Journal of Luminescence 145 (2014) 767773

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Journal of Luminescence
journal homepage: www.elsevier.com/locate/jlumin

Quantitation of species differences in albuminligand interactions

for bovine, human and rat serum albumins using uorescence
spectroscopy: A test case with some Sudlow's site I ligands
Mikls Por a, Yin Li b,c, Gergely Matisz b,c, Lszl Kiss b, Sndor Kunsgi-Mt b,c,
Tams Kszegi a,n
a
b
c

Institute of Laboratory Medicine, University of Pcs, Ifjsg u. 13, Pcs H-7624, Hungary
Department of General and Physical Chemistry, University of Pcs, Pcs H-7624, Hungary
Jnos Szentgothai Research Center, Pcs H-7624, Hungary

art ic l e i nf o

a b s t r a c t

Article history:
Received 17 May 2013
Received in revised form
13 August 2013
Accepted 26 August 2013
Available online 5 September 2013

Albumin, the most abundant plasma protein is an approximately 67 kDa sized water-soluble macromolecule. Since several drugs and xenobiotics circulate in the blood at least partially in albumin-bound
form, albumin plays a key role in the pharmacokinetics/toxicokinetics of these chemicals. Most of the
drugs and xenobiotics are Sudlow's site I ligands. In numerous studies, bovine serum albumin (BSA) is
used for modeling albuminligand interactions and the results are extrapolated to human serum
albumin (HSA). Furthermore, only limited information is available related to albuminligand interactions
of different albumin species. Therefore, in our study, we have focused on the quantication of differences
between bovine, human and rat serum albumin (RSA) using four Sudlow's site I ligands (luteolin,
ochratoxin A, phenylbutazone and warfarin). Interactions were analyzed by uorescence spectroscopy.
Stability constants as well as competing capacities of the ligands were determined, and thermodynamic
study was also performed. Our results highlight that there could be major differences between BSA, HSA
and RSA in their ligand binding properties. Based on our observations we emphasize that in molecular
aspects BSA behaves considerably differently from HSA or from albumins of other species therefore, it is
strongly recommended to apply at least some conrmatory measurements when data obtained from
other species are attempted to be extrapolated to HSA.
& 2013 Elsevier B.V. All rights reserved.

Keywords:
Albuminligand interaction
Sudlow's site I ligands
Species-specic differences
Human serum albumin
Bovine serum albumin
Rat serum albumin

1. Introduction
Albumin is an approximately 67 kDa sized water-soluble macromolecule with a very high biological importance. Albumin is the most
abundant plasma/serum protein; it is responsible for maintenance of
the oncotic pressure in plasma therefore it modulates the uid
distribution among body compartments and, it also shows considerable buffering, antioxidant and pseudo-enzymatic capacities [1].
In addition, albumin provides a large variety of binding and transport
functions therefore it has a major inuence on the pharmacokinetics
of numerous endogenous ligands (like thyroid hormones, fatty acids,
bilirubin etc.) and drugs resulting in the establishment of a depot
effect [2]. Native albumin (without ligands or bound molecules) is
built up from three homologous domains (I, II and III), and all
domains consist of twotwo subdomains (named A and B). There
are numerous binding sites on albumin [1], but drugs and other
exogenous compounds occupy mainly two of them: Sudlow's site I or

Corresponding author. Tel.: 36 72 536 120; fax: 36 72 536 121.

E-mail addresses: koszegit@freemail.hu, tamas.koszegi@aok.pte.hu (T. Kszegi).

0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jlumin.2013.08.059

acidic drug binding site (on subdomain IIA) and Sudlow's site II or
benzodiazepine binding site (on subdomain IIIA) [1,3,4]. Our study
focuses on the investigation of Sudlow's site I ligands (different drugs
and xenobiotics) which are thought to be bound primarily to this
location.
In vivo animal studies are mainly based on rat (or mouse)
models. On the other hand, it is a common practice to use bovine
serum albumin (BSA) for the examination/modeling of albumin
ligand interactions [58] most probably because BSA is signicantly
cheaper than HSA and is much easier to obtain. A few examples
from recent studies are as followings: Shi et al. [5] examined the
effects of avonoids on the albumin (BSA) binding of pantoprazole
and suggest the possibility of fooddrug interaction. Khodarahmi
et al. [6] emphasized the similarity of BSA and HSA when applying
BSA to study non-enzymatic protein glycation and its potential
pharmacological consequences. Bani-Yaseen [7] suggested that BSA
is an extensively characterized protein and its structure is homologous to the structure of HSA the reason why it is commonly
chosen as a model protein to investigate proteindrug interactions.
Therefore BSA was used for studying sulfamethazinealbumin
interaction. Finally, Sarkar et al. [8] drew the attention to the strong

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M. Por et al. / Journal of Luminescence 145 (2014) 767773

binding of alprazolam to albumin and its possible toxicological

results based on his study with BSA. None of the above described
cases was done because of the importance of these interactions in
veterinary research. Although we emphasize the value of these
interesting and useful publications still in our opinion the use of
HSA would have been better in the above investigations. Keeping in
mind the subtle differences among the albumins of varying species
the question arises how the results derived from measurements
for BSA could be extrapolated to HSA or could it be done at all?
Obviously it is true that the size, the number of amino acid
constituents, the biological function, and often the binding sites of
albumins of different species seem to be very similar [9], but it is
important to note that there is a 2530% variability in amino acid
sequences between HSA, BSA and RSA, respectively [1015]. The
available data concerning ligandRSA interactions and especially
comparison of the results with those of HSA are limited [9,16,17].
Panjehshahin et al. [9] found that for bovine albumin the uorescence of bound warfarin could be decreased by phenylbutazone,
while long chain fatty acids enhanced warfarin uorescence. On
the other hand, there are a few data for the different molecular
behavior of rat albumin compared to albumin of the other two
species. The interaction of warfarin and non-steroidal anti-inammatory drugs (NSAIDs) with HSA and RSA was studied by Massolini
et al. [16] and Aurby et al. [17] using HPLC: the stereoselectivity of
drug binding was much lower on RSA than on HSA, therefore
differences in the binding sites were assumed. We have a lot more
information on BSA than RSA; on the other hand relatively few
studies investigate the ligand binding properties of both BSA and
HSA under the same experimental conditions [1824]. Some of the
studied substances show very similar stability constants with BSA
and HSA as well, for example folic acid, 5-uorouracil or 6-azauracil
[18,19]. Slightly stronger binding of folic acid to BSA than HSA was
observed and explained by the interaction of folic acid with both
Trp-132 and Trp-212 tryptophan residues of BSA [18]. Based on
uorescence quenching studies, 5-uorouracil and 6-azauracil have
almost the same binding constants for BSA and HSA. On the other
hand, 6-mercaptopurine and thioimidazole bind stronger to BSA
then HSA, while thiouracil and gemcitabin prefer HSA more than
BSA [19,20]. A further quenching study using the corticosteroid
dexamethasone also resulted in different binding afnities moreover, larger protein unfolding was found for HSA than for BSA [21].
Based on spectroscopic investigations, nortriptyline hydrochloride
and promazine hydrochloride showed much higher afnity towards
BSA than HSA [22]; furthermore, the antithyroid drug methimazole
and the organophosphorous compound methyl parathion also exert
different complex stabilities in the case of HSA and BSA [23,24].
The above listed studies recommend alterations of about 0.20.4 in
log K values depending on the albumin type (BSA or HSA) used
[1924]. In addition, data for other agents suggest even higher
differences, like 6-mercaptopurine (0.6) [19] or the mycotoxin
ochratoxin A ( 1.2) [25]. Being aware of these substantially diverse
results, the question arises: at what extent between-species binding
differences can exist in the case of molecular displacement studies,
where two or more ligands at the same time compete for albumin
binding?
In our study, albumin binding capacity and displacing ability of
four Sudlow's site I ligands were investigated for bovine, human and
rat serum albumin respectively, using steady-state uorescence and
uorescence polarization techniques. The oral anticoagulant warfarin (WAR), the NSAID phenylbutazone (PHEBU), the avonoid
aglycone luteolin (LUT) and the mycotoxin ochratoxin A (OTA) were
examined as model compounds (Fig. 1) [2629]. Our aim was to
compare the binding properties of three types of albumin, and
to give numerical data on the albuminligand interactions quantifying the degree of potential differences among the albumins of
different species. For this reason in the rst step stability constants

Fig. 1. Chemical structure of the studied four different Sudlow's site I (subdomain
IIA) ligands.

of albuminligand complexes were determined. Then displacing

abilities of LUT, PHEBU and WAR were investigated in OTAalbumin
complex systems. Finally thermodynamic measurements were done
in the case of PHEBUalbumin complexes.
It is important to note that instead of the investigation of
structural diversities of the different albumins, the main aim
of this study was to determine quantitative parameters and the
possible displacement reactions related to the albuminligand
interactions and highlighting that the three albumins examined
show quite different binding properties towards biologically active
ligands. Our results strongly suggest that depending on the type of
the ligand there could be major alterations in binding properties
among the three studied albumins.

2. Materials and methods

2.1. Reagents
All reagents and solvents were of analytical grade. Bovine serum
albumin (BSA), human serum albumin (HSA), rat serum albumin
(RSA), ochratoxin A (OTA), phenylbutazone (PHEBU), warfarin (WAR)
(all from Sigma-Aldrich), luteolin (LUT, from Indone) were used as
received. 2500 M stock solutions of ligands were prepared in
dimethyl sulfoxide (DMSO; Reanal, spectroscopic grade) and stored
at 4 1C, protected from light. Phosphate buffered saline (PBS; pH 7.4)
was applied as medium in all analyses to mimic extracellular
physiological conditions.
2.2. Instrumentation
Fluorescence spectra were recorded and steady-state uorescence
polarization values were measured by a Hitachi F-4500 uorescence
spectrophotometer and a Fluorolog 3 (Jobin-Yvon) spectrouorimetric system. All analyses were performed in the presence of air at
25 1C (with the exception of thermodynamic studies).
2.3. Data analysis
Stability constants of albuminligand complexes were evaluated using Hyperquad2006 program package (Protonic Software)
applying a 1:1 stoichiometric model using the following previously published equation [30,31]:
0
s1


I HG I 0 @
1
1 2
H0 G0
I I0
H0 G0 
4H0 G0 A
2H0
K
K
1

M. Por et al. / Journal of Luminescence 145 (2014) 767773

769

where, I denotes the uorescence emission intensity of albumin in

the presence of the ligand; I0 denotes the uorescence emission
intensity of albumin in the absence of the ligand; IHG denotes the
uorescence emission intensity of pure ligandHSA complex,
which is calculated by the Hyperquard2006; K denotes the binding
constant; [H]0 and [G]0 denote the total concentrations of albumin
and the ligand, respectively.
Background correction of uorescence spectra and evaluation
of uorescence intensities were done by OriginPro8 software
(OriginLab Corp., Northampton, MA, USA).
The degree of uorescence polarization was calculated as
P

I V V GI V H
I VV GI V H

where IVV and IVH are the uorescence intensities measured at

vertical excitation polarizer setting and at vertical and horizontal
emission polarizer settings respectively, while G is the actually
measured optical correction factor. The degree of uorescence
polarization was averaged from 30 measuring points.
For determination of the degree of molecular displacements,
the previously described uorescence polarization-based
approach was performed [3234]:

PP f
P b P PP f 

Fig. 2. Fluorescence emission spectra of BSA (5 M), HSA (5 M), RSA (5 M), OTA
(1 M) and WAR (20 M) in PBS (pH 7.4) [excitation wavelengths: BSA, HSA and
RSA: 295 nm; OTA: 380 nm; WAR: 309 nm; excitation and emission slit widths
were both 5 nm, respectively].

where is the bound fraction of the toxin, P is the measured

polarization, Pf and Pb are the uorescence polarization values of
free and bound OTA, respectively. Furthermore

b b
f f

where b and f are the molar absorptivities as well as b and f

are the uorescent quantum yields of the bound and free forms
of OTA.

3. Experimental
3.1. Spectral properties of different albumins and Sudlow's
site I ligands
Emission maximum of all three studied albumins was 342 nm
at 295 nm excitation wavelength (Fig. 2). It means that the
emission maxima of the albumins are very similar however; BSA
shows considerably higher uorescence signal than the same
amount of HSA and RSA.
Among the examined ligands, luteolin and phenylbutazone did
not exert any uorescence, while warfarin (309 nm-389 nm) and
ochratoxin A (380 nm-443 nm) showed strong uorescence properties (Fig. 2). In the presence of albumins (Fig. 3), a major increase in
uorescence intensities and minor changes of wavelength maxima
were observed in the case of OTA and WAR (OTABSA: 393 nm444 nm; OTAHSA: 393 nm-445 nm; OTARSA: 391 nm-442 nm;
WARBSA: 317 nm-380 nm; WARHSA: 317 nm-380 nm; and
WARRSA: 317 nm-372 nm). Excitation and emission slit widths
were 55 nm, respectively in the case of quenching studies while
5 and 10 nm in OTAalbumin and WARalbumin investigations.
3.2. Determination of stability constants
In order to determine the stability constants (log K) of LUT and
PHEBU, the uorescence quenching method was applied. 5 M
albumin solutions were prepared in PBS (pH 7.4) then increasing
ligand concentrations (050 M) were added to the systems in
both cases. Samples were excited at 295 nm and evaluation was
done using 342 nm as emission wavelength (Fig. 4).

Fig. 3. Fluorescence emission spectra of OTA (above; excitation and emission slit
widths: 5 nm) and WAR (below; 5 nm excitation and 10 nm emission slit widths) in
the absence and in the presence of albumin of different species (albumins were
applied above the maximal saturation concentration for the ligands: 1 M
OTA 1.7 M albumin, 1 M WAR 12 M albumin) in PBS (excitation wavelengths:
OTABSA and OTAHSA: 393 nm; OTARSA: 391 nm; and WARBSA, WARHSA
and WARRSA: 317 nm).

Because of their molecular structures, ochratoxin A and warfarin show strong uorescence properties, therefore they can
interfere with uorescence quenching experiments. In order
to eliminate interfering uorescence (and also to apply other

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M. Por et al. / Journal of Luminescence 145 (2014) 767773

sensitivity of the polarization method enable us to detect minor

changes in the motional freedom of the studied uorescent
molecules therefore, quantifying the degree of molecular displacing activities is possible.
As demonstrated in our previous works [33,34] 1 M OTA and
1.7 M albumin (in PBS) were applied in all cases. Fluorescence
polarization measurements were done using the excitation and
emission wavelength maxima of bound OTA (detailed in Section
3.1). Since OTA binds to different types of albumins with a very
high afnity, considerably higher LUT, PHEBU and WAR concentrations (25, 50 and 75 M) were added to the samples. Controls
contained the same volume of the solvent; the nal DMSO
concentration did not exceed 3% (v/v). Neither the three different
albumins nor LUT, PHEBU and WAR showed detectable uorescence signal at the wavelengths applied.
3.4. Thermodynamic studies
Potential differences of thermodynamic parameters were
investigated in the case of PHEBUalbumin complexes. The experimental conditions were the same as shown in Section 3.2, with the
exception of the applied temperatures (20, 25, 30, 35 and 40 1C,
respectively). Entropy and enthalpy changes were calculated using
the Vant Hoff theory.
3.5. Docking studies

Fig. 4. Fluorescence emission spectra and intensities of BSA (5 M) in the absence

and presence of increasing LUT (050 M) concentrations (above; excitation and
emission slit widths: 5 nm); WAR (1 M) in the absence and presence of increasing
RSA (012.5 M) concentrations (below; 5 nm excitation and 10 nm emission slit
widths) in PBS (LUTBSA: 295 nm-342 nm; WARRSA: 317 nm-372 nm).

methods than quenching), when OTAalbumin and WARalbumin

complexes were studied, their uorescence excitation and emission wavelength maxima were used (detailed in Section 3.1) to
quantify log K values (Fig. 4). In this part of our experiments,
increasing serum albumin concentrations were analyzed in the
presence of standard amount of OTA (1 M OTA 02 M albumin) and WAR (1 M WAR 012.5 M albumin) in PBS.
3.3. Competing ability against ochratoxin A
In our study, the displacing capacity of LUT, PHEBU and WAR
was investigated by uorescence polarization technique using the
three different types of OTAalbumin complexes. Molecular displacement from albumin is a very complex problem because log K
values could be overwritten due to steric reasons. The previously
published OTAHSA uorescence polarization-based model system [3234] seems to be very suitable for this type of investigation
because of the following reasons: the binding site of OTA has been
precisely characterized on subdomain IIA [25,29,32]; the model is
independent of the uorescent properties of the albumins (at the
wavelengths used); uorescence polarization technique is also
independent of the concentrations of the uorescent molecules, if
the signal to noise ratio is acceptable (in this way it is independent
of the diverse intensities of different OTAalbumin complexes);
our data show that the saturation function in the case of the three
types of albumins is very similar (Fig. S1); the high precision and

If the three dimensional structure of a protein is available, the

afnity of a ligand of interest could be predicted based solely on
theoretical modeling approaches. The molecular docking studies
have been carried out to predict the binding afnities and binding
sites of the ligands to HSA, BSA and RSA using the AutoDock Vina
program [35]. The crystallographic structures for HSA [3639] and
BSA [40,41] have been used [42], while the structure of RSA has
been approximated by homology modeling using the target
structures of bovine and equine serum albumin [40]. In order to
get improved relative binding afnities of the ligands, the NNScore
1.0 code [43] has been used. The docking studies have been carried
out by selecting the whole protein structure as target, while the
values of the exhaustiveness parameter, which determines the
accuracy of prediction of the binding sites and the associated
binding afnities have been selected in the order of magnitude of a
few hundreds in every cases.

4. Results and discussion

The results of log K calculations (Table 1) highlight that in most
cases signicant differences were observed among the three species. Although LUT did not show disparities in the case of BSA and
HSA, both WAR and PHEBU and especially OTA represented diverse
values. The studied compounds gave 0.30.6 alterations in log K
values between BSA and RSA. WARHSA and WARRSA complexes
show similar log K data; nevertheless PHEBU and OTA indicate very
high differences (0.4 and 1.5, respectively) in these two species
Table 1
Stability constants ( 7 SD) of the different albuminligand complexes.
Stability constants (log K)

Luteolin
OTA
Phenylbutazone
Warfarin

BSA

HSA

RSA

5.247 0.02
6.487 0.22
4.167 0.03
4.94 7 0.06

5.23 7 0.04
7.65 7 0.36
4.337 0.08
5.377 0.05

5.51 70.06
6.17 70.12
4.74 70.05
5.44 70.04

M. Por et al. / Journal of Luminescence 145 (2014) 767773

(HSA and RSA). Our results reveal that there cannot be established a
general rule concerning the binding afnity for the three types of
albumins. The previously published stability constants derived from
uorescence measurements or from equilibrium dialysis are consistent with our data [25,28,32,33,4457].
The major differences among the studied three species were
further supported by investigation of molecular displacement
experiments using OTAalbumin models (Fig. 5 and Table S1). At
the wavelengths used only free OTA and albumin-bound OTA gave
uorescence signal, therefore a decrease in uorescence polarization data indicates an increased concentration of the free form of
OTA (and a reduction in number of the bound form at the same

771

time) thus it reects the molecular displacement of OTA from the

surface of albumin. Fig. 5 represents that LUT is a weaker
competitor in the case of OTAHSA complex; it is not surprising
because of the very high afnity of OTA towards HSA (Table 1).
In the case of PHEBU, the curves of the polarization data (in view
of ligand concentration; Fig. 5) of the three OTAalbumin complexes are very similar however, there are major differences in
competing abilities: considerably higher displacement was
achieved by PHEBU for OTABSA than for OTAHSA or OTARSA
complexes. Furthermore, desorption of OTA from albumin by WAR
is the easiest from the RSA and the most difcult is from the BSA
complex. Our data suggest that there could be more than one

Fig. 5. Fluorescence polarization data (left) and desorbed fractions of OTA (right) in the case of OTAalbumin complexes in the presence of 25, 50 and 75 M competitor
concentrations (1 M OTA, 1.7 M albumin; wavelengths used: OTABSA: 393 nm-444 nm, OTAHSA: 393 nm-445 nm, and OTARSA: 391 nm-442 nm).

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M. Por et al. / Journal of Luminescence 145 (2014) 767773

Table 2
Stability constants of PHEBUalbumin complexes ( 7 SD) at ve different temperatures and calculated entropy and enthalpy changes (using Van't Hoff equation).
log K

BSA
HSA
RSA

20 1C

25 1C

30 1C

35 1C

40 1C

4.147 0.03
4.32 7 0.07
4.737 0.04

4.167 0.03
4.337 0.08
4.747 0.05

4.20 7 0.03
4.34 7 0.07
4.747 0.04

4.26 7 0.03
4.36 7 0.07
4.737 0.04

4.30 7 0.03
4.38 7 0.07
4.707 0.04

subtle difference among the three albumins when competitive

interactions are performed.
Table 2 shows that complex formations are entropy driven
processes in all three PHEBUalbumin complexes. Although the
entropy change associated to the complex formation between the
albumins and PHEBU overcompensates the enthalpy change, a considerable difference exists between RSA, BSA and HSA; RSA shows
attractive, while HSA and BSA show repulsive interaction towards
PHEBU. Since all the enthalpy changes reect weak albuminPHEBU
interaction, very low temperature-dependence of the stability constants is observed. As a result, the afnity of PHEBU towards RSA is
always the strongest; however the temperature dependence of RSAPHEBU is controversial compared to the temperature-dependence of
HSA or BSA PHEBU interaction (Table 2). Furthermore, we have to
highlight another property of these interactions: When the process is
such entropy driven, as seen in our case, the molecular environment
also plays an important role not only in determining the stability [58]
but in dening the kinetics of the molecular interactions as well [59].
Crystal structures for HSA are available through the RCSB
protein data bank [42] based on several studies [3639]. However,
the crystallographic studies related to complexes of HSA with
various ligands are more limited: warfarin (both enantiomers)
[60,61] and phenylbutazone [61] are available but no crystal
structures with ochratoxin A and luteolin could be found. BSA
crystal structures [40,41] have only been published in 2012, largely
because of the difculties of the crystallization process and the
diffraction method as well [40]. Due to these reasons no structure
of BSA complexes with our investigated ligands are available.
Crystal structure for RSA is not available at all, thus the only
possibility to obtain relatively easily an approximate structure is
homology modeling (ModBase database), which requires the
knowledge of the amino acid sequence of the protein in interest
and the availability of the structure of a similar protein (e.g. other
albumin species; from NCBI protein database).
Since only a few modeling studies are available involving the
examined ligands and the three different albumins [6264], with
comparative purposes molecular docking studies have also been
performed for all the possible albuminligand pairs. Essentially
the same trends based on the used scoring function have been
obtained (Table 3) as from the experimental investigations
presented here, taking into account the following notes and
considerations: without the knowledge of a crystallographic
structure of RSA it is expected that the results obtained from
modeling (i.e. ligand docking) is less reliable in the case of rat
albumin. In spite of this inaccuracy, the modeling study gave the
same trend for the binding afnity of OTA towards the albumins,
i.e. HSA 4 BSA4RSA. The approximate match is also true for the
other ligands (for trends see Tables 1 and 3) except for RSA
luteolin complex which can be explained with the partial uncertainty of the scoring function used in the modeling studies or with
the existence of a different binding site for luteolin in RSA.
Finally, the results of our investigation and the previously cited
studies [1624] highlight that the chemical structure of the ligand
and the albumin species itself has a large inuence on stability
constants and also on competing capacity. Most probably these

H (kJ mol  1)

S (J mol  1 K  1)

157 1
57 1
 37 2

1297 4
1007 2
817 6

Table 3
Values of the scoring function related to binding afnities of the ligands towards
albumins, evaluated by the neural network based NNScore 1.0 code [43] which has
been applied on the determined structures obtained by the AutoDock Vina program
[35]. The minimum value of the scoring function is  1.0 while the maximum is 1.0.
The value of 0.0 corresponds to log Ka 4.60. Accordingly, the negative values of the
scoring function correspond to log Ka o 4.60 while for the positive values log
Ka 4 4.60. In the proximity of the 0.0 value of the scoring function [  0.3, 0.5], the
change of log Ka is relatively small (log-sigmoid function).

HSA
BSA
RSA

OTA

Phenylbutazone

(R)-Warfarin

(S)-Warfarin

Luteolin

0.3900
0.3556
0.3283

 0.0368
 0.5315
0.0295

0.1172
 0.2034
0.0196

0.1068
 0.3030
0.1228

 0.0308
 0.0538
 0.2286

factors taken together are responsible for the major differences

seen in our study. Therefore, considering the possible important
role of the molecular environment in the entropy-driven processes, our results strongly suggest that it is an incorrect protocol
to extrapolate data derived from experiments with BSA to humans
or other species without conrmative measurements with the
actual albumin species.

5. Conclusion
In our study albuminligand interactions of Sudlow's site I
ligands in the case of bovine, human and rat serum albumin were
investigated using steady-state uorescence spectroscopy and
uorescence polarization techniques. Our results clearly represent
that there could be major differences between the three species
mainly because of the varying chemical structures of albumins.
Since numerous studies use bovine serum albumin to examine
albuminligand interactions and these results are commonly
extrapolated to human albumin we would like to emphasize that
BSA is not equal to HSA or to albumins of other species. As we have
proven at least some conrmatory measurements are needed
when dealing with albumins other than HSA.

Acknowledgments
Our study was supported by PTE OK-KA-2013/15. Financial
support of the Developing Competitiveness of Universities in the
South Transdanubian Region project (SROP-4.2.1.B-10/2/KONV2010-0002), SROP-4.2.2.A-11/1/KONV-2012-0065 and Supporting
Scientic Training of Talented Youth at the University of Pcs
(SROP-4.2.2/B-10/1-2010-0029) are highly appreciated.

Appendix A. Supplementary materials

Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jlumin.2013.08.059.

M. Por et al. / Journal of Luminescence 145 (2014) 767773

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