Allosteric modulation of myristate and Mn(III)heme
binding to human serum albumin
Optical and NMR spectroscopy characterization
Gabriella Fanali
1
, Riccardo Fesce
1
, Cristina Agrati
1
, Paolo Ascenzi
2,3
and Mauro Fasano
1
1 Dipartimento di Biologia Strutturale e Funzionale, and Centro di Neuroscienze, Universita
`
dell’Insubria, Busto Arsizio (VA), Italy
2 Dipartimento di Biologia, and Laboratorio Interdisciplinare di Microscopia Elettronica, Universita
`
‘Roma Tre’, Italy
3 Istituto Nazionale per le Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani’, Roma, Italy
Human serum albumin (HSA) is the most prominent
protein in plasma, but it is also found in tissues and
secretions throughout the body. HSA abundance (its
concentration being 45 mgÆmL
)1
in the serum of
human adults) contributes significantly to colloid-
osmotic blood pressure. HSA, best known for its
extraordinary ligand binding capacity, is constituted
by a single nonglycosylated all-a chain of 65 kDa con-
taining three homologous domains (labelled I, II, and

the heme site, not only increases optical absorbance of Mn(III)heme-bound
HSA by a factor of approximately three, but also increases the Mn(III)-
heme affinity for the fatty acid binding site FA1 by 10–500-fold. Cooper-
ative binding appears to occur at FAx and accessory myristate binding
sites. The conformational changes of the Mn(III)heme–HSA tertiary struc-
ture allosterically induced by myristate are associated with a noticeable
change in both optical absorbance and NMR spectroscopic properties of
Mn(III)heme–HSA, allowing the Mn(III)-coordinated water molecule to
exchange with the solvent bulk. At pH ¼ 10.0 both myristate affinity for
FAx and allosteric modulation of FA1 are reduced, whereas cooperation
of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme
binds to HSA with higher affinity than at pH 7.0 even in the absence of
myristate, and the metal-coordinated water molecule is displaced. As a
whole, these results suggest that FA binding promotes conformational
changes reminiscent of N to B state HSA transition, and appear of general
significance for a deeper understanding of the allosteric modulation of
ligand binding properties of HSA.
Abbreviations
FA, fatty acid; HSA, human serum albumin; MSE, mean square error; NMRD, nuclear magnetic relaxation dispersion.
4672 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS
responsible for the transport of lipophilic compounds
and drugs and of medium and long chain fatty acids;
among them, myristic acid is a stereotypic ligand to
investigate fatty acid binding and transport properties
of HSA [1–8].
Fatty acids (FAs) are required for the synthesis of
membrane lipids, hormones and second messengers,
and serve as an important source of metabolic
energy. Although the binding of fatty acids to human
and bovine serum albumin has been thoroughly inves-

bridges with His146 and Lys190 residues [6,8].
HSA undergoes pH- and allosteric effector-depend-
ent reversible conformational isomerization(s). Between
pH 2.7 and 4.3, HSA shows a fast (F) form, character-
ized by a dramatic increase in viscosity, much lower
solubility, and a significant loss in helical content.
Between pH 4.3 and 8, in the absence of allosteric
effectors, HSA displays the normal (N) form that is
characterized by heart-shaped structure. Between
pH 4.3 and 8, in the presence of allosteric effectors,
and at pH greater than 8, in the absence of ligands,
HSA changes conformation to the basic (B) form with
loss of a-helix and an increased affinity for some lig-
ands, such as warfarin [5,18–23].
Fatty acids are effective in allosterically regulating
ligand binding to Sudlow’s site I and to the heme cleft.
Myristate regulates HSA binding properties in a com-
plex manner, involving both competitive and allosteric
mechanisms. The structural changes associated with
FAs binding can essentially be regarded as relative
domain rearrangements to the I-II and II-III inter-
faces. This allosteric regulation is not observed for short
FAs (e.g. octanoate) that preferably bind to Sudlow’s
site II and displace the specific ligands (e.g. ibuprofen)
without inducing HSA allosteric rearrangement(s).
This indicates that the hydrophobic interactions
between the long FA polymethylenic tail and HSA
drives allosteric rearrangements. In turn, Sudlow’s site
I ligands (e.g. warfarin) displace FA7, while Sudlow’s
site II ligands (e.g. ibuprofen) displace FA3 and FA4.

the basic (B) state of HSA [5,18,23,29–31].
Here, we report the spectroscopic analysis of the
myristate-dependent conformational changes of the N
and B states of Mn(III)heme–HSA, by optical absorb-
ance spectroscopy and NMR spectroscopy, that show
allosteric interaction(s) between FAs and Mn(III)heme
with HSA. Interestingly, FAs increase Mn(III)heme
affinity to HSA, whereas warfarin and FA7 ligands
were reported to behave in the opposite way with
respect to ferric heme binding to HSA [19,21,31].
Additionally, the affinity of Mn(III)heme for HSA and
the spectroscopic properties of the Mn(III)heme–HSA
adduct in the presence of myristate are similar to those
of the B conformational state of HSA, suggesting that
myristate binding to one or more modulatory sites
possibly drives the N to B state HSA transition.
Results
In the absence of myristate, at pH 7.0 (i.e. HSA in the
N conformational state), Mn(III)heme binds to fatty
acid-free HSA with a dissociation constant K
H
%
2.0 · 10
)5
m (Fig. 2A). Although the binding curve
does not reach saturation and therefore the K
H
value
should be considered as a lower limit, it is worth to
note that it is two order of magnitude larger than that

based on the molar fraction of the Mn(III)heme–HSA
adduct that gave similar spectral data at pH 10.0 (see
below). Furthermore, (c) at intermediate HSA concen-
tration the binding curves rapidly rise and appear to
Fig. 2. (A) Binding isotherms for Mn(III)heme binding to fatty acid-
free HSA and to the HSA–myristate complexes, at pH 7.0 and
25.0 °C; open triangles: no myristate; solid triangles: 5.0 · 10
)6
M
myristate; open circles: 1.0 · 10
)5
M myristate; solid circles:
2.5 · 10
)5
M myristate; crossed diamonds: 5.0 · 10
)5
M myri-
state; open diamonds: 7.5 · 10
)5
M myristate; solid diamonds:
1.0 · 10
)4
M myristate. The continuous lines were obtained by
numerical fitting of the data. Values of the dissociation equilibrium
constants obtained according to Scheme 1 are given in Table 1.
(B) UV-visible spectral changes observed for a solution of
1.0 · 10
)5
M Mn(III)heme titrated with HSA (0–3.0 · 10
)5

*,
K
H
M
, and possibly K
M
M1
K
M
, if myristate binding to
FA1 is also modulated; see Experimental procedures
for explanation of the notations for the equilibrium
constants). However, binding of myristate to addi-
tional FA sites must also be considered, to take into
account the decrease in free myristate concentration at
increasing concentrations of HSA; this requires the
further set of parameters K
M
S1
to K
M
S5
. For the sake
of simplicity, these constants were bound to a fixed
affinity ratio series, with K
M
S1
as a free parameter and
K
M

*). However, this
simplified model did not adequately fit the experimen-
tal data (MSE ¼ 4.9 · 10
)5
); in particular, it could not
reproduce the peak followed by partial decline
observed at intermediate myristate concentrations, par-
ticularly evident for 1.0–5.0 · 10
)4
m myristate, and in
general the right part of the curves (at high [HSA]). In
order to qualitatively reproduce this feature, positive
cooperation must be introduced between at least one
of the additional FA binding sites and FAx, so that
the Mn(III)heme–HSA–myristate adduct releases myri-
state from FAx, as free myristate concentration van-
ishes, and the optical absorbance signal declines.
Several sets of parameters gave good fits to the
experimental data, yielding almost identical curves
(MSE ¼ 3.0 ± 0.1 · 10
)5
): an example of a set of fit-
ting curves is displayed as continuous lines in Fig. 2A.
All these solutions indicate a value for K
M
S1
in the
range between 1.5 · 10
)6
and 3.0 · 10

(Scheme 1) are reported in Table 1 for two nicely
(Myr)P(…)–(…)
K
M
*
↔ (Myr)P(Myr)–(…)
K
M
Sn
↔
↔
(Myr)P(Myr)–(Myr)
n
K
M
K
M
M
K
M
(…)P(…)–(…)
K
M
*
↔ (…)P(Myr)–(…)
K
M
Sn
↔ (…)P(Myr)–(Myr)
n

depending on FAx binding (K
M
M
¼ K
M
), or a similar
change in FA1 affinity for both Mn(III)heme and myr-
istate (K
M
M
⁄ K
M
¼ K
H
M
⁄ K
H
); in both cases 100-fold
decrease was assumed in K
M
* when additional FA site
no. 3 releases myristate. By inspection of the model
parameters (Table 1) it is clear that the assumptions
strongly affect the estimated affinity of FAx for myri-
state (K
M
*) and, as a consequence, the magnitude of
the allosteric modulation of FA1 (K
H
M

H
) increases by at least one order of
magnitude with respect to pH 7.0, but both FAx
affinity and allosteric modulation of FA1 are reduced.
(b) Cooperation of accessory sites and FAx is almost
unaffected. Finally, (c) the asymptotic absorbance of
the Mn(III)heme–HSA complex (A
10
) becomes com-
parable to that of the Mn(III)heme–HSA–(FAx +
myristate) complex (A
10
*), and the latter is not
altered by the change in pH. Again, the occurrence
of well-defined isosbestic points indicate that the
binding equilibrium occurs through only two forms,
the HSA-free and the HSA-bound Mn(III)heme
(Fig. 3B).
Table 1. Values of the thermodynamic dissociation constants (M) for myristate and Mn(III)heme binding to HSA at pH 7.0 and 10.0 (Scheme 1
and see text). Assumptions:
a
K
M
M
¼ K
M
. K
M
* · 100 for unoccupied FAS3.
b

)5
1.1 · 10
)6
K
M
(FA1 + Myr) 3.4 · 10
)7
1.1 · 10
)6
7.2 · 10
)6
1.3 · 10
)5
K
H
M
[FA1 + Heme (FAx bound)] 8.2 · 10
)7
9.3 · 10
)8
8.5 · 10
)8
7.9 · 10
)7
K
M
M
[FA1 + Myr (FAx bound)] 3.4 · 10
)7
1.1 · 10

10
* [Asympt. DA (+ Myr)] 0.030 0.028 0.030 0.028
Mean square error 2.9 · 10
)5
9.2 · 10
)6
2.8 · 10
)5
8.8 · 10
)6
Fig. 3. (A) Binding isotherms for Mn(III)heme binding to fatty acid-
free HSA and to the HSA–myristate complex, at pH 10.0 and
25.0 °C; open triangles: no myristate; solid diamonds: 1.0 · 10
)4
M
myristate. The continuous lines were obtained by numerical fitting
of the data. Values of the dissociation equilibrium constants
obtained according to Scheme 1 are given in Table 1. (B) UV-visible
spectral changes observed for a solution of 1.0 · 10
)5
M Mn(III)-
heme titrated with HSA (0–3.0 · 10
)5
M) in the presence of
1.0 · 10
)4
M myristate, at pH 10.0 and 25.0 °C. The arrows indicate
the increase of HSA concentration.
Myristate and Mn(III)heme binding to HSA–heme G. Fanali et al .
4676 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS

affected, for Mn(III)heme–HSA and Mn(III)heme–
HSA-myristate at 1 : 1 : 3, 1 : 1 : 4.5, and 1 : 1 : 6
molar ratios. Values of pK for the three titration steps,
obtained at the different Mn(III)heme–HSA-myristate
ratios, have been determined using Eqn (2) (Experimen-
tal procedures; Table 2).
Fig. 4. (A) Absorbance change measured at 440 nm for a solution
of 1.0 · 10
)5
M Mn(III)heme–HSA as a function of myristate con-
centration. Data were obtained at pH 7.0 and 25.0 °C. (B) UV-visible
absorption spectra of a solution of 1.0 · 10
)5
M Mn(III)heme–HSA
in the absence (continuous line) and in the presence of
4.5 · 10
)5
M (dotted line) and 1.0 · 10
)4
M myristate (dashed line).
Fig. 5. Change of the relaxivity measured at 10 MHz of a
1.0 · 10
)3
M solution of Mn(III)heme–HSA as a function of myri-
state concentration. Data were obtained at pH 7.0 and 25.0 °C.
Fig. 6. Water proton relaxation rates measured at 10 MHz and
25.0 °C, as functions of pH, for fatty acid-free Mn(III)heme–HSA
(solid squares), Mn(III)heme–HSA–myristate at 1 : 1 : 3 (solid tri-
angles), 1 : 1 : 4.5 (open diamonds), and 1 : 1 : 6 molar ratios (open
circles). The continuous lines were calculated according to Eqn (2).

profiles of Mn(III)heme–HSA as a function of myri-
state concentration were also measured at pH 10.0 in
order to check whether any change occurred for the B
state of HSA as well. As shown in Fig. 7, the NMRD
profiles of Mn(III)heme–HSA at pH 10.0 do not
appear to be affected by myristate.
Optical absorbance spectra are suggestive of different
coordination modes of Mn(III)heme in the different
conformational states of HSA [35], therefore we meas-
ured the paramagnetic contribution to the
17
O-NMR
linewidth at pH 7.0 and 10.0 as a function of myristate
concentration (Fig. 8). For paramagnetic metallopro-
teins, the width of the
17
O NMR resonance is affected
by the presence of the paramagnetic metal through the
exchange of water molecules directly coordinated to the
metal center, according to Eqn (3) (Experimental pro-
cedures) [20]. Unlike protons,
17
O nuclei are negligibly
affected by dipolar coupling with nearby unpaired
electrons, and the paramagnetic broadening of the
17
O
resonance is diagnostic of the occurrence of a direct
coordination bond between water and Mn(III) [20,36].
Table 2. pK values of pH-dependent water proton relaxation rates

Myristate and Mn(III)heme binding to HSA–heme G. Fanali et al .
4678 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS
As shown in Fig. 8, the linewidth change is significant
(about 20 Hz) for the protein in the N state, and
becomes larger (about 40 Hz) in the presence of satur-
ating concentration of myristate. On the other hand,
this contribution is almost negligible for HSA in the B
state, and starts to increase to about 20 Hz in the pres-
ence of high myristate concentration.
Discussion
Myristate binding to HSA affects the Mn(III)heme
binding properties. The results presented here indicate
that current views – seven FA binding sites, FA1
involved in ipsosterical competition with heme binding,
FAx allosterically coupled to FA1, a scale of affinity
ratios of about half a decade among FA sites, with dis-
sociation constants in the range 10
)6
)10
)8
m, and sev-
eral possible allosteric cross interactions among FA
sites [1,3,6,9–16,33] – allow us to numerically model the
experimental results with good accuracy. In particular,
modeling indicates that binding of myristate to FAx
not only increases optical absorbance of Mn(III)heme-
bound HSA by a factor of % 3, but also increases FA1
affinity for Mn(III)heme by 10–500-fold (depending on
the assumptions about possible similar changes in affin-
ity of FA1 for myristate). This brings the value of HSA

Mn(III)heme–HSA-(FAx+myristate) complex (A
10
*),
whereas the latter is not altered by the change in pH.
Taken together, these observations strongly suggest
that the conformational changes produced by changing
the pH from 7.0 to 10.0 (i.e. shifting the HSA confor-
mation from the N to the B state) is very similar to
that induced by myristate binding to site FAx. Indeed,
the HSA affinity for Mn(III)heme and the absorbance
of Mn(III)heme–HSA increase by factors of about 10
and 3, respectively, and myristate effects become much
attenuated. Still, the same interaction(s) that at pH ¼
7.0 produces marked differences among absorbance
curves at various myristate concentrations appear to
fully account for the small reshaping of the curve pro-
duced by 1.0 · 10
)4
m myristate at pH ¼ 10.0.
Myristate binding to HSA determines conformational
changes that open the FA1 cavity allowing Mn(III)-
heme binding and consequently myristate displacement.
Actually, addition of up to three moles of long-chain
FAs is reported to enhance the binding of Sudlow’s site
I (i.e. FA7) ligands, and this behaviour is usually
explained by a cooperative effect established by FA
binding to domain III (i.e. to FA4 and FA5) [26,37–39].
On the other hand, myristate bound at the limit of
subdomain IA (i.e. to FA2) was suggested to be func-
tionally linked to Sudlow’s site I [25]. It should be

indicate that there is a water molecule coordinated at
both pH but that its exchange is limiting the relaxivity.
Therefore, the binding of myristate seems to markedly
increase the exchange rate and induce a relaxivity
G. Fanali et al. Myristate and Mn(III)heme binding to HSA–heme
FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS 4679
enhancement, although at pH 10.0 the possible increase
in the exchange rate by myristate is incapable to
induce a significant increase of the relaxivity.
At pH 10.0 (i.e. when HSA is in the B state),
17
O-NMR linewidth measurements show no evidence of
water molecules coordinated to Mn(III)heme, as already
observed in the case of Fe(III)heme–HSA. Two hypo-
theses should be taken into consideration: either the
absence of water molecules in the coordination sphere
of the metal ion, or the presence of one water molecule
with a very slow exchange rate. Myristate binding to
HSA might increase the exchange rate, thereby produ-
cing a small broadening, but this is only observed
at pH 7.0. The structural similarity of Mn(III)heme
vs. Fe(III)heme and the structural evidence of a penta-
coordinated Fe(III) atom, with no water molecules
coordinated to it, favour the first hypothesis: in this
case, upon deprotonation at pH 10.0 the phenolic
Tyr161 oxygen becomes more nucleophylic and displa-
ces the Mn(III)-coordinated water molecule with the
consequent quenching of the paramagnetic relaxation.
Conclusions
The conformational transition(s) driven by myristate

without further purification. HSA was essentially fatty acid-
free according to the charcoal delipidation protocol [41–43]
and used without any further purification. Absence of signi-
ficant amounts of covalent dimers was checked by
MALDI-TOF mass spectrometry. Mn(III)heme was pre-
pared as previously reported [28]. The actual concentration
of the Mn(III)heme stock solution was checked as bis-
imidazolate complex in sodium dodecyl sulfate micelles
with an extinction coefficient of 10.3 cm
)1
Æmm
)1
(at
556 nm) [44]. Mn(III)heme–HSA was prepared by adding
the appropriate volume of 3.0 · 10
)2
m Mn(III)heme dis-
solved in 1.0 · 10
)1
m NaOH to a 1.0 · 10
)3
m HSA solu-
tion in NaCl ⁄ P
i
(1.0 · 10
)2
m phosphate buffer, 0.15 m
NaCl). The final solution of Mn(III)heme–HSA was
1.0 · 10
)3

)5
m.
This solution was titrated with HSA by adding small
amounts of a 1.0 · 10
)3
m protein solution in the aqueous
buffer and recording the spectrum after incubation for
a few min after each addition. Difference spectra with
respect to Mn(III)heme were taken and the binding iso-
therm was analyzed by plotting the difference of absorb-
ance between the maximum and the minimum of the
two-signed difference spectra against the protein concentra-
tion [27].
Data have been numerically analyzed using the matlab
language (The MathWorks, Natick, MA, USA) according
to Scheme 1, with the following dissociation equilibrium
constants: K
H
for Mn(III)heme binding to site FA1; K
M
for
myristate binding to site FA1 and competing ipsosterically
with Mn(III)heme; K
M
* for myristate binding to site FAx,
allosterically coupled to FA1; K
H
M
for Mn(III)heme bind-
ing to the HSA–myristate complex, with myristate bound

FFC fast field cycling relaxometer (Stelar, Mede, Italy) with
16 experiments in four scans. The reproducibility in T
1
measurements was ± 0.5%.
1
H nuclear magnetic relaxation dispersion (NMRD) pro-
files were recorded at variable concentration of myristate
by measuring water proton longitudinal relaxation rates
(R
1
obs
) at magnetic field strengths in the range from
2.4 · 10
)4
to 0.235 T (corresponding to 0.01–10 MHz pro-
ton Larmor frequencies) with the field cycling relaxometer
described above.
The R
1p
relaxivity values (i.e. paramagnetic contributions
to the solvent water longitudinal relaxation rate referenced
to a 1.0 mm concentration of paramagnetic agent) were
determined by subtracting from the observed relaxation
rate (R
1
obs
) the blank relaxation rate value (R
1
dia
) measured

M
ð1Þ
where s
M
is the exchange lifetime and q is the number of
water molecules close to the metal centre. [M] is the con-
centration of the paramagnetic metal ion, and T
1M
is the
longitudinal relaxation time of localized water protons
[20,36].
Relaxivity of Mn(III)heme–HSA solutions at 25.0 ° Cas
a function of pH was analyzed according to Eqn (2):
R
1p
¼ C
0
þ
X
i
C
i
1 þ½H
þ
=K
i
ð2Þ
where C
0
is the R

17
O signal in the presence
of HSA from the width of the H
2
17
O signal in the
presence of Mn(III)heme–HSA at different myristate con-
centrations. DW values are related to the transverse relaxa-
tion time of the directly coordinated water oxygen (T
O
2M
)
by Eqn (3):
DW ¼
½Mq
55:56
Â
1
T
O
2M
þ s
M
ð3Þ
where s
M
is the exchange lifetime of the metal-coordinated
water molecule, [M] is the concentration of the paramag-
netic metal ion, and q is the number of water molecules
coordinated to it. The oxygen transverse relaxation time

FL, USA.
6 Zunszain PA, Ghuman J, Komatsu T, Tsuchida E &
Curry S (2003) Crystal structural analysis of human
serum albumin complexed with hemin and fatty acid.
Struct Biol 3,6.
G. Fanali et al. Myristate and Mn(III)heme binding to HSA–heme
FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS 4681
7 Petitpas I, Petersen CE, Ha CE, Bhattacharya AA,
Zunszain PA, Ghuman J, Bhagavan NV & Curry S
(2003) Structural basis of albumin–thyroxine inter-
actions and familial dysalbuminemic hyperthyroxinemia.
Proc Natl Acad Sci USA 100, 6440–6445.
8 Wardell M, Wang Z, Ho JX, Robert J, Ru
¨
ker F, Ruble
J & Carter DC (2002) The atomic structure of human
methemalbumin at 1.9 A
˚
. Biochem Biophys Res Commun
291, 813–819.
9 Reed RG, Feldhoff RC, Clute OL & Peters T Jr (1975)
Fragments of bovine serum albumin produced by lim-
ited proteolysis. Conformation and ligand binding.
Biochemistry 14, 4578–4583.
10 Reed RG (1986) Location of long chain fatty acid-bind-
ing sites of bovine serum albumin by affinity labeling.
J Biol Chem 261, 15619–15624.
11 Cistola DP, Small DM & Hamilton JA (1987) Carbon
13 NMR studies of saturated fatty acids bound to
bovine serum albumin. I. The filling of individual fatty

19 Baroni S, Mattu M, Vannini A, Cipollone R, Aime S,
Ascenzi P & Fasano M (2001) Effect of ibuprofen and
warfarin on the allosteric properties of haem–human
serum albumin. A spectroscopic study. Eur J Biochem
268, 6214–6220.
20 Fasano M, Baroni S, Vannini A, Ascenzi P & Aime S
(2001) Relaxometric characterization of human hemal-
bumin. J Biol Inorg Chem 6, 650–658.
21 Mattu M, Vannini A, Coletta M, Fasano M & Ascenzi
P (2001) Effect of bezafibrate and clofibrate on the
heme-iron geometry of ferrous nitrosylated heme-human
serum albumin: an EPR study. J Inorg Biochem 84,
293–296.
22 Fasano M, Mattu M, Coletta M & Ascenzi P (2002)
The heme-iron geometry of ferrous nitrosylated heme-
serum lipoproteins, hemopexin, and albumin: a com-
parative EPR study. J Inorg Biochem 91, 487–490.
23 Ascenzi P, Bocedi A, Bolli A, Fasano M, Notari S &
Polticelli F (2005) Allosteric modulation of monomeric
proteins. Biochem Mol Biol Educ 33, 169–176.
24 Bhattacharya AA, Curry S & Franks NP (2000) Binding
of the general anesthetics propofol and halothane to
human serum albumin. High resolution crystal struc-
tures. J Biol Chem 275, 38731–38738.
25 Petitpas I, Bhattacharya AA, Twine S, East M & Curry
S (2001) Crystal structure analysis of warfarin binding
to human serum albumin: anatomy of drug site I. J Biol
Chem 276 , 22804–22809.
26 Chuang VTG & Otagiri M (2002) How do fatty acids
cause allosteric binding of drugs to human serum albu-

33 Ashbrook JD, Spector AA, Santos EC & Fletcher JE
(1975) Long chain fatty acid binding to human plasma
albumin. J Biol Chem 250, 2333–2338.
Myristate and Mn(III)heme binding to HSA–heme G. Fanali et al .
4682 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS
34 Pedersen AO, Mensberg KD & Kragh-Hansen U (1995)
Effect of ionic strength and pH on the binding of med-
ium-chain fatty acids to human serum albumin. Eur J
Biochem 233, 395–405.
35 Ikezaki A & Nakamura M (2003) Importance of the
C–H

N weak hydrogen bonding on the coordination
structures of manganese (III) porphyrin complexes.
Inorg Chem 42, 2301–2310.
36 Aime S, Botta M, Fasano M, Geninatti Crich S & Ter-
reno E (1999)
1
H and
17
O-NMR relaxometric investiga-
tions of paramagnetic contrast agents for MRI. Clues
for higher relaxivities. Coord Chem Rev 186, 321–333.
37 Wilding G, Feldhoff RC & Vesell ES (1977) Concentra-
tion-dependent effects of fatty acids on warfarin binding
to albumin. Biochem Pharmacol 26, 1143–1146.
38 Rietbrock N, Menke G, Reuter G, Lassmann A &
Schmeidl R (1985) Influence of palmitate and oleate on
the binding of warfarin to human serum albumin:
stopped-flow studies. J Clin Chem Clin Biochem 23,

FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS 4683