American Journal of Analytical Chemistry, 2011, 2, 250-257
doi:10.4236/ajac.2011.22030 Published Online May 2011 (
Copyright © 2011 SciRes. AJAC
Analysis of Binding Interaction between Captopril and
Human Serum Albumin
Xiaoyan Gao, Yingcai Tang, Wanqi Rong, Xiaoping Zhang, Wujie Zhao, Yanqin Zi
Department of Chemistry and Materials Science, Huaibei Normal University, Huaibei, China
Received October 8, 2010; revised January 24, 2011; accepted May 10, 2011
The interaction between captopril, an inhibitor of angiotensin converting enzyme and human serum albumin,
a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condi-
tion by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of
human serum albumin was strongly quenched by captopril. The binding constants and the number of binding
sites can be calculated from the data obtained from fluorescence quenching experiments. The negative value
of G0 reveals that the binding process is a spontaneous process. According to the van’t Hoff equation, the
standard enthalpy change (H0) and standard entropy change (S0) for the reaction were calculated to be
35.98 KJmol–1 and 221.25 Jmol–1 K. It indicated that the hydrophobic interactions play a main role in the
binding of captopril to human serum albumin. In addition, the distance between captopril (acceptor) and
tryptophan residues of human serum albumin (donor) was estimated to be 1.05 nm according to the Förster’s
resonance energy transfer theory. The results obtained herein will be of biological significance in pharma-
cology and clinical medicine.
Keywords: Human Serum Albumin, Captopril, Fluorescence Quenching, Stern-Volmer Equation, The
Förster’s Resonance Energy Transfer Theory
1. Introduction
Captopril, as show in Figure 1, 1-[(2S)-3-mercapto-2-
methyl-1-oxopropyl]-L-proline, contains two centers of
dissymmetry, one associated with the (S)-proline portion
and the other associated with the 3-mercapto-2-methyl-
propionic acid side chain. As an inhibitor of angiotensin
converting enzyme (ACEI) with a short duration of
Figure 1. The structure of captopril.
action [1], it is a drug with a number of cellular actions
and clinical applications, and it now extensively used for
treatment of chronic heart failure and hypertention [2]
for it can catalyze the conversion of angiotensin I to an-
giotensin II. It has been shown to reduce proteinuria [3]
and retard the progression of renal failure in patients with
insulin dependent diabetes mellitus and nephropathy, and
provides the basis of its use against hypertension. The
drug stimulates prostaglandin production and release
IL-2 production [4], and possesses immunosuppressant
activity. Besides its vasodilative effects, it possesses in-
hibiting effects on hypertrophy and remodeling of myo-
cardium without interference with the metabolism of
fatty acid, and can improve the patients’ quality of life [5,
6]. Some experimental studies have suggested that cap-
topril has antiarrhythmic activity, a reduction of ar-
rhythmias induced by isehemia, reperfusion, or digitalis
toxicity [7]. Furthermore, some clinical reports have
showed that captopril can decrease the frequency and
grade of ventricular ectopy in patients with heart failure
or cardiac infarction.
It is well known that protein is an important chemical
X. Y. GAO ET AL.251
substance in almost of the life and the main target of all
medicines in living beings. Human serum albumin (HSA),
the most abundant protein in human plasma, acts as a
carrier for transporting many ligands, including fatty
acids, amino acids, metal ions, and a variety of pharma-
ceuticals [8,9] and disposer of many endogenous and
exogenous compounds [10,15]. It is synthesized in the
liver, and exported as a non-glycosylated protein. HSA is
a single polypeptide chain of 585 amino acid residues
and comprises three structurally homologous domains: I
(residues 1-195); II (196-383); and III (384-585). It is
folded into three homologous domains each of which
contains two subdomains (A and B). HSA has significant
physiological function and is one of the model proteins
commonly used in methodological research on immuno-
assay. It can interact with many endogenous and exoge-
nous substances, and it is to bind a wide variety of en-
dogenous and exogenous substances such as, hormones,
fatty acids, bilirubin, and foreign molecules such as
drugs [16,17]. It is important to study the interaction of
drug with the protein for protein-drug binding plays an
important role in pharmacology and pharmacodynamics.
The information on the interaction of HSA and drug can
help us better understand the absorption and distribution
of the drug.
To better understand the pharmacological activities of
captopril at molecular level, we characterized the inter-
action between captopril and HSA by different spectro-
scopic methods as our previous studies. Efforts were
made to investigate the quenching mechanism, binding
constants, binding sites, binding mode, and binding dis-
tance. Moreover, synchronous fluorescence was employed
to probe conformational changes induced by captopril.
2. Experimental
2.1. Apparatus and Reagents
Absorption spectra were recorded on TU1901 UV/Vis
Spectrophotometer (PGeneral, Beijing, China). An RF-
5301PC fluorescence spectrometer (Jasco, Japan) was
applied to record the fluorescence spectra. The pH values
were measured with a PHS-23 meter (Shanghai Secondly
Analytical Instruments, China).
HSA (1.0 × 10–4 molL–1) was prepared by dissolving
3.3250 g of HSA (66500 Da, Shanghai Wenhao Bio-
chemistry Tech., Shanghai, P. R. China) in 500 ml of
deionized water. Catopril solution (5.0 × 10–3 molL1)
was prepared by dissolving 0.2720 g of captopril tablets
(217.29 Da, Shanghai XuDong hoop pharmaceutical Co.,
LTD., Shanghai, PR China) in 250 mL of water. The so-
lution should be stored in a refrigerator freezer. The
working solutions were obtained by diluting the stock
solution with water. Britton–Robinson buffer solutions
(B-R) were prepared by mixing the mixed acid (com-
posed of 0.04 molL–1 H3PO4, HAc and H3BO3) with 0.2
molL–1 NaOH in proportion. The buffer solutions were
prepared to adjust the acidity of the system. The pH val-
ue of solutions was kept at 7.24.
Deionized water was used for the preparation of some
solutions. All chemicals used were of analytical-reagent
or higher grade.
2.2. General Procedure
All studies were carried out in 10 mL calibrated tubes. In
tubes, 1.00 mL known B-R buffer, 1.0 ml of 1.0 × 10–5
molL–1 HSA solution and a known volume of the stan-
dard captopril solution were added. Then the solution
was diluted to 10 mL with deionized water and mixed
well. After reacted for 20 minutes, the solutions were
taken into the optical cell.
The ultraviolet (UV) absorption spectra were meas-
ured on a TU1901 UV/Vis Spectrophotometer. The fluo-
rescence spectra of the system were recorded on a FP-
6500 fluorescence spectrometer at 300-400 nm. Excita-
tion bandwidth was 3 nm and emission bandwidth was 5
nm, using a 1cm quartz cell. The pH values were meas-
ured with a PHS-23 meter.
3. Results and Discussion
3.1. Fluorescence Quenching Spectra and
Quenching Mechanism of HSA by Captopril
Any process, which decreases the fluorescence intensity
of a sample, is called fluorescence quenching [18]. The
basic principles like excited state reactions, molecular
rearrangements, energy transfer, ground state complex
formation, and collisional quenching involves in mo-
lecular interaction, which can result in quenching.
The interaction of captopril with HSA was evaluated
by monitoring the intrinsic fluorescence intensity changes
of HSA upon addition of captopril. The fluorescence
emission spectra of HSA with various amounts of Cap-
topril were recorded on a FP-6500 fluorescence spec-
trometer at 300 - 400 nm following an excitation at 280
nm. Fluorescence quenching spectra of HSA at the pres-
ence of various concentrations of captopril are shown in
Figure 2.
As seen in Figure 2, the addition of captopril led to a
concentration-dependent quenching of HSA intrinsic
fluorescence intensity along with a slight blue shift of
maximum emission wavelength, implying that the bind-
ing of captopril to HSA occurs and the microenviron-
ment around the chromophore of HSA is changed upon
Copyright © 2011 SciRes. AJAC
Figure 2. Fluorescence emission spectra of HSA in the ab-
sence and presence of increasing amount of captopril, ex =
280 nm, CHSA = 1.0 × 106 molL1; Ccaptopril = (1-8: 0.0, 1.25,
1.5, 1.75, 2.0, 2.25, 2.5 and 2.75) 104molL1.
addition of captopril. With the increasing concentration
of captopril, the fluorescence intensity decreased gradu-
ally. Such strong quenching clearly indicated the binding
of captopril with HSA.
Fluorescence quenching could proceed via different
mechanisms, usually classified as dynamic quenching
and static quenching. Dynamic and static quenching can
be distinguished by their different dependence on tem-
perature. Higher temperatures will result in faster diffu-
sion and hence larger amounts of collisional quenching
and higher temperatures will typically result in the disso-
ciation of weekly bound complexes and hence smaller
amounts of static quenching.
To clarify the fluorescence quenching mechanism in-
duced by HSA, the Stern-Volmer equation Equation (1)
was utilized to process the data [19,20].
 (1)
where 0
and F represent the fluorescence intensities
of HSA in the absence and presence of the quencher
(captopril). is the concentration of the quencher,
and SV
is the dynamic quenching constant, which is
equal to 0q
, q
is the bimolecular quenching rate
constant and 0
is the average lifetime of the molecule
without quencher. In order to confirm the quenching
mechanism, the procedure of the fluorescence quenching
was first assumed to be dynamic quenching. According
to Equation (1), the curve of 0
F versus was
plotted based on the experimental data. The
Stern-Volmer curve was linear when the concentration of
captopril ranged from 1.25 to 2.75 × 104 mo l L1 at
293.15, 300.15 and 310.15 K (see Figure 3). All the
plots show a good linear relationship. As it is known,
linear Stern- Volmer plots represent a single quenching
mechanism, either static (a formation of a complex be-
Figure 3. Plots of 0
F for HSA against [Q] of captopril
at different temperatures: a: 293.15 K; b: 300.15 K; c:
310.15 K.
tween quencher and fluorophore) or dynamic (a colli-
sional process) [21]. In a static quenching process, gen-
erally, a linear Stern- Volmer plot indicates either only
one drug binding site existing in the proximity of fluo-
rophore or more than one binding site being all equally
accessible to quenchers [22,23]. In a dynamic quenching
process, the bimolecular quenching constant KSV is ex-
pected to increase with rising temperature because it is
closely related to diffusions or diffusion coefficients.
In addition, the Stern-Volmer slope is expected to de-
pend on the concentration of donor (HSA) in a static
quenching process, whereas the slope is independent of
the concentration of donor in a dynamic process. Linear
fittings of the experimental data to Equation (1) afforded
KSV and Kq listed in Table 1. Table 1 showed that KSV
decreases with increase in temperature. It indicates that
the fluorescence quenching of HSA by captopril appears
to occur via a dynamic quenching mechanism. Kq in Ta-
ble 1 was calculated by 0qSV
, and generally the
values of 0
for biopolymers were given as 108 s1 [24].
However, q
in Table 1 is of the magnitude of 1011 L
mol 1s1, which is greater than the maximum diffusion
collision quenching rate constant (2.0 × 1010 L mol1 s1)
for a variety of quenchers with biopolymer. Therefore, it
suggests that the fluorescence quenching process of HSA
may be mainly governed by a static quenching mecha-
nism arising from a complex formation rather than a dy-
namic quenching mechanism [25].
3.2. Binding Constant and Number of Binding
In a static quenching process, small molecules will be
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
Table 1. Stern-Volmer quenching constants for the capto-
pril-HSA system at pH 7.24 (293.15 K to 310.15 K).
T (K) Ksv (×103 Lmol1) R Kq (× 1011 Lmol1s1)
293.15 3.157 0.991 3.157
300.15 2.370 0.998 2.370
310.15 2.314 0.996 2.314
independently bound to a set of equivalent sites on a ma-
cromolecule. Thus, the equilibrium between free and
bound molecules is given by References [26,27]. When
small molecules were bound independently to a set of
equivalent sites on a macromolecule, theequilibrium be-
tween free and bound molecules was given by Equation
(2) [28].
Figure 4. Double-lg plot of captopril quenching effect on the
fluorescence of HSA at different temperatures: a 293.15 K;
b 300.15 K; c 310.15 K.
log()loglog[ ]
FFK nQ  (2)
where K
b was the binding constant and n was the
number of binding sites. For the captopril-HSA system in
the lower concentration range, the values for Kb and n at
different temperatures can be derived from the intercept
and slope of Figure 4 based on Equation (2) and pre-
sented in Table 2. Linear regression Equations (4-6) at
290.15, 300.15 and 310.15 K are expressed as follows:
cule and a biomacromolecule include hydrogen bond,
van der Waals force, electrostatic and hydrophobic inter-
actions, etc. Because of the –COOH and –SH in the mo-
lecular of Captopril, these active groups can interact with
the HSA. Thermodynamic parameters for a binding in-
teraction can be used as a major evidence to learn the
nature of intermolecular forces. Thus, the temperature-
dependent thermodynamic parameters for the captopril-
HSA system are used to characterize the intermolecular
forces between captopril and HSA.
log()5.13991.57log[] 293.15 KFFF Q  (3)
log()5.29891.62log[] 300.15 KFFF Q  (4)
log()5.49691.49log[] 310.15 KFFF Q  (5)
Thermodynamic parameters such as free energy (G0),
enthalpy (H0) and entropy (S0) due to complex forma-
tion provide an insight into the binding mode. If an en-
thalpy change (H0) does not vary significantly with tem-
perature, its value and that for an entropy change (S0)
can be determined by the van’t Hoff equation as follows:
respectively. Kb in Table 2 shows that there exist a strong
interaction between captopril and HSA and a complex
formation of captopril with HSA. Furthermore, it can be
inferred from the values of n that there is an independent
class of binding sites on HSA for captopril. Otherwise, it
appears that the binding constants and the number of
binding sites increased with increase in temperature
[29,30]. This may be attributed to that the capacity of
captopril binding to the fact that HSA is increased with
increase in temperature.
ln 0
HRT SR (6)
Consequently, a free energy change (G0) for a bind-
ing interaction at different temperatures can be evaluated:
  (7)
3.3. Mode of Binding from Thermodynamic
Parameters where K is the binding constant and R the gas constant.
The values of G0, H0, S0 for captopril binding to
HSA are listed in Table 2. The negative value of G0
Intermolecular interacting forces between a small mole-
Table 2. Biding constants and thermodynamic parameters of captopril with HSA at different temperatures (293.15 K to
310.15 K).
T (K) Kb (× 105 L mol1) n R H0 (KJmol-1) G0 (KJmol1) S0 (Jmol1 k1)
293.15 1.38 1.49 0.992 35.97 28.84 221.22
300.15 1.99 1.57 0.992 35.97 30.45 221.22
310.15 3.14 1.62 0.991 35.97 32.64 221.22
Copyright © 2011 SciRes. AJAC
reveals that the binding process is a spontaneous process.
The positive value of S0 change arises from water mo-
lecules arranged more random around HSA and drug,
caused by hydrophobic interactions between HSA and
drug molecules. Besides, the positive H0 is considered
as another evidence for hydrophobic interactions. Thus,
positive values for both H0 and S0 indicate hydropho-
bic interactions playing a major role in captopril binding
to HSA [31].
3.4. Conformational Changes Investigated by
Synchronous Fluorescence
Synchronous fluorescence is a very useful tool to inves-
tigate the microenvironments around the fluorophore fun-
ctional groups. It is well known that the fluorescence of
HSA arises from the tyrosine, tryptophan and pheny-
lalanine residues. The change in HSA conformation upon
addition of captopril can be also demonstrated by syn-
chronous fluorescence spectra. As is known, synchro-
nous fluorescence spectra show tyrosine residues of HSA
only at the wavelength interval (Δλ) of 15 nm and tryp-
tophan residues of HSA only at Δλ of 60 nm. The effect
of captopril on synchronous fluorescence spectra was
shown in Figure 5. The synchronous spectra of capto-
pril-HSA system were scanned at Δλ = 60 nm (Figure
5(a)) and Δλ = 15 nm (Figure 5(b)). The fluorescence of
tryptophan (Figure 5(a)) was strong, with addition of
captopril the fluorescence intensity decreased and no
apparent shift occurred in Figure 5(a). It can be seen the
fluorescence spectra of tyrosine (Figure 5(b)) was weak
and addition of captopril resulted in a decrease in inten-
sity and also no shift of maximum emission wavelength
[32]. It indicated that the interaction of captopril with
HSA does not significantly affect the conformation of
tryptophan and tyrosine residue microregions.
3.5. Energy Transform between Captopril and
In order to determine the precise location of captopril in
HSA, the efficiency of energy transfer was studied ac-
cording to the Förster resonance energy transfer theory
[33]. The energy transfer was calculated by determina-
tion the emission fluorescence of donor and the UV
spectra of accepter. The fluorescence quenching of HSA
after binding with captopril indicated the transfer of en-
ergy between captopril and HSA has occurred. The effi-
cient ligand–protein interaction gives rise to energy,
from which the distance between two interacting mole-
cules can be easily evaluated. The efficiency of energy
transfer E, is described by the following equation (8) [34,
Figure 5. Synchronous fluorescence spectra of HSA in the
absence andpresence of increasing amount of captopril. (a):
Δλ = 60 nm; (b): Δλ = 15 nm, T = 300.15 K, pH = 7.24, CHSA
=1.0 × 106 molL1, Ccaptopril = (1-7: 0.0, 1.25, 1.5, 1.75, 2.0,
2.25 and 2.5) 104 molL1, respectively.
1EFFRRR (8)
where F0 and F are the fluorescence intensity of donor in
the absence and presence of acceptor, respectively, R is
the distance between acceptor and donor and R0 is the
critical distance, and the value of R0 is calculated by fol-
lowing equation:
08.8 10RKN
 (9)
where K2 is the spatial orientation factor of the dipole, N
is the refractive index of the medium, is the fluores-
cence quantum yield of the donor, J is the overlap inte-
gral of the fluorescence emission spectrum of the donor
and the absorption spectrum of the acceptor. J is given
()() d()dJF F
 
X. Y. GAO ET AL.255
In this equation, F(
) is the fluorescence intensity of
the fluorescent donor of wavelength,
, ε(
) is the molar
absorption coefficient of the acceptor at wavelength. It
was reported earlier that K2 = 2/3, N = 1.336, and
0.118 [36]. The overlap of UV absorbance spectrum of
captopril with fluorescence spectrum of HSA was shown
in Figure 6. From the above relationships, for the capto-
pril-HSA, the values for E, and R evaluated are the fol-
lowing: J = 1.09 × 1018 cm3Lmol1, = 2.369 ×
1043 cm6, E = 0.15, and R = 1.05 nm for HSA. The dis-
tance R < 8 nm [37] between donor and acceptor indi-
cates that the energy transfer from HSA to captopril oc-
curred with high possibility. This accord with the condi-
tions of Förster’s energy transfer theory indicated the
static quenching interaction between captopril and HSA.
4. Conclusions
The interaction between captopril and HSA has been
investigated by using fluorescence and ultraviolet (UV)
absorption spectra in vitro under a simulated physiologi-
cal condition (pH = 7.24) in this work. The experimental
results suggested that the fluorescence of HSA was
quenched through static mode, and the pharmaceutical
(captopril) can strongly binding to HSA. The synchro-
nous fluorescence spectra showed no significant change
in the conformation of HSA upon addition of captopril
under experimental conditions; the binding distance R of
1.05 nm between captopril and HSA indicated that the
energy transfer from HSA to captopril occurred with
high possibility. In addition, the thermodynamic para-
meter elucidated the binding reaction was mainly driven
by hydrophobic interaction.
The determinations performed herein may provide an
approach to evaluate the toxic effects of chemicals on
target proteins and the molecular mechanism of toxicity.
Figure 6. The overlap of the UV absorption spectrum of
captopril (a) with the fluorescence emission spectrum of
HAS (b) when the molar ratio is 1:1, CHSA = CCaptopril = 1.0 ×
106 molL1, T = 300.15 K, pH = 7.24.
The results obtained herein will be of biological signifi-
cance in pharmacology and clinical medicine.
5. Acknowledgements
We thank the National Key Technology R&D Program
of China (No.2008BAJ08B13) for financially supporting
this work.
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