Interaction of Antithyroid Drugs With Bovine Serum
Albumin: Electrophoretic and Fluorimetric Study
MARGARITA S. CHERNOV YANTS, ANDREY O. DOLINKIN, ANATOLY V. CHERNYSHEV, EVGENIY V. KHOHLOV,
ELIZAVETA G. GOLOVANOVA
Department of Analytical Chemistry, Southern Federal University, Rostov-on-Don 344090, Russia
Received 18 May 2009; revised 17 July 2009; accepted 28 July 2009
Published online 22 September 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21915
ABSTRACT: The pre-equilibrium capillary zone electrophoretic (pre-eq CZE) method to
determine association constants of active anionic forms of antithyroid drugs: 6-n-propyl-
2-thiouracil (PTU), 6-methyl-2-thiouracil (MTU), 2-thiouracil (TU) with bovine serum
albumin (BSA) under physiological pH 7.4 has been developed for the first time. Using
the decrease of the selective electrochromatographic peak area of a drug anionic form
due to binding with BSA the association constants K of the binary BSA complexes were
calculated. It has been found that the binding constants (log K) of BSA with TU, MTU,
and PTU are equal to 2.99, 1.85, and 2.11, respectively. The interaction of PTU, MTU,
TU, 2-mercapto-1-methylimidazole (MMI), and ethyl-3-methyl-2-thionoimidazoline-1-
carboxylate (Carb), which is considered to be a prodrug for MMI, with BSA has been
investigated under physiological conditions by means of fluorescence spectroscopy.
Fluorescence emission spectra of BSA in the presence of thioamides recorded at
295 nm excitation wavelength clearly show that the studied drugs act as quenchers,
except MMI, which acts as quencher when being excited at 280 nm. The 295 nm light
excites tryptophan residues, while the 280 nm light excites both tryptophan and tyrosine
residues. The binding constants (log K) of BSA with PTU, MTU, TU, MMI, and Carb
have been found to be 4.51, 4.30, 4.30, 2.64, and 4.32, respectively. ß 2009 Wiley-Liss, Inc.
and the American Pharmacists Association J Pharm Sci 99:1567 1573, 2010
Keywords: heteroaromatical thioamides; antithyroid drugs; serum albumin drug
interactions; binding constants; affinity capillary electrophoresis (ACE); fluorescence
spectroscopy
INTRODUCTION thyroid gland. Metabolized by conjugation with
glucoronic acid and sulfate.1
The traditional treatments given for Grave s Among the four aspects of pharmacokinetics
disease are antithyroid drugs: PTU, MTU, TU, (absorption, distribution, metabolism, and excre-
MMI, and Carb (Fig. 1). These drugs act by tion), distribution is the one that is controlled by
depressing the formation of the thyroid hormones, serum protein because most drugs that travel in
triiodothyronine and thyroxine. Antithyroid drugs plasma bind to protein. Binding strength to
are rapidly and almost completely absorbed plasma proteins is an important factor to be
after oral administration and concentrated in the considered in drug development. The proteins in
the serum mainly responsible for transporting
these drugs are the albumins. Serum albumin
Correspondence to: Margarita S. Chernov yants (Telephone:
is one of the most widely studied and applied
þ7-8632975152; Fax: þ7-8632675185;
proteins in biochemistry.2,3 It is the most abun-
E-mail: [e-mail])
dant protein in mammalian blood circulation and
Journal of Pharmaceutical Sciences, Vol. 99, 1567 1573 (2010)
ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association is characterized as the major transport protein
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010 1567
1568 CHERNOV YANTS ET AL.
spectroscopy. Therefore, studies on the binding
of drug with protein will increase interpretation of
the metabolism and transporting process of
antithyroid drugs and will help to explain the
relationship between the structure and the
function of the protein. BSA has been studied
extensively, partly due to of its structural
homology with human serum albumin (HSA).14,15
Figure 1. Structures of antithyroid drugs.
due to its ability to bind a diverse complement of EXPERIMENTAL
endogenous and exogenous ligands including fatty
acids, amino acids and a host of pharmaceuti- Reagents and Chemicals
cals.4 20 Smaller and less hydrophobic compounds
6-n-Propyl-2-thiouracil (Propylthiouracil, PTU),
are held less strongly, but their binding can still be
2-thiouracil (TU) were obtained from Sigma Aldrich
highly specific.3 The interaction between bovine
Rus (Moscow, Russia), 6-methyl-2-thiouracil (MTU)
serum albumin or human serum albumin and
and ethyl 3-methyl-2-thionoimidazoline-1-carboxy-
drugs has been previously investigated by visible
late (Carb) were purchased from Alfa Aesar GmbH
absorption,13,21 fluorescence spectroscopies,14 20
& Co (Karlsruhe, Germany), and 2-mercapto-1-
and by capillary electrophoresis.22 28 Although
methylimidazole (methimazole, MMI) was obtained
medically important, serum albumin owes much
from Lancaster Synthesis (OOO   S-Reakor  , Moscow,
of this interest to its ready availability and
Russia).
unusual ligand-binding properties.
Crystallized and lyophilized bovine serum
Crystallographic studies have shown that many
albumin (99% purity BSA), fraction V was
of the ligands are bound primarily within three of
purchased from Sigma Aldrich (A 3059).
the six subdomains, namely domains IB, IIA, and
IIIA. The structures of the responsible amino
acids have been previously reported.29 31 Analysis
Fluorescence Procedure
of HSA and BSA interactions within IB, IIA, and
Fluorescence emission spectra were recorded on
IIIA ligand-binding structural domains may allow
a Varian Cary Eclipse spectrofluorimeter at
suggesting that the binding site for antithyroid
lexc ź 280 and lexc ź 295 nm using 1 cm cuvette.
drugs is located in subdomains IB and IIA. HSA
All quenching experiments were performed at
includes one tryptophan residue (Trp 214) in
room temperature using protein solutions of
subdomain IIA, whereas BSA possesses two
invariable concentration 1.0 10 5 M. The con-
tryptophan moieties (Trp 134 and Trp 213) located
centrations of antithyroid drugs were varied from
in subdomain IB and IIA respectively.32,33 It is
deficiency to 20-fold excess (the MMI concentra-
well recognized that the pharmacological activity
tion was varied from deficiency to 200-fold excess)
of a drug is related to the free drug concentration
comparing to BSA concentration. The phosphate
in the blood. In order to be able to adjust the
buffer (0.05 M sodium phosphate, pH 7.4) was
optimum therapeutic dose of a drug, it is therefore
used.
necessary to know the extent of drug protein
The fluorescent emission spectra of BSA
binding.3
recorded at lexc ź 280 nm exhibit maximum at
The antithyroid drugs thiouracil derivatives
340 nm; the maximum of fluorescent emission
(PTU, MTU, TU) under physiological pH present
spectra of BSA obtained at lexc ź 295 nm is located
partly in anionic pharmacological active forms
at 350 nm.
(about 10%). No attempts had been made so far to
investigate the binding parameters, such as
binding constants, binding sites of these forms
Electrophoretic Procedure
with BSA. In this paper the novel pre-eq CZE
technique for determination of binding constants Kapel-103R high-efficiency capillary electrophor-
of antithyroid drug s active anionic forms with esis (CE) instrument with an UV detector
BSA was developed. The interaction between (l ź 253.7 nm) was employed for capillary deter-
molecular forms of antithyroid drugs with BSA mination of antithyroid drug anionic forms and
was investigated by means of fluorescence their associates with albumin. Data acquisition
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010 DOI 10.1002/jps
INTERACTION OF ANTITHYROID DRUGS WITH BOVINE SERUM ALBUMIN 1569
and processing were conducted using PC supplied
with Multichrom software. To measure electro-
phoretic mobility of antithyroid drugs, BSA
in phosphate electrophoresis buffer (pH 7.4)23
and nonbinding electroosmotic flow marker (EOF,
benzyl alcohol) were injected into capillary
filled with neat buffer and were driven through
by a potential of 15 kV. BSA and buffer
solutions were filtered through 0.45 mm pore
size filter before use. Samples were injected
hydrodynamically at a pressure of 30 mbar
within 5 s. Hydrodynamic injection is preferred
over the electrokinetic injection for protein bind-
ing studies as it is reproducible, robust, and
unbiased.34
Figure 2. Fluorescence spectra of a BSA PTU sys-
The capillary was prepared to the experiments
tem: C(BSA) ź 1.0 10 5 M; C(PTU) ź 0, 3.0 10 6,
by washing with 1 M NaOH solution for 2 min,
6.0 10 6, 1.0 10 5, 2.0 10 5, 4.0 10 5, 6.0
with water to remove NaOH traces, and with the
10 5, 1.0 10 4, 2.0 10 4 (from up to bottom). The
buffer solution for 15 min. Between the experi- arrow indicates the decrease of fluorescence intensity
upon drug concentration increases.
ments, the capillary was washed with 1 M NaOH
solution, with water and with the electrophoresis
buffer for 30 min to minimize protein adsorb-
tion.35 Uncoated fused silica capillary with an
For static quenching, the relationship between
inner diameter of 75 mm, a length of 60 cm, and a
fluorescence intensity quenching and the concen-
distance from the sample injection port to the
tration of quenchers can be described by Eq. (2):
detector window of 50 cm (Ldet/Ltot ź 50/60 cm)
Imax Ii
was used. Each measurement was repeated three
log ź log K þ n log½LŠ (2)
Ii
times. All ACE experiments were performed at
room temperature (228C, air thermostatic capil-
where Ii and Imax are the fluorescence intensity in
lary) using invariable concentration of drugs
the presence of a quencher and without quencher
(2.14 10 4 M). The concentrations of BSA were
respectively, [L] is the unbound antithyroid agent
varied from deficiency to 10-fold excess comparing
concentration. Using the fluorescence decrease
to a drug concentration.
the association constants (or log K) and the
RESULTS AND DISCUSSION
Fluorescence spectra of BSA (1.0 10 5 M) and
BSA-PTU complex in the phosphate buffer (pH
7.4) are shown in Figure 2. Fluorescence intensity
decay observed upon addition of increasing
amounts of antithyroid drugs (antithyroid drug
concentration was ranged from 3.0 10 6 to
2.0 10 4 M) is presented in Figure 3.
Calculation of Binding Constants
The interaction between albumin (Alb) and
antithyroid drugs (L) can be described by the
binding constant (K) formula:
Figure 3. Quenching effect of PTU on BSA fluores-
cence: protein concentration is 1.0 10 5 M; lexc ź
½Alb LnŠ
295 nm. Points are experimental data, continuous line
Alb þ nL ź Alb Ln; K ź (1)
½LŠn½AlbŠ
is fitting by Eq. (3).
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010
1570 CHERNOV YANTS ET AL.
Table 1. Binding Parameters of the Systems of binary complexes association constants were
Antithyroid Drugs/BSA Estimated from Fluorescence
calculated by the nonlinear least-squares method
Data
using Eq. (3):
0 sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1
ffi
Linear
2
1 1 1
Method Number of Nonlinear Imax@CAlb CL þ CL CAlb þ þ4 CAlbA
K K K
Ligand (log K) Binding Sites (n) Method
Ii ź
2CAlb
PTU 4.71 0.11 1.03 4.51 0.25
(3)
MTU 4.17 0.15 0.98 4.30 0.29
TU 4.21 0.14 0.96 4.30 0.17
where CAlb and CL are the total concentrations of
MMI 2.54 0.14 1.06 2.64 0.10
albumin and antithyroid agent respectively.
Carb 4.28 0.16 1.08 4.32 0.23
The binding parameters found for the same
ligands and protein from fluorescence data are
summarized in Table 1. The results obtained by
number of binding sites (n) for the complexes23 of linear and nonlinear least-squares calculations
antithyroid drugs with BSA were calculated by are in good agreement.
the linear least-squares method. The results Some electropherograms of the PTU-BSA sys-
indicate that binding ratio of antithyroid drugs tem are presented in Figure 4. An addition of BSA
with BSA extremely access to 1:1 in the complex to a drug solution results in a decrease of drug s
(Tab. 1). Since BSA forms with antithyroid drugs anion form peak area and in an increase of
Figure 4. Electropherograms of the PTU BSA system: C(PTU) ź 2.14 10 4 M;
C(BSA): 2.14 10 4 M (I), 4.29 10 4 M (II), 8.57 10 4 M (III), 2.14 10 3 M (IV).
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010 DOI 10.1002/jps
INTERACTION OF ANTITHYROID DRUGS WITH BOVINE SERUM ALBUMIN 1571
summary peak area of binary complex and free conditions were estimated. It is known that
albumin. Using the decrease of the electrochro- tryptophan and tyrosine included in the protein
matographic peak area of a drug anionic form the structures can act as intrinsic fluorescence
association constants K of the binary BSA probes.36,37 The portion of the subdomain IIA
complexes were calculated according to Eq. (4) that binds the hydrophobic part of antithyroid
drugs contains Trp 213. A comparison of quench-
n
log ź log K þ log½AlbŠ (4)
ing effects when protein is excited at 295 and
ð1 nÞ
280 nm, reveals that the tryptophanyl groups
where n is the average number of thioamides (excited at 295 nm) interact with PTU, MTU, TU,
anions associated with BSA molecule, n ź Carb, but does not interact with MMI. These
ððSmax SiÞ=SmaxÞ (0< n <1), Smax, Si is the peak observations agree with the findings of Singh and
area for initial and current concentrations of Kishore.38 While the tyrosinyl group (excited at
anionic forms of thioamides respectively; [Alb] is 280 nm) weakly interacts with MMI. MMI acts as
the unbound concentration of BSA, ½AlbŠ ź quencher only when lexc ź 280 nm is used.39 40
CAlb CL þ½L Š, where ½L Š źðSi=SmaxÞCL , Therefore, the MMI BSA binary complex binding
CL is measured according to equation CL ź constant calculated from fluorescence data should
ðCL=ð1 þ 10pKa pHÞÞ; pKa for TU, MTU, and PTU be low. The extents of albumin-binding for MMI
are equal to 8.20, 8.40, and 8.37, respectively;23 and its prodrug Carb in vivo, estimated from
½L Š and CL are unbound and total concen- binding constant data significantly differ (20%
trations of antithyroid agent anionic forms, and 92%, respectively). Thus, N,N-disubstituted
respectively. compound Carb is rapidly distributed and con-
The linear regression method has been used in verted to MMI.
order to determine binding constant from ACE From association constants presented in
experimental data. The binding constants of the Table 1 it has been shown that PTU binds BSA
anionic forms of ligands with protein found from more stably. PTU being a hydrophobic molecule
electrophoretic data are summarized in Table 2. (log P(octanol/water)1.0) can better penetrate into
the hydrophobic subdomains, where Trp 213 or
Trp 134 is located. The other antithyroid drugs
CONCLUSIONS (except MMI) exhibit similar affinity to BSA.
PTU, MTU, TU, and Carb containing electro-
The interaction between molecular and active negative oxygen atoms would preferentially inter-
anionic forms of antithyroid drugs: PTU, MTU, act with tryptophan and tyrosine residues due to a
TU, MMI, Carb, and BSA has been investigated by hydrogen bond. Analysis of interaction of BSA
means of fluorescence spectroscopy and ACE, with MMI allows making a suggestion that drug
respectively. can form hydrogen bonds with ionized carboxylic
After oral administration, the antithyroid drugs groups of Asp and Glu residues. Since MMI acts as
get concentrated in the thyroid gland where they quencher when being excited at 280 nm, it is
inhibit the biosynthesis of thyroid hormones. possible that MMI binds only tyrosine residues of
Binding constants of molecular and anionic protein.
(except MMI, Carb) forms of antithyroid drugs Under physiological pH (0.05 M sodium
with drug transport BSA under physiological phosphate buffer, pH 7.4), bovine serum albumin
Table 2. Electrophoretic Characteristics and Binding Constant Values of
Pyrimidine Derivatives Anionic Forms With BSA
Migration Electrophoretic Binding
Ligand (L ) Time, tL (s) Mobilitya, mL (cm2 kV 1 s 1) Constants (log K)
PTU 452 2.1 2.99 0.01
MTU 560 7.2 1.85 0.04
TU 498 4.5 2.11 0.02
a
Electrophoretic mobility of PTU, MTU, and TU anion forms is normally calculated using the
following equation mL źðLcLd=VÞð1=tL 1=teoÞ, where V is the potential, teo is the migration
time (s) of the EOF marker, and tL is the migration time (s) of the anion.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 3, MARCH 2010
1572 CHERNOV YANTS ET AL.
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