Báo cáo khoa học: Ibuprofen binding to secondary sites allosterically modulates the spectroscopic and catalytic properties of human serum heme–albumin doc
Ibuprofenbindingtosecondarysites allosterically
modulates thespectroscopicandcatalytic properties
of humanserum heme–albumin
Alessandra di Masi
1
, Francesca Gullotta
2,3
, Alessandro Bolli
1
, Gabriella Fanali
4
, Mauro Fasano
4
and
Paolo Ascenzi
1
1 Department of Biology, and Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Italy
2 Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Italy
3 Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy
4 Department of Structural and Functional Biology, and Center of Neuroscience, University of Insubria, Busto Arsizio (VA), Italy
Introduction
Human serum albumin (HSA), the most abundant
protein in plasma, provides a depot and carrier for
many endogenous and exogenous compounds. Among
other roles, HSA affects the pharmacokinetics of many
drugs, holds some ligands in a strained orientation that
results in their metabolic modification, renders poten-
tial toxins harmless by transporting them to disposal
sites, accounts for most ofthe antioxidant capacity of
human serum, and displays (pseudo)enzymatic proper-
ties [1–8].
HSA is a single nonglycosylated all-a-chain protein,
composed of 585 amino acids, and containing three
homologous domains (labeled I, II, and III). Each
domain is composed of two separate subdo-
mains (named A and B) connected by random coils.
Interdomain helical regions link subdomain IB to
Keywords
allostery; humanserum heme–albumin;
ibuprofen binding; modulation of reactivity
and spectroscopic properties; recombinant
truncated humanserum heme–albumin
(Asp1–Glu382)
Correspondence
P. Ascenzi, Department of Biology, and
Interdepartmental Laboratory of Electron
Microscopy, University Roma Tre, Viale
Guglielmo Marconi 446, I-00146 Rome, Italy
Fax: +39 06 5733 6321
Tel: +39 06 5733 3200(2)
E-mail: ascenzi@uniroma3.it
(Received 14 July 2010, revised 24
November 2010, accepted 6 December
2010)
doi:10.1111/j.1742-4658.2010.07986.x
The ibuprofen primary binding site FA3–FA4 is located in domain III of
human serum albumin (HSA), thesecondary clefts FA2 and FA6 being
sited in domains I and II. Here, the thermodynamics ofibuprofen binding
to recombinant Asp1–Glu382 truncated HSA (tHSA)–heme-Fe(III) and
nitrosylated tHSA–heme-Fe(II), encompassing domains I and II only, is
reported. Moreover, the allosteric effect ofibuprofen on the kinetics of
tHSA–heme-Fe(III)-mediated peroxynitrite isomerization and nitrosylated
tHSA–heme-Fe(II) denitrosylation has been investigated. The present data
indicate, for the first time, that the allosteric modulation of tHSA–heme
and HSA–heme reactivity by ibuprofen depends mainly on drug binding to
the FA2 and FA6 secondarysites rather than drug association with the
FA3–FA4 primary cleft. Thus, tHSA is a valuable model with which to
investigate the allosteric linkage between the heme cleft FA1 and the
ligand-binding pockets FA2 and FA6, all located in domains I and II of
(t)HSA.
Abbreviations
FA, fatty acid; HSA, humanserum albumin; HSA–heme-Fe, humanserum heme-albumin; HSA–heme-Fe(III), ferric HSA–heme-Fe;
HSA–heme-Fe(II), ferrous HSA–heme-Fe; HSA–heme-Fe(II)-NO, nitrosylated HSA–heme-Fe(II); tHSA, truncated HSA; tHSA–heme-Fe(III),
ferric heme tHSA; tHSA–heme-Fe(II), ferrous tHSA–heme-Fe; tHSA–heme-Fe(II)-NO, nitrosylated tHSA–heme-Fe(II).
654 FEBS Journal 278 (2011) 654–662 ª 2011 The Authors Journal compilation ª 2011 FEBS
subdomain IIA, and subdomain IIB to subdo-
main IIIA (Fig. 1) [8,9].
The structural organization of HSA provides several
ligand-binding sites (Fig. 1). HSA has seven binding
clefts hosting chemically diverse ligands, including
fatty acids (FAs), that are labeled FA1–FA7 (Fig. 1).
In particular, FA1 (located in subdomain IB) has
evolved to specifically bind heme, FA3 and FA4 make
up the so-called Sudlow site II (located in subdo-
main IIIA), which preferentially recognizes aromatic
carboxylates with an extended conformation, and FA7
is the so-called Sudlow site I (located in subdo-
main IIA), which binds, in particular, bulky heterocy-
clic anions. Remarkably, warfarin, a coumarinic
anticoagulant drug, and ibuprofen, a nonsteroidal
anti-inflammatory drug, are considered to be stereotyp-
ical ligands for Sudlow sites I and II, respectively. In
contrast to warfarin, ibuprofen has been reported to
also bind tosecondarysites in HSA domains I and II
that have been characterized by X-ray crystallographic
and solution spectroscopic studies [1,2,6,8–20].
Recently, the recombinant truncated form of HSA
(tHSA), encompassing residues Asp1–Glu382 (corre-
sponding to domains I and II, containing only the ibu-
profen secondarybinding sites), has been preliminarily
characterized [19]. Here, ibuprofenbindingto tHSA–
heme-Fe(III) and tHSA–heme-Fe(II) is reported.
Moreover, the allosteric effect ofibuprofen on tHSA–
heme-Fe(III)-mediated peroxynitrite isomerization and
denitrosylation of tHSA–heme-Fe(II)-NO has been
investigated. The present data indicate, for the first
time, that the allosteric modulation of tHSA–heme
and HSA–heme reactivity by ibuprofen depends
mainly on drug bindingtothesecondarysites FA2
and FA6 rather than drug association with the FA3–
FA4 primary cleft.
Results
Ibuprofen bindingto tHSA–heme-Fe(III) and
HSA–heme-Fe(III)]
Figure 2A shows thebinding isotherm for ibuprofen
binding to tHSA–heme-Fe(III) and HSA–heme-Fe(III).
FA1
FA2
FA7
FA6
Glu382
FA3-FA4
FA5
Fig. 1. HSA structure. The six subdomains of HSA are colored as
follows: IA, blue; IB, cyan; IIA, dark green; IIB, light green; IIIA,
orange; IIIB, red. The heme (red) fits the primary cleft in subdo-
main IB, corresponding to FA1. Sudlow site I (in subdomain IIA,
corresponding to FA7) is occupied by warfarin (purple). Glu382 is
highlighted. Sudlow site II (in subdomain IIIA, corresponding to
FA3–FA4) and FA6 (in subdomain IIB) are occupied by ibuprofen
(magenta). Sites FA2 (at the subdomain I–IIA interface) and FA5 (in
subdomain IIIB) are occupied by myristate (orange). Atomic coordi-
nates were taken from Protein Data Bank entries 1O9X [40], 2BXD,
and 2BXG [6]. For details, see text.
Fig. 2. Ibuprofenbindingto tHSA–heme-Fe(III) and HSA–heme
Fe(III). (A) Thermodynamics ofibuprofenbindingto tHSA–heme-
Fe(III). The continuous line was calculated according to Eqn (1) by
non-linear regression curve fitting with the following parameters:
K
2
= (1.7 ± 0.2) · 10
)5
M; K
3
= (8.9 ± 0.9) · 10
)4
M; a = 0.18 ±
0.03; and 1 – a = 0.82 ± 0.04. [tHSA–heme-Fe(III)] = 1.9 · 10
)6
M.
(B) Thermodynamics ofibuprofenbindingto HSA–heme-Fe(III). The
continuous line was calculated according to Eqn (1) by non-linear
regression curve fitting with the following parameters: K
2
=
(6.9 ± 0.7) · 10
)6
M; K
3
= (8.1 ± 0.7) · 10
)4
M; a = 0.14 ± 0.03; and
1 ) a = 0.86 ± 0.04. [HSA–heme-Fe(III)] = 2.2 · 10
)6
M. The ibupro-
fen concentration corresponds to that ofthe free ligand. Where not
shown, the standard deviation is smaller than the symbol. All data
were obtained at pH 7.0 and 20.0 °C. For further details, see text.
A. di Masi et al. Ibuprofenbindingto truncated HSA secondary sites
FEBS Journal 278 (2011) 654–662 ª 2011 The Authors Journal compilation ª 2011 FEBS 655
The analysis ofthe data given in Fig. 2A,B, according
to Eqn (1), allowed the determination of K
2
and K
3
values for ibuprofenbindingto tHSA–heme-Fe(III)
(1.7 · 10
)5
and 8.9 · 10
)4
m, respectively) and to
HSA–heme-Fe(III) (6.9 · 10
)6
and 8.1 · 10
)4
m,
respectively). Thespectroscopic contributions of ibu-
profen bindingtothe high-affinity and low-affinity
sites of tHSA–heme-Fe(III) and HSA–heme-Fe(III)
[represented by a and (1 – a), respectively, in Eqn (1)]
are 0.18 and 0.82, and 0.14 and 0.86, respectively
(Table 1).
Effect ofibuprofen on peroxynitrite isomerization
by tHSA–heme-Fe(III)
In the absence and presence of tHSA–heme-Fe(III),
the kinetic data of peroxynitrite isomerization were fit-
ted to a single-exponential decay for more than 95%
of their course (Eqn 2). According tothe literature
[21], this indicates that the formation ofthe transient
tHSA–heme-Fe(III)-OONO species represents the
rate-limiting step in catalysis, the conversion of the
tHSA–heme-Fe(III)-OONO complex to tHSA–heme-
Fe(III) and NO
À
3
being faster by at least one order of
magnitude.
In the absence and presence of ibuprofen, the
observed rate constants for tHSA–heme-Fe(III)-cata-
lyzed isomerization of peroxynitrite (l
obs
) increased
linearly with the tHSA–heme-Fe(III) concentration
(Fig. 3A). The analysis of data reported in Fig. 3A,
according to Eqn (3), allowed the determination of val-
ues ofthe second-order rate constant for peroxynitrite
isomerization by tHSA–heme-Fe(III) (l
on
= 4.3 · 10
5
m
)1
Æs
)1
, corresponding tothe slope ofthe linear plots)
and ofthe first-order rate constant for peroxynitrite
isomerization in the absence of tHSA–heme-Fe(III)
(l
0
= 2.4 · 10
)1
s
)1
, corresponding tothe y-intercept
of the linear plots).
Ibuprofen dose-dependently impairs tHSA–heme-
Fe(III)-mediated isomerization of peroxynitrite
(Fig. 3A,B). Indeed, values of l
on
for tHSA–heme-
Fe(III)-catalyzed isomerization of peroxynitrite
decreased from 4.3 · 10
5
m
)1
Æs
)1
in the absence of ibu-
profen [l
on(top)
] to 5.8 · 10
4
m
)1
Æs
)1
at [ibupro-
fen] = 1.0 · 10
)2
m (Fig. 3A,B). On the other hand,
values of l
0
were unaffected by ibuprofen, being depen-
dent only on CO
2
(Fig. 3A,C). The values of l
on
and
l
0
for tHSA–heme-Fe(III)-catalyzed isomerization of
peroxynitrite determined here are very similar to those
previously reported for HSA–heme-Fe(III)-mediated
peroxynitrite isomerization [21].
The analysis ofthe dependence of l
on
on ibuprofen
concentration for tHSA–heme-Fe(III)-catalyzed isom-
erization of peroxynitrite (Fig. 3B), according to
Eqn (4), allowed determination ofthe value of the
dissociation equilibrium constant for ibuprofen binding
to tHSA–heme-Fe(III) (K
3
= 9.3 · 10
)4
m). Under
conditions where [ibuprofen] >> K
3
, tHSA–heme-
Fe(III) did not catalyze the isomerization of peroxy-
nitrite. Indeed, the value of l
obs
(2.6 · 10
)1
s
)1
) was
independent ofthe tHSA–heme-Fe(III) concentration,
and corresponded to that obtained in the absence of
tHSA–heme-Fe(III) (l
0
= 2.4 · 10
)1
s
)1
).
Ibuprofen bindingto tHSA–heme-Fe(II)-NO
Figure 4 shows thebinding isotherm for ibuprofen
binding to tHSA–heme-Fe(II)-NO. Analysis of the
dependence ofthe molar fraction ofthe ibuprofen-
bound tHSA–heme-Fe(II)-NO (Y) on the ibuprofen
concentration (Fig. 4), according to Eqn (5), allowed
to determine the value ofthe dissociation equilibrium
Table 1. Values ofthe dissociation equilibrium constants for ibuprofenbindingto tHSA–heme and HSA–heme derivatives obtained by differ-
ent experimental methods. For consistency with previous studies [20,21], equilibrium dissociation constants and Hill coefficients are indi-
cated as K
1
, K
2
, and K
3
, and as n
1
, n
2
, and n
3
, respectively.
Protein Experimental method K
1
(M) n
1
K
2
(M) n
2
K
3
(M) n
3
tHSA–heme-Fe(III)
a
Absorbance spectroscopy –
b
–
b
1.7 · 10
–5
0.98 8.9 · 10
–4
1.01
tHSA–heme-Fe(III)
a
Peroxynitrite isomerization –
b
–
b
–
b
–
b
9.3 · 10
–4
0.99
tHSA–heme-Fe(II)-NO
a
Absorbance spectroscopy –
b
–
b
–
b
–
b
2.1 · 10
–3
0.99
tHSA–heme-Fe(II)-NO
a
Denitrosylation kinetics –
b
–
b
1.3 · 10
–4
0.99 2.5 · 10
–3
1.01
HSA–heme-Fe(III)
a
Absorbance spectroscopy –
b
–
b
6.9 · 10
–6
1.00 8.1 · 10
–4
0.99
HSA–heme-Fe(III)
c
Peroxynitrite isomerization –
b
–
b
–
b
–
b
9.7 · 10
–4
–
d
HSA–heme-Fe(II)-NO
e
Absorbance spectroscopy –
b
–
b
–
b
–
b
2.6 · 10
–3
–
d
HSA–heme-Fe(II)-NO
e
Denitrosylation kinetics 3.1 · 10
–7
–
d
1.7 · 10
–4
–
d
2.2 · 10
–3
–
d
a
pH 7.0 and 20.0 °C. Present study.
b
Not applicable; see text.
c
pH 7.2 and 22.0 °C. From [21].
d
Although values of n
1
, n
2
and n
3
have not
been reported [20,21], the data fit to simple equilibria, implying n
1
=1,n
2
= 1, and n
3
=1.
e
pH 7.0 and 10.0 °C. From [20].
Ibuprofen bindingto truncated HSA secondarysites A. di Masi et al.
656 FEBS Journal 278 (2011) 654–662 ª 2011 The Authors Journal compilation ª 2011 FEBS
constant for drug bindingto tHSA–heme-Fe(II)-NO
(K
3
= 2.1 · 10
)3
m) (Fig. 4 and Table 1).
Effect ofibuprofen on tHSA–heme-Fe(II)-NO
denitrosylation
In the absence and presence of ibuprofen, the time
course for NO dissociation from tHSA–heme-Fe(II)-
NO conformed to a single-exponential decay for more
than 95% of its course (Eqn 6). According to the
literature [20], this indicates that the formation of
the transient tHSA–heme-Fe(II) species represents the
rate-limiting step in tHSA–heme-Fe(II)-CO formation.
Values ofthe first-order rate constant for NO dissocia-
tion from tHSA–heme-Fe(II)-NO (k
off
) were indepen-
dent of wavelength and [CO] in the presence of excess
dithionite.
Values of k
off
for tHSA–heme-Fe(II)-NO denitrosy-
lation increased from 1.5 · 10
)4
s
)1
in the absence of
ibuprofen (k
þ
off
in Eqn 7) to 8.5 · 10
)3
s
)1
in the pres-
ence of 1.0 · 10
)2
m ibuprofen (Fig. 5). The k
off
value
for tHSA–heme-Fe(II)-NO denitrosylation determined
here in the absence ofibuprofen is very similar to that
previously obtained for HSA–heme-Fe(II)-NO denitro-
sylation [20,22].
Analysis ofthe dependence of k
off
for tHSA–heme-
Fe(II)-NO denitrosylation on theibuprofen concentra-
tion (Fig. 5), according to Eqn (7), allowed us to
determine the values ofthe dissociation equilibrium
constants for drug bindingto tHSA–heme-Fe(II)-NO
(K
2
= 1.3 · 10
)4
m and K
3
= 2.5 · 10
)3
m) (Fig. 5).
The values of k
off(2)
and k
off(3)
for tHSA–heme-Fe(II)-
NO denitrosylation determined here (1.1 · 10
)3
and
9.1 · 10
)3
s
)1
, respectively) are very similar to those
previously obtained for HSA–heme-Fe(II)-NO denitro-
sylation [20].
Fig. 4. Ibuprofenbindingto tHSA–heme-Fe(II)-NO. The continuous
line was calculated according to Eqn (5) with K
3
= (2.1 ± 0.3) ·
10
)3
M). The tHSA–heme-Fe(II)-NO concentration was 4.8 · 10
)6
M.
Where not shown, the standard deviation is smaller than the
symbol. All data were obtained at pH 7.0 (1.0 · 10
)1
M phosphate
buffer) and 20.0 °C. For further details, see text.
Fig. 3. Ibuprofen inhibits peroxynitrite isomerization by tHSA–
heme-Fe(III). (A) Dependence ofthe pseudo-first-order rate
constant for peroxynitrite isomerization (l
obs
) on the tHSA–heme-
Fe(III) concentration. The peroxynitrite concentration was
2.5 · 10
)4
M. Theibuprofen concentrations were 0.0 M (trace a),
1.5 · 10
)3
M (trace b), and 7.5 · 10
)3
M (trace c). The continuous
lines in (A) were calculated according to Eqn (3) with the following
parameters: trace a, l
on
= (4.3 ± 0.4) · 10
5
M
)1
Æs
)1
and
l
0
= (2.4 ± 0.3) · 10
)1
s
)1
; trace b, l
on
= (1.8 ± 0.2) · 10
5
M
)1
Æs
)1
and
l
0
= (2.8 ± 0.3) · 10
)1
s
)1
; and trace c, l
on
= (6.8 ± 0.4) · 10
4
M
)1
Æs
)1
and l
0
= (2.7 ± 0.3) · 10
)1
s
)1
. (B) Effect ofibuprofen concentration
on the second-order rate constant for tHSA–heme-Fe(III)-catalyzed
isomerization of peroxynitrite (l
on
). The filled symbol on the ordinate
indicates the l
on
value obtained in the absence ofibuprofen [l
on(-
top)
= (4.3 ± 0.4) · 10
5
M
)1
Æs
)1
]. The continuous line was calculated
according to Eqn (4) with l
on(top)
= (4.3 ± 0.4) · 10
5
M
)1
Æs
)1
and
K
3
= (9.3 ± 1.0) · 10
)4
M. (C) Effect ofibuprofen concentration on
the first-order rate constant for tHSA–heme-Fe(III)-catalyzed isomer-
ization of peroxynitrite (l
0
). The filled symbol on the ordinate indi-
cates the l
0
value obtained in the absence of ibuprofen
[(2.4 ± 0.3) · 10
)1
s
)1
]. Values of l
0
are independent of ibuprofen
concentration; the average l
0
value is (2.6 ± 0.3) · 10
)1
s
)1
. Where
not shown, the standard deviation is smaller than the symbol. All
data were obtained at pH 7.0 (1.0 · 10
)1
M phosphate buffer) and
20.0 °C. For further details, see text.
A. di Masi et al. Ibuprofenbindingto truncated HSA secondary sites
FEBS Journal 278 (2011) 654–662 ª 2011 The Authors Journal compilation ª 2011 FEBS 657
Discussion
The present results highlight the rol e ofibuprofen second-
ary sites in modulating HSA–heme spectroscopic p roper-
ties and reactivity. Indeed, values ofthe diss ociation
equilibrium constants for ibuprofen b inding to secon dary
sites present in tHSA–heme (present study) are in excel-
lent agreement with those for drug bindingto full-
length HSA–heme (Table 1). However, the abse nce of
domain III in tH SA–heme precludes thebinding of
ibuprofen to its primary clef t [19–21] (Table 1).
The analysis of thermodynamic parameters reported
in Table 1 allows the following conclusions to be drawn.
l
Ibuprofen binds to two secondarysitesof tHSA–
heme-Fe(III) and HSA–heme-Fe(III) (present study),
affecting the absorption spectra. However, only ibupro-
fen bindingtothe (t)HSA–heme-Fe(III) secondary site
showing the lowest drug affinity allosterically modulates
peroxynitrite isomerization (present study and [21]).
l
Ibuprofen affects the absorption spectra of tHSA–
heme-Fe(II)-NO (present study) and HSA–heme-
Fe(II)-NO [20] by bindingto only one secondary site.
However, the allosteric modulation of tHSA–heme-
Fe(II)-NO denitrosylation (present study) reflects ibu-
profen bindingto two secondary sites. By contrast,
HSA–heme-Fe(II)-NO denitrosylation is allosterically
modulated by ibuprofenbinding not only to both
secondary sites located in domains I and II, but also
to the primary cleft sited in domain III [20].
l
Values of K
2
and K
3
for ibuprofenbinding to
tHSA–heme-Fe(III) and HSA–heme-Fe(III) (present
study) are lower than those for drug bindingto tHSA–
heme-Fe(II)-NO (present study) and HSA–heme-
Fe(II)-NO [20]. This indicates that the redox and the
(un)ligated state ofthe heme Fe atom allosterically
affects ibuprofenbindingto (t) HSA–heme.
l
Values of K
2
and K
3
for ibuprofenbinding to
tHSA–heme-Fe (present study) are in excellent agree-
ment with those reported for drug bindingto HSA–
heme species (present study and [20,21]). Also, the
kinetic parameters for ibuprofen-mediated peroxyni-
trite isomerization by (t)HSA–heme-Fe(III) and for
(t)HSA–heme-Fe(II)-NO denitrosylation are very simi-
lar (present study and [20,21]). This indicates that the
removal of domain I from HSA does not significantly
affect the functional and structural properties of
domains I and II [i.e. ofthe 1–382 region of (t)HSA].
l
The values ofthe Hill coefficients (n
1
, n
2
, and n
3
) for
ibuprofen bindingto tHSA–heme and HSA–heme
derivatives range between 0.98 and 1.01 (present study
Table 1), indicating that drug association with (t)HSA–
heme is a non-cooperative event, under all the experi-
mental conditions.
l
The ibuprofen-dependent (t)HSA–heme-Fe(III)-
mediated peroxynitrite isomerization and (t)HSA–
heme-Fe(II)-NO denitrosylation reflect drug-dependent
structural changes occurring at the heme-binding
pocket (FA1). Indeed, ibuprofenbindingto HSA–heme
has been reported to induce the hexa-coordination of
the heme Fe atom [18,21,23]. Thus, peroxynitrite can-
not bind tothe heme Fe(III) atom of hexa-coordinated
HSA–heme-Fe(III), and therefore cannot undergo facil-
itated isomerization [21]. Moreover, the increase in k
off
for NO dissociation from HSA–heme-Fe(II)-NO upon
stabilization ofthe hexa-coordinated heme-Fe(II)-NO
atom is reminiscent of what has been reported
for abacavir-induced and warfarin-induced hexa-
coordination of HSA–heme-Fe(II)-NO [22], and for
1-methyl-imidazole-mediated hexa-coordination of the
heme-Fe(II)-NO model compound [24].
The ibuprofen primary binding cleft (i.e. Sudlow
site II, formed by the FA3 and FA4 sites) and the
drug secondary pocket (i.e. the FA6 region) have been
substantiated by X-ray crystallography [6]. The third
low-affinity ibuprofen-binding site has been tentatively
identified with the FA2 site. Indeed, it acts as
the modulatory site that controls the FA-induced
conformational switch [18,21,25,26]. Ibuprofen binding
to the third site induces the stabilization of the
hexa-coordinate derivative ofthe HSA–heme-Fe(III)
and HSA–heme-Fe(II)-NO species, which are instead
predominantly penta-coordinated in the absence of
allosteric effectors. Indeed, high (> 1.0 · 10
)3
m) ibu-
profen concentration clearly induces the coordination
of a histidine nitrogen as the sixth coordination posi-
Fig. 5. Effect ofibuprofen on tHSA–heme-Fe(II)-NO denitrosylation.
The continuous line was calculated according to Eqn (7) with
k
off(2)
= (1.1 ± 0.1) · 10
)3
s
)1
, K
2
= (1.3 ± 0.2) · 10
)4
M, k
off(3)
=
(9.1 ± 1.0) · 10
)3
s
)1
, K
3
= (2.5 ± 0.3) · 10
)3
M), and k
þ
off
=
(1.5 ± 0.2) · 10
)4
s
)1
. The tHSA–heme-Fe(II)-NO concentration was
2.9 · 10
)6
M. Where not shown, the standard deviation is smaller
than the symbol. All data were obtained at pH 7.0 (1.0 · 10
)1
M
phosphate buffer) and 20.0 °C. For further details, see text.
Ibuprofen bindingto truncated HSA secondarysites A. di Masi et al.
658 FEBS Journal 278 (2011) 654–662 ª 2011 The Authors Journal compilation ª 2011 FEBS
tion ofthe heme Fe, as the result ofthe conforma-
tional transition following ligand bindingto FA2 [18].
The present considerations appear to apply only to
ibuprofen, as other Sudlow site II ligands, such as FAs,
diazepam, and diflunisal, display different binding
properties. FAs bind to multiple sites with different
affinities and functional effects. In particular, the FA6
(corresponding tothe highest-affinity site for ibuprofen
in tHSA) andthe FA7 (also named Sudlow site I and
corresponding tothe warfarin-binding pocket) sites
show low affinity for FAs, whereas the FA1 (also named
the heme site) andthe FA2 (corresponding to the
regulatory site ofthe neutral-to-basic conformational
transition) pockets display high affinity for FAs [17].
Therefore, the effect of FAs is opposite to that of
ibuprofen [8]. Moreover, FA bindingtothe FA1 site
induces heme dissociation from HSA–heme, thus
impairing HSA–heme reactivity [25]. Diazepam has
been reported to bind only to Sudlow site II [17], so it
does not bind to tHSA, which is deprived of domain III
(Fig. 1). Finally, the FA6 site is also thesecondary bind-
ing cleft for diflunisal, another Sudlow site II ligand.
However, diflunisal binds not only tothe FA6 site
(corresponding tothe highest-affinity site for ibuprofen
in tHSA), but also tothe FA7 cleft (corresponding to
the warfarin pocket) [17], thus inducing mixed effects,
combining the actions ofibuprofenand warfarin [8].
It is worth noting that the modular architecture of
HSA allows the removal of domain III without this
affecting the conformational stability ofthe remaining
protein scaffold. Moreover, lost contacts between par-
alogous HSA domains do not impair the correct fold-
ing of single domains, as shown by the agreement
between thermodynamic and kinetic parameters in
HSA and tHSA derivatives. As a whole, the present
data: (a) demonstrate unequivocally, for the first time,
that ibuprofenallostericallymodulatesthe spectro-
scopic and reactivity propertiesof tHSA–heme and
HSA–heme by bindingtothe low-affinity secondary
sites FA2 and FA6 rather than associating with the
primary-high affinity cleft FA3–FA4; and (b) reinforce
the idea that HSA could be taken as the prototype of
monomeric allosteric proteins [8,19,27,28]. Finally,
tHSA is a valuable model with which to investigate the
allosteric linkage between ligand-binding pockets
located in domains I and II of HSA.
Experimental procedures
Chemicals
HSA (‡ 96%, essentially FA-free), hemin [Fe(III)-proto-
porphyrin IX] chloride andibuprofen were purchased
from Sigma-Aldrich (St Louis, MO, USA). Recombinant
tHSA was expressed and purified as previously reported
[19]. NO (Aldrich Chemical Co., Milwaukee, WI,
USA) was purified by flowing it through an NaOH
column in order to remove acidic nitrogen oxides. CO
was purchased from Linde AG (Ho
¨
llriegelskreuth,
Germany).
tHSA–heme-Fe(III) and HSA–heme-Fe(III) were pre-
pared by adding a 0.8 mol heme-Fe(III) per mol tHSA and
HSA (1.0 · 10
)1
m sodium phosphate buffer, pH 7.2) at
20.0 °C [19–21]. The final tHSA–heme-Fe(III) and HSA–
heme-Fe(III) concentrations ranged between 1.9 · 10
)6
and
5.0 · 10
)5
m, and between 2.2 · 10
)6
and 5.0 · 10
)5
m,
respectively. tHSA–heme-Fe(II)-NO (final concentration,
4.8 · 10
)6
m) was obtained, under anaerobic conditions, by
blowing purified NO over the ferrous heme–protein solu-
tion (1.0 · 10
)1
m sodium phosphate buffer, pH 7.0) at
10.0 °C [20].
The ibuprofen stock solution (1.0 · 10
)1
m) was prepared
by dissolving the drug in 1.0 · 10
)1
m phosphate buffer
(pH 7.0) at 20.0 °C [23]. The final ibuprofen concentration
ranged between 1.0 · 10
)6
and 1.0 · 10
)2
m.
Peroxynitrite was synthesized from KO
2
and NO or from
HNO
2
and H
2
O
2
, and stored in small aliquots at )80.0 °C
[29,30]. The peroxynitrite stock solution (2.0 · 10
)3
m) was
diluted immediately before use with degassed 5.0 · 10
)2
m
NaOH to achieve the desired concentration [21,31–35].
Nitrate and nitrite contamination levels were in the ranges
of 0–7% and 8–19% ofthe peroxynitrite concentration,
respectively [21]. The concentration of peroxynitrite was
determined spectrophotometrically prior to each experiment
by measuring the absorbance at 302 nm (e
302 nm
= 1.705 ·
10
3
m
)1
Æcm
)1
) [29,30].
The CO solution was prepared by keeping the
1.0 · 10
)1
m phosphate buffer solution (pH 7.0) in a
closed vessel with CO at a pressure of 760.0 mmHg, under
anaerobic conditions, at 20.0 °C [20,36].
All ofthe other chemicals were obtained from Sigma-
Aldrich and Merck AG (Darmstadt, Germany). All prod-
ucts were of analytical or reagent grade, and were used
without further purification.
Ibuprofen bindingto tHSA–heme-Fe(III) and
HSA–heme-Fe(III)
Values ofthe dissociation equilibrium constants for ibupro-
fen bindingto tHSA–heme-Fe(III) and HSA–heme-Fe(III)
(i.e. K
2
and K
3
) were obtained spectrophotometrically,
at pH 7.0 (1.0 · 10
)1
m phosphate buffer) and 20.0 °C.
Ibuprofen-dependent absorbance changes were recorded
between 350 and 450 nm. Small aliquots ofthe ibuprofen
(1.0 · 10
)1
m) stock solution were added tothe tHSA–
heme-Fe(III) (1.9 · 10
)6
m) and HSA–heme-Fe(III)
(2.2 · 10
)6
m) solutions, andthe ibuprofen-dependent
absorbance changes of tHSA–heme-Fe(III) and HSA–
A. di Masi et al. Ibuprofenbindingto truncated HSA secondary sites
FEBS Journal 278 (2011) 654–662 ª 2011 The Authors Journal compilation ª 2011 FEBS 659
heme-Fe(III) were recorded after incubation for 10 min,
after each addition [19,21]. Test measurements performed
after 2 h excluded slow kinetic events.
Ibuprofen bindingto tHSA–heme-Fe(III) and HSA–
heme-Fe(III) was analyzed by plotting the molar fraction of
drug–tHSA–heme-Fe(III) and drug–HSA–heme-Fe(III)
complexes (Y) as a function ofthe free ibuprofen concen-
tration (ranging between 1.0 · 10
)6
m and 1.0 · 10
)2
m).
Data were analyzed according to Eqn (1) [36]:
Y ¼½a Âf½ibuprofen=ðK
2
þ½ibuprofenÞg
þ½ð1 À aÞÂf½ibuprofen=ðK
3
þ½ibuprofenÞg
ð1Þ
where a and 1 ) a are the relative spectroscopic contri-
butions tothe total electronic absorbance change of ibu-
profen bindingtothe high-affinity and low-affinity sites,
respectively.
Effect ofibuprofen on peroxynitrite isomerization
by tHSA–heme-Fe(III)
Kinetic data for peroxynitrite isomerization in the absence
and presence of tHSA–heme-Fe(III) andibuprofen were
recorded with the SMF-100 rapid-mixing stopped-flow
apparatus (Bio-Logic SAS, Claix, France). The light path
of the observation cuvette was 10 mm, andthe dead time
was 1.4 ms. The kinetics were monitored at 302 nm, the
characteristic absorbance maximum of peroxynitrite
(e
302 nm
= 1.705 · 10
3
m
)1
Æcm
)1
) [29,30,35,37]. Kinetic data
were obtained in the absence and presence of tHSA–heme-
Fe(III) (final concentration, 5.0 · 10
)6
to 5.0 · 10
)5
m) and
ibuprofen (final concentration, 1.0 · 10
)6
to 1.0 · 10
)2
m),
by rapid mixing ofthe protein-buffered solution with the
peroxynitrite solution (final concentration, 2.5 · 10
)4
m).
Kinetic data were obtained at pH 7.0 (1.0 · 10
)1
m
phosphate buffer) and 20.0 °C; no gaseous phase was
present [21].
The kinetics of peroxynitrite isomerization by tHSA–
heme-Fe(III), in the absence and presence of ibuprofen,
were analyzed in the framework ofthe following minimum
reaction scheme [21]:
tHSAÀhemeÀFeðIIIÞþHOONO
!
l
on
tHSAÀhemeÀFeðIIIÞÀ OONO þ H
þ
tHSAÀhemeÀFeðIIIÞÀOONO
!
fast
tHSAÀhemeÀFeðIIIÞþNO
3
À
Values ofthe pseudo-first-order rate constant for tHSA–
heme-Fe(III)-mediated peroxynitrite isomerization (l
obs
)
were determined, in the absence and presence of ibuprofen,
at pH 7.0 (1.0 · 10
)1
m phosphate buffer) and 20.0 °C,
from the analysis ofthe time-dependent absorbance
decrease at 302 nm, according to Eqn (2) [21]:
½peroxynitrite
t
¼½peroxynitrite
i
 e
Àl
obs
Ât
ð2Þ
Values ofthe second-order rate constant for tHSA–
heme-Fe(III)-mediated peroxynitrite isomerization (l
on
) and
of the first-order rate constant for peroxynitrite isomeriza-
tion in the absence of tHSA–heme-Fe(III) (l
0
) were deter-
mined, in the absence and presence of ibuprofen, at pH 7.0
and 20.0 °C, from the linear dependence of l
obs
on the
tHSA–heme-Fe(III) concentration, according to Eqn (3)
[21,32,38,39]:
l
obs
¼ l
on
½tHSAÀhemeÀFeðIIIÞ þ l
0
ð3Þ
The value ofthe dissociation equilibrium constant for
ibuprofen bindingto tHSA–heme-Fe(III) (K
3
) was deter-
mined, at pH 7.0 (1.0 · 10
)1
m phosphate buffer) and
20.0 °C, from the dependence of l
on
on the free drug con-
centration (ranging between 1.0 · 10
)6
and 1.0 · 10
)2
m).
The effect ofthe drug concentration on l
on
was analyzed
according to Eqn (4) [21,32,38,39]:
l
on
¼ l
onðtopÞ
Àfðl
onðtopÞ
½ibuprofenÞ = ðK
3
þ½ibuprofenÞg ð4Þ
where l
on(top)
represents the asymptotic value of l
on
under
conditions where [ibuprofen] = 0 (i.e. l
on(top)
= l
on
).
Ibuprofen bindingto tHSA–heme-Fe(II)-NO
The value ofthe dissociation equilibrium constant for ibu-
profen bindingto tHSA–heme-Fe(II)-NO (K
3
) was deter-
mined spectrophotometrically, at pH 7.0 (1.0 · 10
)1
m
phosphate buffer) and 20.0 °C. Ibuprofen-dependent absor-
bance changes were recorded between 350 and 450 nm. Small
aliquots oftheibuprofen (1.0 · 10
)1
m) stock solution were
added tothe tHSA–heme-Fe(II)-NO (4.8 · 10
)6
m) solution,
and the ibuprofen-dependent absorbance changes of tHSA–
heme-Fe(II)-NO were recorded after incubation for 10 min,
after each addition [20]. Test measurements performed after
2 h excluded slow kinetic events.
Ibuprofen bindingto tHSA–heme-Fe(III) was analyzed by
plotting the molar fraction ofthe drug–tHSA–heme-Fe(II)-
NO complex (Y) as a function ofthe free ibuprofen concen-
tration (ranging between 1.0 · 10
)6
and 1.0 · 10
)2
m). Data
were analyzed according to Eqn (5) [20]:
Y ¼½ibuprofen=ðK
3
þ½ibuprofenÞ ð5Þ
Effect ofibuprofen on tHSA–heme-Fe(II)-NO
denitrosylation
Values ofthe first-order rate constant for NO dissociation
from tHSA–heme-Fe(II)-NO (i.e. for NO replacement by
CO; k
off
) were obtained by mixing the tHSA–heme-Fe(II)-
NO (final concentration, 2.9 · 10
)6
m) solution with the CO
(final concentration, 1.0 · 10
)4
to 5.0 · 10
)4
m) solution in
the presence of dithionite (final concentration, 1.0 · 10
)2
m),
under anaerobic conditions, at pH 7.0 (1.0 · 10
)1
m sodium
Ibuprofen bindingto truncated HSA secondarysites A. di Masi et al.
660 FEBS Journal 278 (2011) 654–662 ª 2011 The Authors Journal compilation ª 2011 FEBS
phosphate buffer) and 20.0 °C [20], in the absence and pres-
ence ofibuprofen (final concentration, 1.0 · 10
)7
to
1.0 · 10
)2
m). The excess of NO was pumped off gently
before recording of ligand dissociation kinetics [20].
The kinetics were monitored between 350 and 460 nm.
Absorbance electronic spectra were collected every 30 s.
The time course for tHSA–heme-Fe(II)-NO denitrosylation
was fitted to a single-exponential process according to the
minimum reaction mechanism represented by the following
scheme [20]:
tHSAÀhemeÀFeðIIÞÀNO þ CO
!
k
off
tHSAÀhemeÀFeðIIÞÀCO þ NO
Values of k
off
were determined from data analysis
according to Eqn (6) [20]:
½HSAÀhemeÀFe ðIIÞÀNO
t
¼½HSAÀhemeÀFeðIIÞÀNO
i
 e
Àk
off
Ât
ð6Þ
Values ofthe dissociation equilibrium constants for ibu-
profen bindingto tHSA–heme-Fe(II)-NO (K
2
and K
3
) were
obtained from the dependence of k
off
on the free ibuprofen
concentration. Values of K
2
and K
3
were determined from
data analysis, according to Eqn (7) [20]:
k
off
¼ðk
offð2Þ
Âð½ibuprofen=ðK
2
½ibuprofenÞ
þðk
offð3Þ
Âð½ibuprofen=ðK
3
½ibuprofenÞÞ þ k
þ
off
ð7Þ
where k
off(2)
and k
off(3)
indicate values of k
off
occurring at
K
2
< [ibuprofen] < K
3
, and at K
2
< K
3
< [ibuprofen],
respectively, and k
þ
off
is the k
off
value obtained in the
absence of ibuprofen.
Data analysis
Kinetic and thermodynamic data were analyzed with the
matlab program (The Math Works, Natick, MA, USA).
The results are given as mean values of at least four experi-
ments plus or minus the corresponding standard deviation.
Acknowledgements
This work was partially supported by grants from
the Ministero dell’Istruzione, dell’Universita
`
e della
Ricerca of Italy (PRIN 2007ECX29E_002 and Univer-
sity Roma Tre, CLAR 2009, to P. Ascenzi).
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662 FEBS Journal 278 (2011) 654–662 ª 2011 The Authors Journal compilation ª 2011 FEBS
. Ibuprofen binding to secondary sites allosterically
modulates the spectroscopic and catalytic properties
of human serum heme–albumin
Alessandra di. according to Eqn (7) [20]:
k
off
¼ðk
offð2Þ
Âð ibuprofen =ðK
2
 ibuprofen Þ
þðk
offð3Þ
Âð ibuprofen =ðK
3
 ibuprofen ÞÞ þ k
þ
off
ð7Þ
where k
off(2)
and k
off(3)
indicate