Effect of ibuprofen and warfarin on the allosteric properties of
haem–human serum albumin
A spectroscopic study
Simona Baroni
1
, Marco Mattu
2
, Alessandro Vannini
2
, Rita Cipollone
2
, Silvio Aime
1
, Paolo Ascenzi
2
and
Mauro Fasano
3
1
Department of Chemistry ‘IFM’, University of Torino, Italy;
2
Department of Biology, University ‘Roma Tre’, Rome, Italy;
3
Department of
Structural and Functional Biology, University of Insubria, Italy
Haem binding to human serum albumin (HSA) endows the
protein with peculiar spectroscopic properties. Here, the
effect of ibuprofen and warfarin on the spectroscopic
properties of ferric haem–human serum albumin (ferric
HSA–haem) and of ferrous nitrosylated haem –human
serum albumin (ferrous HSA–haem-NO) is reported. Ferric

M in the presence of ibuprofen
and warfarin. Ferrous HSA – haem-NO is a five-coordinated
haem-iron system. Drug binding to the warfarin primary site
of ferrous HSA–haem-NO induces the transition towards
the six-coordinated haem-iron species, the haem-iron atom
being bonded to His105. Remarkably, the ibuprofen primary
cleft appears to be functionally and spectroscopically
uncoupled from the haem site of HSA. Present results
represent a clear-cut evidence for the drug-induced shift of
allosteric equilibrium(a) of HSA.
Keywords: allostery; haem–human serum albumin; human
serum albumin; ibuprofen; warfarin.
Human serum albumin (HSA), the most prominent protein
in plasma, is best known for its exceptional ligand binding
capacity, the most strongly bound compounds being
hydrophobic organic anions of medium size, long-chain
fatty acids, haem and bilirubin. Smaller and less
hydrophobic compounds (e.g. tryptophan) are held less
strongly, but their binding can still be highly specific. For
many compounds, HSA provides a depot so they will be
available in quantities well beyond their solubility in
plasma. Moreover, HSA abundance (concentration of
45 mg
:
mL
21
in the serum of human adults) makes it an
important determinant of the pharmacokinetic behaviour of
many drugs. In other cases, HSA holds some ligands in a
strained orientation, allowing their metabolic modification,

Note: S. Baroni and M. Mattu contributed equally to this work.
(Received 2 July 2001, revised 27 September 2001, accepted 3 October
2001)
Abbreviations: HSA, human serum albumin; ferric HSA –haem, ferric
haem– human serum albumin; ferrous HSA –haem-NO, ferrous
nitrosylated haem–human serum albumin.
Eur. J. Biochem. 268, 6214–6220 (2001) q FEBS 2001
vicinity of the binding site(s), affecting both the structure
and the ligand binding properties of the whole HSA
molecule [1– 11].
The interaction of ligands with HSA occurs mainly in two
regions. According to the Sudlow’s nomenclature, bulky
heterocyclic anions bind to site I (located in subdomain IIA),
whereas site II (located in subdomain IIIA) is preferred by
aromatic carboxylates with an extended conformation.
Remarkably, ibuprofen, a nonsteroidal anti-inflammatory
agent [12], and warfarin, an anticoagulant drug [12], are
considered as stereotypical ligands for Sudlow’s site II and
Sudlow’s site I, respectively [1,9,11,13,14]. Ibuprofen binds
to Sudlow’s site II with K
d
¼ 3.7 Â 10
27
M [1,15], whereas
warfarin binds to Sudlow’s site I with K
d
¼ 3.0 Â 10
26
M
[1,16–18]. Secondary binding clefts have been found for

Chemical Co. All the other products were from Merck AG.
All chemicals were of analytical or reagent grade and used
without further purification.
Ferric HSA–haem was prepared by adding 0.83-
M defect
of ferric haem, dissolved in 1.0 Â 10
21
M KOH, to an HSA
solution, in 1.0 Â 10
21
M phosphate buffer plus
1.0 Â 10
21
M NaCl, pH 7.0 [8,21]. Both in the absence
and presence of ibuprofen and warfarin, haem binds to
HSA mostly at the high affinity site and virtually no free
haem is present in solution (see [1,8,21], and present study).
Values of the apparent dissociation equilibrium constant
(K
d
) for haem binding to the HSA primary site are
1.3 ^ 0.2 Â 10
28
M in the absence of drugs, and
1.5 ^ 0.2 Â 10
27
M in the presence of ibuprofen and
warfarin (5.0 Â 10
22
M) (see [1,22,23] and the present

21
M NaCl, pH 7.0 [8,21].
HSA solutions were prepared by dissolving the protein in
1.0 Â 10
21
M phosphate buffer plus 1.0 Â 10
21
M NaCl, at
pH 7.0 and 25.0 8C. Haem solutions were prepared in
1.0 Â 10
21
M KOH. Ibuprofen solutions were prepared by
dissolving the drug in 1.0 Â 10
21
M phosphate buffer plus
1.0 Â 10
21
M NaCl, at pH 7.0 and 25.0 8C. Warfarin
solutions were prepared by stirring the drug in 1.0 Â 10
21
M
phosphate buffer plus 1.0 Â 10
21
M NaCl at pH 12.0
until it dissolved, then adjusting to pH 7.0 with HCl (at
25.0 8C).
Binding of ibuprofen and warfarin to ferric HSA –haem
was followed by electronic absorption spectroscopy at
pH 7.0, in 1.0 Â 10
21

M phosphate buffer plus 1.0 Â 10
21
M NaCl)
and 25.0 8C.
1
H-NMR relaxometry of ferric HSA–haem
(1.0 Â 10
23
M) in the absence and presence of ibuprofen
and warfarin (1.0 Â 10
24
M to 1.0 Â 10
21
M) was investi-
gated on a Stelar SpinMaster Spectrometer (Stelar S.n.c.,
Mede, PV, Italy). Water proton relaxation rate (R
1
)
measurements were obtained at 0.47 T (i.e. at 20 MHz
proton Larmor frequency) by means of the Inversion-
Recovery technique (16 experiments, four scans). Magneti-
zation values were obtained by averaging the first 128 data
points of the Free Induction Decay. A phase cycle (1x, –x,
–x, 1x) was applied on the 908 observation pulse to cut
off the y-scale receiver offset. A typical 908 pulse width
was 3.5 ms. The
t
-values were increased linearly from a
starting value corresponding to one-seventh of the estimated
null-point (0.693/R

Test measurements performed after 2 h excluded slow
kinetic effects [7].
Haem binding to HSA in the absence and presence of
ibuprofen and warfarin was followed by electronic
absorption spectroscopy (between 350 nm and 450 nm) at
pH 7.0 (1.0 Â 10
21
M phosphate buffer plus 1.0 Â 10
21
M
NaCl) and 25.0 8C [22]. The HSA concentration ranged
between 3.0 Â 10
28
M and 2.0 Â 10
26
M, the haem
concentration was 1.0 Â 10
27
M, and the ibuprofen and
warfarin concentrations were 5.0 Â 10
22
M. The electronic
absorption spectra were recorded in a 10-cm path length
cuvette. The haem-induced electronic absorption spectro-
scopic transition of HSA was complete within the time
to achieve the sample preparation (, 10 min). Test
measurements performed after 2 h excluded slow kinetic
effects.
Binding of ibuprofen and warfarin to ferrous HSA– haem-
NO was followed by X-band EPR spectroscopy at pH 7.0 in

change in the haem-iron atom coordination number, nor in
the spin state of the metal centre, is observed. Spectra shown
in Fig. 2 are indicative of a high-spin state of the haem-iron.
Actually, even the minor low-spin component [22] seems to
diminish in the presence of either drug. In fact, the Soret
band is blue-shifted, the charge transfer band is red-shifted
and the a band is decreased in intensity with respect to the
b band. The spectral features shown in Fig. 2 are indicative
of a drug-dependent conformational transition(s) that does
not affect the inner coordination sphere of the haem iron
atom.
Fig. 2. Effect of ibuprofen and warfarin on the electronic
absorption spectroscopic properties of ferric HSA–haem. Elec-
tronic absorption spectra of ferric HSA–haem were obtained in the
absence (spectrum a) and in the presence of 5.0 Â 10
22
M ibuprofen
(spectrum b, continuous line) and 5.0 Â 10
22
M warfarin (spectrum b,
filled circles) at pH 7.0 and 25.0 8C. The electronic absorption spectra
of ferric HSA –haem in the presence of ibuprofen and warfarin are
superimposable. The ferric HSA–haem concentration was
8.4 Â 10
26
M. The electronic absorption spectra were recorded in a
1-cm path length cuvette.
6216 S. Baroni et al. (Eur. J. Biochem. 268) q FEBS 2001
Fig. 3 shows the binding isotherms of ibuprofen and
warfarin to ferric HSA–haem, at pH 7.0 and 25.0 8C. Data

drug, HSA, HSA : ibuprofen, or HSA : warfarin) concen-
tration in the forms indicated by subscripts T (total) and b
(bound), respectively. [L]
b
was calculated according to
Eqn (2) [25]:
½L
b
¼ K
d
1 n
:
½Q
T
1 ½L
T
2
p
ðK
d
1 n
:
½Q
T
1 ½L
T
Þ
2
ÈÈ
24K

value
for ibuprofen binding to ferric HSA –haem is higher than
those reported for drug binding to the ibuprofen primary site
(K
d
¼ 3.7 Â 10
27
M, at pH 7.4 and 37.0 8C) [1,15] and to
the ibuprofen secondary cleft (K
d
< 4 Â 10
25
M at pH 7.4
and 37.0 8C) [1,15] of haem-free HSA. Also the K
d
value for
warfarin binding to ferric HSA–haem is higher than that
reported for drug binding to the warfarin primary site
(K
d
¼ 3.0 Â 10
26
M at pH 7.4 and 25.0 8C) [1,16 – 18] of
haem-free HSA.
Fig. 4 shows the binding isotherms of ferric haem to HSA
in the absence and presence of ibuprofen and warfarin, as
obtained by electronic absorption spectroscopy at pH 7.0
and 25.0 8C. Data analysis according to Eqn (1) allowed the
determination of values of the apparent dissociation
equilibrium constant (K

d
¼ 5.4 ^ 1.1 Â 10
24
M and
n ¼ 1.9 ^ 0.1 for ibuprofen binding, and K
d
¼ 2.1 ^ 0.4 Â 10
25
M
and n ¼ 2.7 ^ 0.1 for warfarin binding. The ferric HSA–haem
concentration was 1.5 Â 10
24
M and 1.0 Â 10
23
M for electronic
absorption spectroscopic and
1
H-NMR relaxometric experiments,
respectively. The electronic absorption spectra were recorded in a
1-mm path length cuvette.
Fig. 4. Haem binding to HSA. Electronic absorption spectroscopic
binding isotherms of haem to HSAwere obtained in the absence (A) and
presence of 5.0 Â 10
22
M ibuprofen (K) and 5.0 Â 10
22
M warfarin
(W) at pH 7.0 and 25.0 8C. The continuous lines were obtained by using
Eqn (1). Best fitting parameters for ferric HSA–haem formation are
K

warfarin primary cleft (i.e. Sudlow’s site I), corresponding
to the ibuprofen secondary site [1,3,11]. Then, ibuprofen
and warfarin impair ferric HSA– haem formation. Remark-
ably, the ibuprofen primary cleft (i.e. Sudlow’s site II)
appears to be functionally and spectroscopically uncoupled
to the haem site of HSA.
Ferric HSA–haem has been widely investigated by
1
H-NMR relaxometry [7]. The high value of the paramagnetic
contribution to the water relaxation rate (R
1p
)of
hexacoordinated ferric HSA– haem (¼ 4.8 m
M
21
:
s
21
at
20 MHz, pH 7.2 and 25 8C) has been ascribed to the
occurrence of slowly exchanging water molecules in the
surroundings of the paramagnetic ferric haem center [7]. In
the presence of saturating amounts of ibuprofen and
warfarin, the R
1p
value of hexacoordinated ferric HSA–
haem decreases to 0.4 m
M
21
:

M ibuprofen (spectrum b, continuous line) and
5.0 Â 10
22
M warfarin (spectrum b, filled circles) at pH 7.0 and
2173 8C. The X-band EPR spectra of ferrous HSA–haem-NO in the
presence of ibuprofen and warfarin are superimposable. The ferrous
HSA–haem-NO concentration was 3.0 Â 10
24
M.
Table 1. X-band EPR parameters and the haem-iron coordination state of HSA–haem-NO. Values listed are for
14
N systems. Experimental
conditions were pH 7.0 and 2173 8C. US, unresolved signal.
Conditions A
3
(mT) g
1
g
2
g
3
Coordination state
Stripped
a
1.65 2.095 2.060 2.010 Five
Bezafibrate
b
US 2.064 1.983 2.005 Six
Clofibrate
b

3
¼ 2.010) (see Fig. 6, spectrum a, and Table 1). This
X-band EPR spectrum has been associated with the five-
coordinate haem-iron state of ferrous HSA– haem-NO [8],
in agreement with data reported for several ferrous
nitrosylated haemoglobin systems [8,29–34]. Addition of
either ibuprofen or warfarin to ferrous HSA–haem-NO
induces the transition towards a species characterized by an
X-band EPR spectrum with a rhombic shape and a weak
superhyperfine pattern in the g
z
region (see Fig. 6, spectrum
b, and Table 1). Such behaviour is similar to that observed in
the presence of bezafibrate and clofibrate, which has been
attributed to the shift of the conformational equilibrium
towards the six-coordinated haem-iron state of ferrous
HSA–haem-NO [8]. The His242 residue has been
postulated to be a likely candidate for the sixth axial ligand
of the haem iron in ferrous HSA–haem-NO in the presence
of bezafibrate and clofibrate [8]. However, more recent
results are consistent with the suggestion that His105 might
be responsible for the sixth axial bonding of the haem iron
[7,10,11]. Remarkably, His242 has been shown to be
hydrogen-bonded to warfarin [14]. As the binding of
warfarin to Sudlow’s site I does not affect the coordination
state of haem iron (see Fig. 2), His242 does not appear to be
a likely candidate for the axial haem bonding.
CONCLUSIONS
The effect of ibuprofen and warfarin on the electronic
absorption spectroscopic,

to S. A.), as well as from the National Research Council of Italy (CNR,
Target oriented project ‘Biotecnologie’ to P. A. and M. F.). S. B. wishes
to thank Consorzio Interuniversitario di Ricerca in Chimica dei Metalli
nei Sistemi Biologici (CIRCMSB) for a doctoral studentship.
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