ORIGINAL PAPER

Analysis of Binding Interaction Between AntibacterialCiprofloxacin and Human Serum Albumin by SpectroscopicTechniques

Ankita Varshney • Yunus Ansari • Nida Zaidi •

Ejaz Ahmad • Gamal Badr • Parvez Alam •

Rizwan Hasan Khan

� 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) mea-

surements. 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. Further-

more, 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

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 subdo-

mains 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 suc-

cessful 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 cross-

resistance 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, gastrointes-

tinal, and respiratory tracts [11]. Chemically, CFX is a

1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-quinolone-

3-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 non-

covalent interactions may play a decisive role in its

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: rizwanhkhan@hotmail.com; rizwanhkhan1@gmail.com

G. Badr

Laboratory of Immunology & Molecular Biology, Zoology

Department, Faculty of Science, Assiut University,

Assiut 71516, Egypt

123

Cell Biochem Biophys

DOI 10.1007/s12013-014-9863-1

mechanism of action. Because of its pharmacological

activity, the investigation of the interactions between this

compound and serum albumins is very important [12]. Fur-

thermore, 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 mole-

cules 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 bind-

ing to HSA. The probable binding site of CFX on HSA is

also predicted from marker displacement experiment.

Materials and Methods

Materials

HSA (A1887; [96 %), CFX (17850; [98.0 %), warfarin

(A2250; [98 %), hemin (Hem) (H5533; [80 %), diaze-

pam (DIA) (D0406; [98 %), and chloroform (C2432;

>99.5 %) purchased from Sigma-Aldrich. Bilirubin (BR)

was purchased from Hemedia. The number in the paren-

thesis corresponds to the purity of the compounds. All of

the other reagents were of analytical grade.

Methods

Protein Concentration Determination

Protein concentration was determined spectrophotometri-

cally 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 Seph-

acryl-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 Shima-

dzu spectrofluorimeter, model RF-5301 PC. The fluores-

cence 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.

Calculations/Data Analysis

The quenching equation is presented by

F0=F ¼ 1 þ kqs0 Q½ � ¼ 1 þ Ksv Q½ �; ð1Þ

Fig. 1 Chemical structure of ciprofloxacin

Cell Biochem Biophys

123

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 bio-

molecule 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

Log ½ðF0 � FÞ=F� ¼ log Kb þ n log Q½ �; 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 moni-

toring 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 fluores-

cence 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

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.

Binding of Bilirubin in the Absence and Presence

of Ciprofloxacin

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).

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 measure-

ments. 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

Cell Biochem Biophys

123

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 sug-

gestive 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 chloro-

form (Fig. 4; Tables 1, 2).

The approach used allows direct monitoring of anes-

thetic binding to the protein and, in addition, provides

information about the location of the anesthetic in the

protein matrix. The results indicate that chloroform occu-

pies the same binding site on this model mammalian pro-

tein, HSA, as that occupied by CFX.

0

50

100

150

200

250

Wavelength (nm)F

luor

esce

nce

Inte

nsit

y

0

50

100

150

200

250

Wavelength (nm)

Flu

ores

cenc

e In

tens

ity

Fo/

F

0

2

4

6

8

Hemin [ M]

C

-1.2

-0.8

-0.4

0

0.4

0.8

1.2

300 350 400 300 350 400

0 1 2 3 4 -7 -6.5 -6 -5.5 -5

Log [Hemin]

D

Log

[(F

o-F

)/F

]

BA

µ

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

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

Bilirubin HSA 0.4 0.4 0.9902

HSA–CFX 0.2 0.2 0.9572

Chloroform HSA 0.02 9 10-3 0.02 9 10-3 0.9992

HSA–CFX 0.01 9 10-3 0.01 9 10-3 0.9951

Diazepam HSA 0.09 0.092 0.9962

HSA–CFX 0.06 0.06 0.9969

a 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 DG0 [kJ mol-1]

Hemin HSA 3.01 9 108 1.40 -48.30

HSA–CFX 29.37 9 108 1.60 -53.93

Bilirubin HSA 0.1088 9 108 1.24 -40.08

HSA–CFX 20.73 9 108 1.68 -53.07

Chloroform HSA 18.13 1.03 -7.16

HSA–CFX 11.98 1.08 -6.14

Diazepam HSA 3.48 9 104 0.93 -25.87

HSA–CFX 3.09 9 104 0.94 -25.57

a The mean value of 4 individual experiments with standard deviation

±0.06–±0.11 %

Cell Biochem Biophys

123

0

80

160

240

320

300 350 4000

50

100

150

200

250

0.8

1.2

1.6

2

C

-2

-1.6

-1.2

-0.8

-0.4

0

300 350 400

0 0.5 1 1.5 2 2.5 -6.6 -6.4 -6.2 -6 -5.8 -5.6

Log

[(F

o-F

)/F

]

D

Flu

ores

cenc

e In

tens

ity

Flu

ores

cenc

e In

tens

ity

Wavelength [nm] Wavelength [nm]

a

j

a

j

Fo/

F

Bilirubin [ M] Log [Bilirubin]

A B

µ

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

0

20

40

60

80

100

-1.6

-1.2

-0.8

-0.4

0

0.4

0

20

40

60

80

100

300 350 400

Wavelength [nm] Wavelength [nm]

Flu

ores

cenc

e In

tens

ity

Flu

ores

cenc

e In

tens

ity

a

j

a

j

0.8

1.2

1.6

2

2.4

300 350 400

-2.2 -1.8 -1.4 -10 20 40 60 80 100

CHCl3 [mM] Log [CHCl3]

Fo/

F

Log

[(F

o-F

)/F

]

BA

DC

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

Cell Biochem Biophys

123

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 com-

plex (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.

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 conforma-

tion and conformational change of proteins and polypep-

tides in solution [27]. Figure 6a, b shows the far-UV CD

spectra of the HSA–marker and HSA–marker–CFX com-

plexes obtained at pH 7.4 at room temperature, respec-

tively. As expected for a protein that is predominantly a-

helical, the CD spectrum of HSA shows a strong negative

ellipticity at 208 and 222 nm (Fig. 6a, curve 3). The rea-

sonable explanation is that the negative peaks at 208 and

222 nm are both contributed as a result of n ? K* tran-

sition 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.

0

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100

150

200

250

0

50

100

150

200

250

300 350 400

-1.2

-0.9

-0.6

-0.3

0

Wavelength [nm]Wavelength [nm]F

luor

esce

nce

Inte

nsit

y

Flu

ores

cenc

e In

tens

ity

a

j

a

j

0.9

1.2

1.5

1.8

2.1

2.4

300 350 400

-6.3 -5.8 -5.3 -4.80 3 6 9 12 15

Fo/

F

Log

[(F

o-F

)/F

] Diazepam [µM] Log [Diazepam]

A B

C D

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

Cell Biochem Biophys

123

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 rel-

ative to each other by the protein sites, results in one or

more ICD bands with different shapes, signs and intensi-

ties. These extrinsic Cotton effects present in light-

absorbing 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 pre-

sence 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

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.

Probing the Binding Site of Ciprofloxacin on HSA

by Ligand Displacement Experiments

The finding that the CFX–HSA complex exhibits induced a

CD couplet in the visible spectral region provides a sen-

sitive 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 albu-

mins. 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

200 250 210 220 230 240 -90

-50

0

20

21

345

B

0

200 210 220 230 240-90

-50

20

12345

250

A

CD

[m

deg]

1 HSA: Hemin= 1:1 2 HSA:Bilirubin=1:1 3 Free HSA / HSA-CFX=1:1 (b& d) 4 HSA:Diazepam=1:1 5 HSA:Chloroform=1:1

CD

[m

deg]

Wavelength [nm]

Wavelength [nm]

Fig. 6 Far-UV CD signals of HSA–marker complex (1:1) a in the

absence and b in presence of equimolar CFX

Cell Biochem Biophys

123

the site where chloroform binds. Our work not only pro-

vides the multiplicity of binding sites on HSA and dem-

onstrates the conformational plasticity of HSA on drug

binding, but it may also provide structural information for

the modulation of HSA–drug interaction by various spec-

troscopic approaches based on HSA–drug interaction.

The binding of small molecules to proteins and protein–

protein interactions are key processes in cell biochemistry.

-13

12

-10

0

320250 260 280 300

DI: HSA=0.1:1 DI: CFX: HSA=0.1:1:1

DI: CFX: HSA=1:1:1 DI: HSA=1:1

C

-6

-4

-2

0

2

4

350

A

HE: HSA=0.1:1 HE: CFX: HSA=0.1:1:1

HE: CFX: HSA=0.1:1:1 HE: HSA=1:1

CD

[m

deg]

Wavelength [nm]

CD

[m

deg]

Wavelength [nm]

-40

-20

0

20

40

CD

[m

deg]

BR: HSA=0.1:1 BR: CFX: HSA=0.1:1:1 BR: HSA=1:1 BR: CFX: HSA=1:1:1

B

Wavelength [nm]

-6

-3

0

3

6

9

12

400 450 500 350 400 450 500 550

300 350 400 450 500

D

CHCl3: HSA=0.1:1 CHCl3: CFX: HSA= 0.1:1:1

CHCl3: CFX: HSA= 1:1:1 CHCl3: HSA= 1:1

Wavelength [nm]

CD

[m

deg]

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

Fig. 8 A pictorial model

indicating the binding of

domain-specific ligands and

predicting the binding site of

ciprofloxacin on human serum

albumin

Cell Biochem Biophys

123

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 com-

plete 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 Par-

vez Alam thank the Council of Scientific and Industrial Research,

New Delhi and Yunus Ansari thanks the department of Biotechnol-

ogy, Govt. of India for financial assistance.

References

1. Bhattacharya, A. A., Grune, T., & Curry, S. (2000). Crystallo-

graphic analysis reveals common modes of binding of medium

and long-chain fatty acids to human serum albumin. Journal of

Molecular Biology, 303, 721–732.

2. Varshney, A., Sen, P., Ahmed, E., Rehan, M., & Subbarao, N.

(2010). Ligand binding strategies of human serum albumin: how

can the cargo be utilized. Chirality, 22, 77–87.

3. Gulam, J., Zunszain, P. A., Petipas, I., Bhattacharaya, A. A.,

Otagiri, M., & Curry, S. (2005). Structural basis of the drug-

binding specificity of human serum albumin. Journal of Molec-

ular Biology, 353, 38–52.

4. Zaidi, N., Ahmad, E., Rehan, M., Rabbani, G., Ajmal, M. R.,

Zaidi, Y., et al. (2013). Biophysical insight into furosemide

binding to human serum albumin: a study to unveil its impaired

albumin binding in uremia. Journal of Physical Chemistry B, 117,

2595–2604.

5. Zaidi, N., Ajmal, M. R., Rabbani, G., Ahmad, E., & Khan, R. H.

(2013). A comprehensive insight into binding of hippuric acid to

human serum albumin: a study to uncover its impaired elimina-

tion through hemodialysis. PLoS One, 8, e71422.

6. Peters, T. (1996). All about albumin. Biochemistry, genetics and

medical applications. San Diego: Academic Press.

7. Gabor, M., & Fred, K. (1957). The disulfide bonds of human

serum albumin and bovine c-globulin. Journal of American

Chemical Society, 79, 134–139.

8. Sugio, S., Kashima, A., Mockizuki, S., Noda, M., & Kobayashi,

K. (1999). Crystal structure of human serum albumin at 2.5 A

resolution. Protein Engineering, 12, 439–446.

9. Vorobey, P., Steindal, A. E., Vorobey, A., & Moan, J. (2006).

Influence of human serum albumin on photodegradation of folic

acid in solution. Journal of Photochemistry Photobiology, 82,

817–822.

10. Bhagvan, N. V., & Ha, C. E. (2013). Novel insight into the

pleiotropic effects of human serum albumin in health and disease.

Biochemica et Biophysica Acta General subjects, 12, 5486–5493.

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 Pharmacoki-

netics, 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 Living-

stone Inc.

16. Khan, M. Y. (1986). Direct evidence for the involvement of

domain III in the N-F transition of bovine serum albumin. Bio-

chemical 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 chem-

istry of human serum albumin. Nature, 358, 209–215.

19. Wallevik, K. (1973). Reversible denaturation of human serum

albumin by pH, temperature, and guanidine hydrochloride fol-

lowed 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 chlo-

roform 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. Bio-

macromolecules, 7, 1350–1356.

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