* transition for the peptide bond of a-helix [22]. As shown in Fig. 6b curve 3, binding of CFX to HSA decreases its helical content. The binding of chloroform to HSA–CFX complex induces increase in negative ellipticity as shown in Fig. 6b that might be due to shielding of the peptide strand due to the increase in hydrophobicity on binding. This conclusion agrees with the result of fluorescence quenching experiment.
Cell Biochem Biophys 20
characteristic CD of BR bound to HSA undergoes a remarkable sign inversion on the addition of CFX. This sign inversion reflects a pronounced conformational change of the bound ligand; probably a complete inversion of chirality. The observation suggests that association of ligand with proteins can markedly alter the internal topography of receptor sites and potentially influence the stereoselectivity of ligand binding.
A
CD [mdeg]
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1 2 3 4 5
-50
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Probing the Binding Site of Ciprofloxacin on HSA by Ligand Displacement Experiments
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-90 200
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Wavelength [nm] 1 2 3 4 5
HSA: Hemin= 1:1 HSA:Bilirubin=1:1 Free HSA / HSA-CFX=1:1 (b& d) HSA:Diazepam=1:1 HSA:Chloroform=1:1
Fig. 6 Far-UV CD signals of HSA–marker complex (1:1) a in the absence and b in presence of equimolar CFX
Chiroptical Properties of the Ciprofloxacin–HSA Complexes at pH 7.4 The method of ICD is based on the observation that an optical activity arises from asymmetry in the ligand induced by its binding to the protein, since the free ligand has either no asymmetric center or, therefore, gives no signal in solution. Chiral conformation of the ligand due to conformational adaptation to its binding site, or interaction between ligand molecules held in chiral arrangement relative to each other by the protein sites, results in one or more ICD bands with different shapes, signs and intensities. These extrinsic Cotton effects present in lightabsorbing region of the optically active or inactive ligand molecules give qualitative and quantitative information of the binding process. Figure 7a–d shows the ICD spectra for marker complexed with protein in the absence and presence of CFX. A molar ratio of 0.1 and 1 was selected for both the conditions to ensure that all of the ligand is bound to the primary binding site, avoiding interactions with secondary, weaker binding sites. In Fig. 7a, the
The finding that the CFX–HSA complex exhibits induced a CD couplet in the visible spectral region provides a sensitive tool for studying the binding location of CFX on HSA. In the presence of a compound having the same binding site as CFX, amplitudes of the induced secondary structure should decrease due to competition. Therefore, CD displacement experiments were performed using domain-specific ligands. Primary binding location of Hem on HSA is domain IB with Ka = 1.1 9 108 M-1, BR binds at the interface of domain I and II with Ka = 9.5 9 107 M-1 [25], diazepam (Ka = 3.8 9 105 M-1) binds at subdomain IIIA, and high-affinity binding site of chloroform (Ka = 3.8 9 105 M-1) is on domain IIA of BSA.
Conclusion Ciprofloxacin binds to site I of HSA; in addition it is also a promising molecular probe to study biologically important, induced conformational polymorphisms of serum albumins. Pharmacological and pharmacodynamic properties of biologically active natural and synthetic compounds are crucially determined via their binding to proteins of the human serum. This paper is aimed to survey competitive binding of drug in the presence of domain-specific ligands and the results investigate the influence of domain-specific ligands in the presence of CFX binding on HSA and how the drug molecules can influence or compete with other ligand molecules bound to the protein. The experimental results revealed that CFX has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. All these experimental results and other data clarified that CFX could bind to HSA and be effectively transported and eliminated in body, which could be a useful guideline for further drug design. On the basis of the above-discussed results, a pictorial model depicting the binding of various domain specific ligands have been formulated (Fig. 8) which also predicts that the binding site of CFX exists on domain IIA as reported earlier by our group [28] in the close vicinity of
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40
A
B
2
CD [mdeg]
20
CD [mdeg]
Fig. 7 ICD (expressed in mdeg) of marker ligands: hemin (a), bilirubin (b), diazepam (c), and chloroform (d) bound to HSA and HSA–CFX at a molar ratio of 0.1 and 1. Line symbols are shown in inset. Protein concentration was 10 lM in 0.06 M sodium phosphate buffer, pH 7.4
0 -2 HE: HSA=0.1:1 HE: CFX: HSA=0.1:1:1 HE: HSA=1:1 HE: CFX: HSA=0.1:1:1
-4 -6 350
400
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0
BR: HSA=0.1:1 BR: CFX: HSA=0.1:1:1 BR: HSA=1:1 BR: CFX: HSA=1:1:1
-20
-40 350
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Wavelength [nm] 12
400
450
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550
Wavelength [nm] 12
C
D CD [mdeg]
CD [mdeg]
9
0 DI: HSA=0.1:1 DI: CFX: HSA=0.1:1:1 DI: HSA=1:1 DI: CFX: HSA=1:1:1
280
300
Wavelength [nm]
3 0
CHCl3: HSA=0.1:1 CHCl3: CFX: HSA= 0.1:1:1 CHCl3: HSA= 1:1 CHCl3: CFX: HSA= 1:1:1
-3
-10 -13 250 260
6
320
-6 300
350
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Wavelength [nm]
Fig. 8 A pictorial model indicating the binding of domain-specific ligands and predicting the binding site of ciprofloxacin on human serum albumin
the site where chloroform binds. Our work not only provides the multiplicity of binding sites on HSA and demonstrates the conformational plasticity of HSA on drug binding, but it may also provide structural information for
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the modulation of HSA–drug interaction by various spectroscopic approaches based on HSA–drug interaction. The binding of small molecules to proteins and protein– protein interactions are key processes in cell biochemistry.
Cell Biochem Biophys
The usual paradigm is that ligand binding induces a change in the conformation of the target protein which, in turn, produces a given response and fundamental importance. Using the binding of various domain-specific ligands to HSA, we have concluded that CFX interacts with domain II of HSA in the close vicinity to chloroform. Recent advances of gene cloning, together with complete understanding of albumin structure and function, provide for a greater abundance of future applications. Accordingly, our information on the relative stability of HSA and its domains should provide a basis for drug design, as they possess binding sites for a variety of exogenous and endogenous ligands. Acknowledgments Facilities provided by A.M.U are gratefully acknowledged. Ankita Varshney, Ejaz Ahmad, Nida Zaidi, and Parvez Alam thank the Council of Scientific and Industrial Research, New Delhi and Yunus Ansari thanks the department of Biotechnology, Govt. of India for financial assistance.
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