Research Communication

Drug Binding to Sudlow’s Site I Impairs Allosterically Human SerumHeme-Albumin-Catalyzed Peroxynitrite Detoxification

Paolo Ascenzi1, Alessandro Bolli1, Francesca Gullotta2,3, Gabriella Fanali4 and Mauro Fasano41Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Roma, Italy2Department of Experimental Medicine and Biochemical Sciences, University of Roma ‘‘Tor Vergata’’, Roma, Italy3Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy4Department of Structural and Functional Biology, and Center of Neuroscience, University of Insubria, Busto Arsizio, Italy

Summary

Heme endows human serum albumin (HSA) with globin-likereactivity and spectroscopic properties. Here, the effect ofchlorpropamide, digitoxin, furosemide, indomethacin, phenylbu-tazone, sulfisoxazole, tolbutamide, and warfarin on peroxynitriteisomerization to NO3

– by ferric HSA-heme (HSA-heme-Fe(III)) isreported. Drugs binding to Sudlow’s site I impair dose-depend-ently peroxynitrite isomerization by HSA-heme-Fe(III). The allo-steric modulation of HSA-heme-Fe(III)-mediated peroxynitriteisomerization by drugs has been ascribed to the pivotal role ofTyr150, a residue that either provides a polar environment inSudlow’s site I or protrudes into the heme cleft (i.e., the fattyacid site 1, FA1), depending on ligand occupancy of eithersites. � 2010 IUBMB

IUBMB Life, 62(10): 776–780, 2010

Keywords human serum heme-albumin; peroxynitrite isomerization;

drug binding to Sudlow’s site I; kinetics; allosteric inhibi-

tion.

Abbreviations FA, fatty acid; heme-Fe(III), ferric heme; HSA,

human serum albumin; HSA-heme, human serum

heme-albumin; HSA-heme-Fe(III), ferric HSA-heme.

Human serum albumin (HSA), the most abundant protein in

plasma (�7.0 3 1024 M), shows an extraordinary ligand bind-

ing capacity, providing a depot and carrier for endogenous and

exogenous compounds [1–5]. Bulky heterocyclic anions bind

preferentially to Sudlow’s site I (corresponding to the fatty acid

site 7; FA7), whereas Sudlow’s site II (composed by FA3 and

FA4 sites) is preferred by aromatic carboxylates with an

extended conformation. Remarkably, warfarin and ibuprofen are

considered as stereotype ligands for Sudlow’s site I and II,

respectively [1, 4–8]. The FA1 binding site has evolved to

selectively bind the heme with a high affinity, so that HSA par-

ticipates physiologically to heme scavenging [3, 5, 9]. In turn,

heme endows HSA with reactivity and spectroscopic properties

similar to those of hemoglobin and myoglobin. Remarkably,

both ferric heme [heme-Fe(III)] binding to HSA and human se-

rum heme-albumin (HSA-heme) reactivity are modulated allos-

terically [5, 10–17].

Here, chlorpropamide, digitoxin, furosemide, indomethacin,

phenylbutazone, sulfisoxazole, tolbutamide, and warfarin are

reported to impair allosterically peroxynitrite isomerization to

NO�3 by ferric HSA-heme [HSA-heme-Fe(III)]. The effect of

drugs has been ascribed to the pivotal role of Tyr150, a residue

that either provides a polar environment in Sudlow’s site I or

protrudes into the heme cleft (i.e., the FA1 site), depending on

ligand occupancy of either sites.

MATERIALS AND METHODS

HSA (‡96%, essentially fatty acid free), hemin [Fe(III)-pro-

toporphyrin IX] chloride, chlorpropamide, digitoxin, furosemide,

indomethacin, phenylbutazone, sulfisoxazole, tolbutamide, and

warfarin (Supporting Information Fig. 1 SI) were obtained from

Sigma-Aldrich (St. Louis, MO). All the other products were

purchased from Merck AG (Darmstadt, Germany).

HSA-heme-Fe(III) (2.0 3 1024 M) was prepared by adding

a 0.8-molar equivalent of the heme-Fe(III) solution (1.0 3 1022

M NaOH) to the HSA solution (1.0 3 1021 M sodium phos-

phate buffer, pH 7.0), at 20.0 8C [17]. The HSA-heme-Fe(III)

concentration was determined spectrophotometrically at 403 nm

(e403 nm 5 1.1 3 105 M21 cm21) [18].

Peroxynitrite was synthesized from KO2 and NO or HNO2 and

H2O2 and stored at 280.0 8C. The concentration of peroxynitrite

Address correspondence to: Paolo Ascenzi, Department of Biology and

Interdepartmental Laboratory for Electron Microscopy, University Roma

Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy. Tel: 139-06-

5733-3494; Fax: 139-06-5733-6321. E-mail: ascenzi@uniroma3.it

Received 22 July 2010; accepted 30 August 2010

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.381

IUBMB Life, 62(10): 776–780, October 2010

was determined spectrophotometrically by measuring the absorb-

ance at 302 nm (e302 nm 5 1.705 3 103 M21 cm21) [19, 20].

The warfarin stock solution (1.0 3 1022 M) was prepared

by dissolving the drug in water at pH 10.0, then adjusting pH

to 7.0 [21]. The chlorpropamide, digitoxin, furosemide, indo-

methacin, phenylbutazone, sulfisoxazole, and tolbutamide stock

solutions (1.0 3 1022 M) were prepared by dissolving the drugs

in 1.0 3 1021 M sodium phosphate buffer, pH 7.0 [12].

Kinetics of peroxynitrite isomerization by HSA-heme-Fe(III)

in the absence and presence of drugs (5.0 3 1026 M to 1.0 3

1023 M) was recorded at 302 nm (e302 nm 5 1.705 3 103 M21

cm21) by rapid mixing the protein solution (5.0 3 1026 to 4.0

3 1025 M) with the peroxynitrite solution (2.5 3 1024 M).

The light path of the observation cuvette was 10 mm, and the

dead-time was 1.4 ms. No gaseous phase was present [17].

Kinetics of peroxynitrite isomerization by HSA-heme-Fe(III),

in the absence and presence of drugs, was analyzed in the

framework of the minimum reaction Scheme 1 [17].

Values of the pseudo-first-order rate constant for HSA-heme-

Fe(III)-mediated peroxynitrite isomerization in the absence and

Figure 1. Peroxynitrite isomerization by HSA-heme-Fe(III). Normalized averaged time courses of peroxynitrite isomerization by

HSA-heme-Fe(III) (A). The HSA-heme-Fe(III) concentration was 2.5 3 1025 M (trace a), 5.0 3 1025 M (trace b), and 2.0 31024 M (trace c). The time course analysis according to Eq. (1) allowed the determination of the following values of kobs: trace a,

kobs 5 2.0 3 101 s21; trace b, kobs 5 6.7 3 101 s21; and trace c, kobs 5 9.8 3 101 s21. Dependence of kobs, in the absence (open

circles; a) and presence (filled circles; b and c) of warfarin, on the HSA-heme-Fe(III) concentration (B). The continuous lines were

calculated according to Eq. (2) with the following parameters: a—kon 5 4.1 3 105 M21 s21 and k0 5 2.8 3 1021 s21; b—kon 52.6 3 105 M21 s21 and k0 5 2.9 3 1021 s21; and; c—kon 5 7.6 3 104 M21 s21 and k0 5 3.2 3 1021 s21. The warfarin concen-

tration was 0.0 M (a), 2.0 3 1025 M (b), and 2.0 3 1024 M (c). Dependence of kon on the warfarin concentration (C). The open

circle on the ordinate indicates the kon value obtained in the absence of warfarin (5 4.1 3 105 M21 s21). The continuous line was

calculated according to Eq. (3) with L 5 2.3 3 1025 M. Dependence of k0 on the warfarin concentration (D). The open square on

the ordinate indicate the k0 value obtained in the absence of the drug (5 3.1 3 1021 s21). The average k0 value is 2.9 3 1021

s21. All data were obtained at pH 7.0 and 20.0 8C. Where not shown, standard deviation is smaller than the symbol. For details,

see text.

Scheme 1. Minimum reaction mechanism for peroxynitrite isomerization by HSA-heme-Fe(III).

777DRUGS IMPAIR HSA-HEME REACTIVITY

presence of drugs (kobs) have been determined from the analysis

of the time-dependent absorbance decrease at 302 nm, accord-

ing to Eq. (1) [17]:

peroxynitrite½ �t¼ peroxynitrite½ �i3e�kobs3t (1)

In the absence and presence of drugs, values of the second-

order rate constant for HSA-heme-Fe(III)-mediated peroxynitrite

isomerization (kon) and of the first-order rate constant for perox-

ynitrite isomerization in the absence of HSA-heme-Fe(III) (k0)

have been determined from the linear dependence of kobs on the

HSA-heme-Fe(III) concentration, according to Eq. (2) [17]:

kobs ¼ kon3½HSA-heme-FeðIIIÞ� þ k0 (2)

Values of the dissociation equilibrium constant for chlorpro-

pamide, digitoxin, furosemide, indomethacin, phenylbutazone,

sulfisoxazole, tolbutamide, and warfarin binding to HSA-heme-

Fe(III) (L) were determined from the dependence of kon on the

drug concentration (ranging between 5.0 3 1026 M and 1.0 31023 M), according to Eq. (3) [17]:

kon ¼ konðtopÞ � ððkonðtopÞ3½drug�Þ=ðLþ ½drug�ÞÞ (3)

where kon(top) represents the value of kon under conditions where

[drug] 5 0.

All data were obtained at pH 7.0 (1.0 3 1021 M phosphate

buffer) and 20.0 8C.NO�

2 and NO�3 analysis was carried out spectrophotometri-

cally at 543 nm by using the Griess reagent and VCl3 to catalyze

the conversion of NO�3 to NO�

2 , as described previously [17].

Kinetic and thermodynamic data were analyzed using the

MatLab program (The Math Works, Natick, MA). The results

are given as mean values of at least four experiments plus or

minus the corresponding standard deviation.

Automatic flexible ligand docking simulation for chlorpropa-

mide, digitoxin, furosemide, indomethacin, phenylbutazone, sul-

fisoxazole, tolbutamide, and warfarin binding to HSA was per-

formed by using Autodock 4.0 and the graphical user interface

AutoDockTools, and values of DGcalc were obtained accord-

ingly [22–24]. The structure of HSA-heme-Fe(III) was down-

loaded from the Protein Data Bank (PDB code: 1O9X) [11].

Chlorpropamide, digitoxin, furosemide, sulfisoxazole, and tolbu-

tamide structures were calculated using the Dundee PRODRG

server [25]. Warfarin, indomethacin, and phenylbutazone geo-

metries in complex with HSA were downloaded from the Pro-

tein Data Bank (PDB codes: 2BXD, 2BXM, and 2BXC, respec-

tively) [4]. The analysis of the conformational space was re-

stricted to a cubic box of 70 A edge centered on the

coordinates of Sudlow’s site I. Monte Carlo simulated annealing

was performed by starting from a temperature of 900 K with a

relative cooling factor of 0.95/cycle, to reach the temperature of

5 K in 100 cycles. Single bonds were allowed to rotate freely

during the Monte Carlo simulated annealing procedure. Intermo-

lecular binding energies DGcalc were obtained as the difference

between docking energy and internal energy components (final

total internal energy and torsional-free energy) [22–24].

RESULTS AND DISCUSSION

Kinetics of peroxynitrite isomerization by HSA-heme-Fe(III),

both in the absence and presence of drugs, was fitted to a sin-

gle-exponential decay for more than 95% of its course [see Eq.

(1)]. This indicates that no intermediate species [e.g., HSA-

heme-Fe(III)-OONO; see Scheme 1] accumulate(s) in the course

of peroxynitrite isomerization, the formation of the transient

HSA-heme-Fe(III)-OONO species representing the rate limiting

step in catalysis.

Both in the absence and presence of drugs, values of kobs for

HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite

increase linearly with the HSA-heme-Fe(III) concentration (Fig.

1). The analysis of data according to Eq. (2) allowed the deter-

mination of values of kon (corresponding to the slope of the lin-

ear plots) and k0 (corresponding to the y intercept of the linear

plots). Values of kon and k0 obtained in the absence of drugs

(4.1 3 105 M21 s21 and 3.1 3 1021 s21, respectively) are in

good agreement with those reported in the literature [17].

As shown in Fig. 1 and in Supporting Information Fig. 2 SI,

drugs impair in a dose-dependent fashion HSA-heme-Fe(III)-

mediated isomerization of peroxynitrite; indeed, values of kondecrease on increasing the drug concentration. The analysis of

data according to Eq. (3) allowed the determination of values of

L (Table 1). Values of L here obtained (see Table 1) are in

Figure 2. Superimposition of the three-dimensional structures of

HSA in the presence of heme (PDB code:1O9X [11], in red) and

in the presence of warfarin (PDB code: 2BXD [4], in green).

The Tyr150 residue is labeled in both structures. Heme-Fe(III)

and warfarin are rendered as sticks. For further details, see text.

778 ASCENZI ET AL.

excellent agreement with those reported in the literature [7, 12,

13]. Under conditions where L � [drug], values of kobs corre-

spond to that of k0 [see Eq. (2)], which is drug-independent

[Fig. 1 and Supporting Information Fig. 3 SM].

Values of the experimental free energy (i.e., DGexp) for

chlorpropamide, furosemide, indomethacin, phenylbutazone, sul-

fisoxazole, tolbutamide, and warfarin binding to HSA-heme-

Fe(III) are in good agreement with those calculated by auto-

matic flexible ligand docking simulation (i.e., DGcalc; Table 1).

Digitoxin could only partially enter into Sudlow’s site I, thus

preventing the determination of the docking energy for the

whole molecule. Nevertheless, favorable values of DGcalc for

binding of the oligosaccharide and the aglycon moieties of digi-

toxin were obtained (218.0 kJ mol21 and 216.6 kJ mol21,

respectively). These values are in agreement with that experi-

mentally determined (i.e., DGexp; Table 1), and suggest that

both ends of the digitoxin molecule could enter Sudlow’s site I.

According to literature [17], the isomerization of peroxyni-

trite yielded 74 6 6% NO�3 and 25 6 3% NO�

2 , in the absence

of HSA-heme-Fe(III). In the presence of HSA-heme-Fe(III), the

NO�3 and NO�

2 yields increased (90 6 5%) and decreased (11

6 4%), respectively. However, drugs do not significantly affect

the NO�3 and NO�

2 yields (Supporting Information Table 1 SM).

Chlorpropamide, digitoxin, furosemide, indomethacin, phe-

nylbutazone, sulfisoxazole, tolbutamide, and warfarin modulate

allosterically not only peroxynitrite isomerization by HSA-

heme-Fe(III) (present study) but also heme-Fe(III) binding to

HSA [12, 13]. Moreover, warfarin facilitates the denitrosylation

of ferrous nitrosylated HSA-heme [16]. These data highlight the

role of heterotropic ligands on modulating the HSA(-heme-Fe)

reactivity [3, 5, 12, 13, 16, 26].

Allosteric inhibition of the HSA-heme-Fe(III)-mediated per-

oxynitrite isomerization by Sudlow’s site I ligands mirrors

structural changes occurring at the heme-binding pocket.

Indeed, the allosteric modulation of HSA-heme-Fe(III)-mediated

peroxynitrite isomerization by warfarin reflects the pivotal role

of Tyr150, a residue that provides a polar environment in

Sudlow’s site I (i.e., the warfarin binding pocket; Fig. 2). In

this context, occupancy of the Sudlow’s site I by chlorpropa-

mide, digitoxin, furosemide, indomethacin, phenylbutazone, sul-

fisoxazole, tolbutamide, and warfarin forces Tyr150 to point

toward the ligand with a consequent distortion of the heme ge-

ometry, eventually impairing peroxynitrite isomerization to NO�3

These results (i) highlight the role of drugs in modulating

HSA functions, (ii) indicate that HSA acts not only as a heme

carrier but also displays heme-based properties, and (iii) open

the scenario toward the possibility of a time- and metabolite-

dependent multiplicity of roles for HSA.

ACKNOWLEDGEMENTS

Authors thank Prof. Massimo Coletta (University of Roma ‘‘Tor

Vergata’’, Italy) for helpful discussions. This work was partially

supported by grants from the Ministero dell’Istruzione, dell’Uni-

versita e della Ricerca of Italy (PRIN 2007ECX29E_002 and

University Roma Tre, CLAR 2009, to P.A.). Dr. Francesca Gul-

lotta fellowship was supported by the Interuniversity Consor-

tium for the Research on the Chemistry of Metals in Biological

Systems, I-70126 Bari, Italy.

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