10442
J. Phys. Chem. B 2010, 114, 10442–10450
Reverse Micelle Induced Flipping of Binding Site and Efficiency of Albumin Protein with
an Ionic Styryl Dye
Dibakar Sahoo, Prosenjit Bhattacharya, and Sankar Chakravorti*
Department of Spectroscopy, Indian Association for the CultiVation of Science, JadaVpur,
Kolkata 700032, India
ReceiVed: April 1, 2010; ReVised Manuscript ReceiVed: July 6, 2010
The effect of reverse micelle environment on the binding mechanism of 2-(4-(dimethylamino)styryl)-1methylpyridinium iodide (DASPMI) with Bovine Serum Albumin (BSA) compared with that in buffer solution
has been investigated in this paper with the help of steady state and time-resolved emission spectroscopy
along with molecular docking to have a correct picture about binding. The binding of DASPMI with attachment
efficiency of 30% and 70% at site I (subdomain IIA) and site II (subdomain IIIA) of BSA, respectively, in
buffer solution gets reversed inside a reverse micelle. The bigger cavity size of site II in buffer solution
ushers the dye with increased attachment efficiency and in reverse micelle change in π-stacking and hydrophobic
interaction control the attachment efficiency. The calculated Förster distance gets curtailed as the environment
changes from buffer to reverse micelle. The binding becomes stronger with a smaller gap between the probe
and Trp-214 inside the reverse micelle than that in buffer solution.
Introduction
Reverse micelles are self-organized aggregates formed by
surfactants in organic solvent, and nanometer-sized water pools
are formed by the solubilization of water in their polar cores.
The reverse micelles that are mostly with spherical shape are
usually formed in ternary surfactant-water-organic solvent
mixtures including surfactants (<10%), water (0-10%), and
organic solvent (80%-90%), so the reverse micelles are also
called water-in-oil emulsions or Winsor II emulsions.1 Reverse
micelles are generally smaller than their hydrophilic counterparts
(micelles), and their aggregation number is commonly lower
than 50.2 Moreover, reverse micellar systems are colloidal
solutions, so characteristic properties of these systems are
thermodynamic stability (no phase separation with time),
spontaneous formation, low interfacial tension (<10-2 mN · m-1),
transparent nature (nanometer size < 100 nm), large surface area
(102-103 m2 · cm-3), viscosity comparable with pure organic
solvents, and highly dynamic (constantly a collision and a fusion
with each other, and occasionally the fusion surfactant molecules
and the contents inside reverse micelles exchange).3 The
microwater pools inside reverse micelles are stabilized by a
surfactant monolayer within an organic continuum, which can
solubilize hydrophilic biomolecules such as proteins, enzymes,
DNA, and amino acids. In reverse micellar systems, the
biomolecules inside the polar core of the surfactant monolayer
are protected from denaturation by organic solvent. Therefore,
protein solubilization in reverse micelles plays a key role in a
number of topics of biotechnology research. Currently, reverse
micelles are used as reaction systems for enzymatic catalysis,4
as models of membrane system separation of proteins,5 for
solvent-based extraction of proteins,6 as a microsurrounding for
protein structure discovery,7 and for protein refolding.8,9 In the
past three decades, the application of reverse micelles for
bioseparation has attracted considerable attention because the
technique is considered to be potentially useful in downstream
processing for a large-scale separation of biomolecules from
* Corresponding author. E-mail: [email protected].
fermentation mixtures. The encapsulation of molecules and
particles by reverse micelles can provide separation media, and
study of these systems is useful for information in drug
designing and delivery.10 Proteins and enzymes are solubilized
into reverse micelles, maintaining their activities and native
structures, and can be back-extracted.11,12 It has been found that
the factors such as water content (the molar ratio of water to
surfactant, namely, W0) and micelle size,13 aqueous phase pH
and ionic strength,14 surfactant type and concentration,15 and
cosurfactant16 affect the protein solubilization and characteristics
based on the interactions between reverse micelles and proteins.
Moreover, adding proteins to the reverse micelles can alter
surfactant self-assembly and phase behavior which can affect
the protein efficiency. Therefore, it is intriguing to explore the
nature of the protein kinetics in the well-known reverse micellar
systems. The qualitative and quantitative detection of binding
characteristic in reverse micelles may help in designing efficient
drug sensitizers for photodynamic therapy (PDT).17 The extended photodynamic action depends on the biodistribution of
the probe molecule in the cytoplasmic and mitochondrial
membranes,18,19 the retention, and the nature of the binding
inside the cell.
Albumins being the most abundant proteins in plasma have
the ability to carry drugs20 as well as to be used as endogenous
and exogenous substances. Bovine Serum albumin (BSA) has
three major domains, each with two subdomains (Figure 1).
Major binding sites, namely, site I and site II, are located at
subdomains IIA and IIIA.21 BSA has three intrinsic fluorophores,
tryptophan (Trp), tyrosine (Tyr), and phenylalanine, of which
Trp 214 is located in site I (subdomain IIA) and Tyr 411 in site
II (subdomain IIIA). Of the three chromophores of BSA, Trp
is the key player; phenylalanine has feeble fluorescence; and
the fluorescence of Tyr is almost totally quenched if it is ionized
or near an amino group, a carboxyl group, or a tryptophan
residue.22,23
Ionic styryl dyes are known to respond to changes in
transmembrane potential by a fast electrostatic mechanism.24
The shift of charge of the ionic dyes from ground state to excited
10.1021/jp102937y  2010 American Chemical Society
Published on Web 07/22/2010
Albumin Protein with an Ionic Styryl Dye
J. Phys. Chem. B, Vol. 114, No. 32, 2010 10443
CHART 1: Molecular Structure of DASPMI
Figure 1. Crystal structure of HSA (which is like BSA) and the
location of different domain binding sites. The location of hydrophobic
binding sites (site I and site II) is indicated. The position of the
Tryptophan residue (Trp-214) is shown.
state coupled with electric field within a cell membrane results
in electrochromism.25,26 Among these dyes, the fluorescence
intensity of 2-(4-(dimethylamino)styryl)-1-methylpyridinium
iodide (DASPMI) is a dynamic measure for the membrane
potential of mitochondria27,28 in living cells. DASPMI has an
interesting multibond rotation involved intramolecular charge
transfer photophysical property,29,30 which is dependent on both
polarity and microviscosity of the medium.31,32 This dye has
also been used in polymer science and in cell biology33-35
because of the strong dependence of its photophysics on
viscosity and polarity. DASPMI can interact with DNA as a
groove binder.36 One would think here of investigating the nature
of binding of DASPMI with protein environment.
Considering interesting photophysical properties of DASPMI
and its possible interaction with protein, we intend to explore
in this article the binding sites and attachment efficiencies. It is
also envisaged to monitor the effect of reverse micelle, a
biomimetic for cells, on the DASPMI-protein binding mechanism and efficiency of attachment once it is inserted inside
the reverse micelle.
Experimental Section
Materials and Methods. 2-(4-(Dimethylamino)styryl)-1methylpyridinium iodide (DASPMI) (Chart 1) was received
from Aldrich Chemical, USA, and purified by column chromatography (on silica gel 60-120; 5% ethyl acetate in
petroleum ether). The purity of the compound was checked by
thin layer chromatography (TLC). The compound was then
subjected to vacuum sublimation before use. n-Heptane (spectroscopy grade), from Aldrich, and AOT (ultra grade) from
Sigma were used as received. The molar ratio of residual water/
AOT, as determined by Karl Fischer titration, was found to be
0.1. Millipore water was used in the preparation of water-in-oil
microemulsion.
The absorption spectra were taken with a Shimadzu UV-vis
absorption spectrophotometer model UV-2401PC. The fluorescence spectra were obtained with a Hitachi F-4500 fluorescence
spectrophotometer. Quantum yields were determined by using
the secondary standard method (φf ) 0.23) with recrystallized
β-naphthol in MCH (methylcyclohexane), and details of the
process are described elsewhere.37,38 For lifetime measurement,
the sample was excited (at 440 nm: optical density ∼0.15) with
a picosecond diode (IBH Nanoled-07). The time-correlated
single photon counting (TCSPC) setup consists of Ortec 9327,
TBX-04 detector, DataStation measurement software, and DSA6
Foundation Package. The data were collected with a DAQ card
as a multichannel analyzer. The typical fwhm of the system
response is about 80 ps. Typical slit width ∼30 nm, monochromator type Jobin-Yvon, number of channels 4000 (6 ps per
channel), window width ∼24.5 ns, and number of counts
∼10 000 were used in taking decay profiles. Data analysis was
carried out using the curve-fitting program supplied by the
manufacturer. The quality of the fit was determined by the
reduced χ2 and a high Durbin-Watson parameter (>1.7).39
For measurement of fluorescence, depolarization decays
parallel (IVV) and perpendicular (IVH) were collected in an
alternating manner for equal amounts of time until at least
10 000 fluorescence counts were collected in the peak channel
of IVV. The time -dependent fluorescence anisotropy, r(t) was
then calculated from the above data using the following relation
r(t) ) [IVV - GIVH]/[IVV + 2GIVH]
(1)
where G is the ratio between the fluorescence intensity at parallel
and perpendicular polarizations of the emission with respect to
the excitation beam. The value of G has been used as 0.56 as
identified for the instrument.
Sample Preparation. Microemulsion solutions of desired w0
were obtained by adding concentrated protein solutions, drug
solutions, or plain buffer (pH 7.0) to a 0.1 M AOT solution in
n-heptane. Volume of additivity was assumed in calculating
AOT concentration and w0 values. The samples were gently
shaken until complete clarification. The final sample concentration was calculated according to the total volume of the
microemulsion. A solution of DASPMI (5 × 10-5 M) was
prepared in n-heptane. Binary mixtures of n-heptane/AOT were
prepared by adding the required amount of AOT into 5 mL
each of the second stock solution. We used the stock solution
containing 5 × 10-5 M dye in a binary solution of 0.1 M AOT
in n-heptane for the preparation of n-heptane/AOT/water
mixtures. For a different value of W, the solution was prepared
by adding an appropriate amount of Millipore water, using
calibrated micropipets, to the binary stock solution. To get a
homogeneous mixture, we sonicated the solutions for 3-5 min,
and the samples were kept for 15-20 h at room temperature
before carrying out all the measurements.
Calculation of Binding Constant. The binding constant
values (total) due to attachment with Trp (site I) and Tyr (site
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J. Phys. Chem. B, Vol. 114, No. 32, 2010
Sahoo et al.
[
knr ) kr
Figure 2. Emission spectra of DASPMI as a function of BSA
concentration (1) 0 µM, (2) 2 µM, (3) 7 µM, (4) 10 µM, (5) 14 µM,
(6) 15 µM, and (7) 17 µM. Inset shows the plot of (FR - F0)/(Fx - F0)
against [L]-1 for BSA.
II) have been determined from the fluorescence intensity data
of DASPMI considering the following rearranged equation of
the original one developed by Benesi and Hildebrand based on
1:1 probe protein complexation40
[(F∞ - F0)/(Fx - F0)] - 1 ) (Kb[L])-1
(2)
where F0, Fx, and F∞ are the fluorescence intensities of DASPMI
in the absence of protein, at an intermediate protein concentration, and at a protein concentration when the interaction is
complete, respectively. Kb is the binding constant, and [L] is
the free concentration of protein.
A plot of [(F∞ - F0)/(Fx - F0)] against [L]-1 for BSA shows
linear variations (Figure 2), justifying the validity of the above
equation and hence confirming one-to-one interaction between
the probe and proteins. The binding constant is determined from
the slope of the plot. To get the free protein concentration from
total protein concentration, we adopted a self-consistent approach similar to the one used during the orbital calculations.
Calculation of Thermodynamic Parameters. The thermodynamic parameter associated with the complexation between
SQ and BSA was determined using the following equations41
lnK ) -∆H/(RT) + ∆S/R
(3)
∆G ) ∆H - T∆S ) -RTln K
(4)
]
1
-1
φf
(6)
Cyclic voltammograms (CVs) were recorded on a BAS-CV50W
cyclic voltameter. Circular dichroism (CD) spectra were recorded on a Jasco Corporation, J-815, spectrophotometer. The
fluorescence picosecond lifetime measurement was done with
a Horiba Jobin Yvon Fluoro Cube 01-NL time-resolved
Fluorescence Lifetime Spectrometer with TBX-04 detector,
DataStation measurement software, and DSA6 Foundation
Package, and the excitation was done at 440 nm (diode laser).
Millipore water was used in all the studies. All experiments
were carried out at room temperature (26 ( 1 °C).
Nuclear magnetic resonance data were taken with Bruker
Avance DPX 300 in deuterium oxide as solvent.
Scanning Electron Microscopy (SEM). BSA solution in the
presence and absence of DASPMI was placed on sample studs
and coated with platinum by ion sputtering. FESEM images
were obtained on a JSM-6700F, from JEOL, scanning electron
microscope with an accelerating voltage of 15 KV. The sample
(DASPMI interacted with BSA) was dried in a vacuum and
kept overnight before taking the SEM.
The crystal structure of HSA is taken from the Brookhaven
Protein Data Bank (entry code 1H9Z)43 as it gives a nearly
similar structural nature to BSA (Figure 1). The potential of
the 3D structure of HSA was assigned according to the Amber
4.0 force field with Kollman-all-atom charges. We took help
from molecular modeling software Sybyl 6.944 for generating
the initial structure of all molecules. The geometry of the
molecule was subsequently optimized to minimal energy using
the Tripos force field with Gasteiger-Marsili charges. The
FlexX program was used to build the interaction modes between
DASPMI and HSA.
Results and Discussion
The effect of BSA on the fluorescence of DASPMI in 0.01
M phosphate buffer at 26 °C is shown in Figure 2. The increase
in DASPMI fluorescence as a function of protein concentration
reaches a plateau, which indicates the increment of rigidity of
the surrounding microenvironments. The BSA binding sites are
very effective in preventing “free rotator motions” in the
DASPMI moiety. The excited state of DASPMI when complexed with BSA is less stabilized than the corresponding ground
state compared to that in uncomplexed form in water, resulting
in an increase of energy gap between the excited state and
where ∆H is enthalpy change; ∆S is entropy change; ∆G is
change in free energy; and K is the binding constant.
Calculation of Radiative and Nonradiative Rate Constant.
To calculate the radiative decay rate constant (kr), eq 5 was
used42
kr )
φf
τf
(5)
where kr is radiative decay rate constant; φf ) fluorescence
quantum yield of DASPMI; and τf is lifetime of DASPMI.
Nonradiative decay rate constant knr can be determined using
eq 6
Figure 3. Time-resolved fluorescence decays of DASPMI, in aqueous
buffer, with BSA in AOT solution, in BSA of buffer solution [BSA]
) 15 µM.
Albumin Protein with an Ionic Styryl Dye
J. Phys. Chem. B, Vol. 114, No. 32, 2010 10445
TABLE 1: Photophysical Properties and Binding Constant of DASPMI in Buffer and AOT Solution in the Absence and
Presence of BSA ([BSA] ) 15 µM)
DASPMI
DASPMI + BSA
in buffer
DASPMI + BSA
in AOT
τ1
τ2
Kr × 10-9
Knr × 10-9
Kb
Φf
(ns)
(ns)
(S-1)
(S-1)
(M-1)
0.018
0.04
1.4 ( 0.03 (20%)
4.8 ( 0.05 (30%)
0.06 ( 0.05 (80%)
2.4 ( 0.1 (70%)
0.056
0.013
3.06
0.3
(2.15 ( 0.2) × 104
0.06
4.2 ( 0.07 (60%)
2.2 ( 0.09 (40%)
0.017
0.27
(5.1 ( 0.3) × 105
TABLE 2: Thermodynamic Parameters of the System
BSA-DASPMI in Buffer Solution at Different Temperatures
T
binding constant
(K)
(Kb × 10 )
(kJ mol )
(kJ mol )
(J mol-1 K-1)
298
303
310
315
2.15 ( 0.2
1.56 ( 0.1
1.31 ( 0.3
1.21 ( 0.1
-14.54
-24.81
-24.78
-24.74
-24.72
34.34
4
∆H
∆G
-1
∆S
-1
ground state and a consequent blue shift. This increase of energy
gap also decreases the efficiency of radiationless deactivation
of DASPMI, a process involving a low-lying twisted intramolecular charge transfer state (TICT),45 and leads to a remarkable
enhancement in its fluorescence quantum yield. Picosecond timeresolved fluorescence analysis indicates that in buffer, at pH 7,
DASPMI exhibits a biexponential decay with a lifetime of 1.4
ns (20%) and 0.06 ns (80%), but addition of BSA causes a
change in decay time at 4.8 ns (30%) and 2.4 ns (70%) (Figure
3). The computed nonradiative and radiative decay constants
of DASPMI from the quantum yield and lifetimes are found to
be 3.06 × 109 and 0.056 × 109 in the absence of BSA, and in
the presence of BSA, they are 0.3 × 109 and 0.012 × 109,
respectively (Table 1).
Considering 1:1 stoichiometry for the complex formation, the
binding constant between DASPMI and protein (BSA) is
calculated to be 2.15 × 104 M-1, and the corresponding free
energy change is -24.81 kJ mol-1. The ∆H0 and ∆S0 values
for the binding reaction between DASPMI and BSA were found
to be -14.54 kJ mol-1 and 34.34 J mol-1 K-1 (Table 2). The
negative ∆G means that the binding process was spontaneous,
and formation of the DASPMI-BSA coordination compound
was an exothermic reaction accompanied by a positive ∆S0
value. However, ∆H0 might play a role in the electrostatic
reaction. So, the binding process of DASPMI to BSA involves
hydrophobic interaction strongly as evidenced by positive values
of ∆S0, but electrostatic interaction could also not be excluded.
The binding of DASPMI with BSA is further confirmed by
Field Emission Scanning Microscope (FESEM), Cyclic Voltametry (CV), Circular Dichroic Spectra (CD), and 1H NMR
techniques. The FESEM images show that BSA alone has a
regular structure, and this drastically changes upon addition of
DASPMI, which is reflected in a drastic increase of width of
the FESEM image of BSA alone of 2 ( 0.1 nm to a mean
width of 70 ( 10 nm upon complexation of BSA with DASPMI
(Figure 4). In the present case, the repulsion between DASPMI
molecules is less as the cationic dye DASPMI loses its positive
charges after interaction with negative residue Glu 292 and Glu
450 in site I and site II of BSA. The spots observed in the
FESEM image point to the binding of protein and dye which is
very similar to the binding of protein and DP/DNSA (Figure
4). In CV there is a noticeable decrease in current intensity (314
µA) upon addition of BSA with DASPMI compared to that of
DASPMI alone (Figure 5). In CD spectra the significant decrease
of negative ellipticity after addition of DASPMI into BSA
indicates that the binding of DASPMI induces the R-helical
structure of protein. The decrement of R-helices by 20% from free
BSA in buffer to bound BSA with DASPMI suggests that binding
of DASPMI to BSA brings forth an alteration of the secondary
structure of the protein significantly (Figure 6). An upfield shift of
about ∆δ 0.03 ppm due to the H atom of CdC in the 1H NMR
spectrum was observed in the presence of BSA (Supporting
Information). All of these above-mentioned phenomena confirm
the formation of a stable and noncovalent complex between
DASPMI and BSA. In all the measurements, we have done the
control experiments with site-selective binding ligands, viz., DP
and DNSA. In BSA, when we add DP or DNSA, we get the same
data/images as that with DASPMI which confirms binding of the
probe with BSA (Supporting Information).
The anisotropy measurement of DASPMI in varying concentration of BSA also gives an indication of binding with BSA.
An increment in anisotropy value of the emission of DASPMI
with BSA concentration (Figure 7) implies an imposed motional
restriction on the fluorophore in the protein environment, leading
to the reduction of tumbling motion and greater binding
interaction between the probe and BSA (Table 3).
Exploring the binding site of any biologically active probe in
proteins is the crucial factor for understanding the efficiency of
the probe as a therapeutic agent. To know the sites in which the
dye gets attached, we determined the micropolarity around the
probe in different states of the proteins, that is, at native (N),
intermediate (Int), and unfolded (U) states. Figure 8 and Table 4
show that the difference in micropolarity values for the N-Int
transition (involving domains I and II) is nearly same as for the
Int-U transition (involving domain II). The micropolarity of
domain II and III is about 51.7 in terms of ET(30),46 which is very
close to our measured values. So it may be inferred that the probe
is located in domains II and III, as they are more hydrophobic
than domain I.47 As Trp is the most active moiety in BSA, the
Förster resonance energy transfer (FRET) from the Tryptophan
(Trp) moiety in BSA to DASPMI has been computed as 27%,
which suggests that the probe is located near the Trp moiety of
domain II.
Quenching of BSA fluorescence in buffer as well as in AOT
reverse micelles (to be discussed in a later section) indicates
that the probe molecule binds in site I. The distance between
the donor (BSA) and acceptor (probe) can be calculated
according to Förster’s theory for resonance energy transfer
(FRET).48 The efficiency of energy transfer, E, is related to the
distance (rAD) between the donor and acceptor probe by
E)
R60
6
R60 + rAD
)1-
( )
F
F0
(7)
where R0 is the Förster distance (critical distance) when the
efficiency of energy transfer is 50%. F and F0 are the
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J. Phys. Chem. B, Vol. 114, No. 32, 2010
Sahoo et al.
Figure 4. SEM images of (a) BSA (15 µM) alone and (b) BSA (15 µM) with DASPMI and (c) BSA (15 µM) with DNSA.
Figure 5. Cyclic voltammogram (CV) of (1) DASPMI (0.05 mM)
alone and (2) DASPMI in the presence of BSA (15 µM).
Figure 7. Variation of fluorescence anisotropy (r) of DASPMI with
increasing concentration of BSA.
TABLE 3: Fluorescence Anisotropy (r) of DASPMI in
Different Concentration of BSA in Buffer
concn of BSA (µM)
anisotropy value (r)
2
0.12
4
0.17
7
0.22
10
0.26
15
0.3
where F(λ) is the fluorescence intensity of the donor at the
wavelength λ, and ε(λ) is the molar absorption coefficient of the
acceptor at wavelength λ.
J can be evaluated by integrating the overlapped portion of
the spectra in Figure 9, and the value of κ2 equals 2/349 if both
the donor and acceptor tumble rapidly and free to assume to
any orientation. Here n ) 1.4 for proteins generally, and φD )
Figure 6. CD spectra of BSA in buffer solution as a function of
DASPMI concentration at (a) 0 mM, (b) 0.02 mM, (c) 0.07 mM, and
(d) DASPMI alone.
fluorescence intensities of BSA in the presence and absence of
quencher, respectively. The value of R0 can be calculated from
R0 ) 0.211(κ2n-4φDJ)1/6
(8)
where κ2 is the special orientation factor between the emission
dipole of the donor and the absorption dipole of the acceptor; n is
the refractive index of the medium; φD is the fluorescence quantum
yield of the donor; and J is the overlap integral of the fluorescence
emission spectrum of the donor and the absorption spectrum of
the acceptor and is given by
J)
∑ F(λ)ε(λ)λ4
∆λ
∑ F(λ)∆λ
(9)
Figure 8. Fluorescence spectra of BSA-bound DASPMI as a function
of added urea. Curves 1f6 correspond to 0.0, 3.0, 5.0, 7.0, 8.0, and
9.0 M urea. Inset shows the variation of emission maximum (λemmax)
of DASPMI in different solvents against ET(30). (1), (2), and (3) give
the interpolated λemmax values of native (N), intermediate (int), and
unfolded (U) states of BSA.
Albumin Protein with an Ionic Styryl Dye
J. Phys. Chem. B, Vol. 114, No. 32, 2010 10447
TABLE 4: Micropolarity Values in Terms of ET(30) at
Different States
different states of protein
BSA
native (N)
intermediate (int)
unfolded (U)
51.8
54.8
59.1
0.11 using the above-mentioned values R0 ) 1.1 nm, E ) 27%,
and rAD) 1.2 nm in buffer solution (Table 5).
The donor to acceptor distance in both environments is less
than 7 nm, indicating a static quenching interaction between
the donor and acceptor according to Förster’s nonradiative
energy transfer theory.48
To understand and confirm the site-selective binding of
DASPMI with BSA, known site selective binding ligands,
dansylamide (DNSA) for site I (subdomain IIA) and dansylproline (DP) for site II (subdomain IIIA)50 were used. Initial
addition of DNSA in the BSA-DASPMI complex showed a
gradual decrease in fluorescence intensity and reached saturation
at 0.7 mM (Figure 10a). In this process, DNSA effectively
displaced 30% of DASPMI from the BSA-DASPMI complex.
A similar experiment was done on the BSA-DASPMI complex
with DP which showed a displacement of 70% of DASPMI
from the BSA-DASPMI complex by DP (Figure 10b). No
noticeable change in intensity due to the titration of DNSA and
DP with DASPMI (Figures 11a and b) indicates all the changes
that were observed earlier were due to displacement of DASPMI
from the BSA-DASPMI complex by binding ligands. The
above observation unambiguously helps us to conclude that the
dye can bind with site I as well as site II of BSA. This is further
corroborated by the observation of the biexponential lifetime
having values 4.8 ns (30%) and 2.4 ns (70%).
The increase in fluorescence quantum yield along with a
hypsochromic shift of emission spectra with the addition of BSA
reflects that the microenvironments around the fluorophore in
the protein solutions are quite different from those in pure
aqueous solution. Possibly the small cavity size (2.53 Å) of the
Figure 10. (a) Fluorescence spectra of DASPMI in buffer solution
with the addition of BSA (1) 0 µM, (2) 2 µM, (3) 7 µM, (4) 10 µM,
and (5) 15 µM followed by the addition of DNSA (6) 0.01 mM, (7)
0.03 mM, (8) 0.05 mM, (9) 0.07 mM, (10) 0.08 mM, and (11) 0.09
mM. (b) Fluorescence spectra of DASPMI in buffer solution with the
addition of BSA (1) 0 µM, (2) 2 µM, (3) 7 µM, (4) 10 µM, and (5) 15
µM followed by the addition of DP (6) 0.01 mM, (7) 0.03 mM, (8)
0.05 mM, (9) 0.07 mM, (10) 0.08 mM, and (11) 0.09 mM. (c)
Percentage of DASPMI attached in site I and site II of BSA in buffer
solution.
Figure 9. Overlap of the fluorescence spectrum of BSA (dotted line)
with absorption spectra (solid line) of DASPMI. The concentration of
BSA and DASPMI are 15 µM and 5 × 10-5 M, respectively.
TABLE 5: Calculated Parameters for the BSA/DASPMI
Complex in Buffer Solution and in the Reverse Micelle
parameters
in buffer
in reverse micelle
E
R0 (nm)
rAD (nm)
0.27
1.1
1.2
0.36
0.94
0.99
site (I) causes the formation of a tight complex with DASPMI
having π stacking and hydrophobic interactions. The aromatic
rings form hydrophobic interaction with residues Leu 219, Phe
223, Leu 234, Leu 238, Leu 260, Ala 261, Ile 264, Ile 290, Ala
291, and the hydrocarbon chain of Glu 292.51,52 Also from
lifetime data the 30% longer lifetime (4.8 ns) indicates that
DASPMI binds with BSA in site I by 30%. This is further
confirmed by binding of the ligand DNSA by displacing 30%
of DASPMI from the BSA-DASPMI complex. The relatively
larger cavity size of 2.6 Å of site II involves binding with
DASPMI by hydrogen bonding and hydrophobic and electrostatic interaction. Site II binds DASPMI twice as much as site
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J. Phys. Chem. B, Vol. 114, No. 32, 2010
Figure 11. (a) Emission spectra of DASPMI as a function of
dansylamide (DNSA) concentration (1) 0 mM, (2) 0.4 mM, and (3)
0.7 mM. (b) Emission spectra of DASPMI as a function of dansylproline
(DP) concentration (1) 0 mM, (2) 0.4 mM, and (3) 0.7 mM.
I does because site II contains tyrosine residue. The aromatic
ring of tyrosine contains a hydroxyl group. This hydroxyl group
is very much reactive; however, in site I the tryptophan has no
hydroxyl group, so the side chain is inert. In site II, the binding
Figure 12. Molecular docking of the DASPMI-BSA complex.
Sahoo et al.
pocket of the hydrophobic portion of the aromatic ring of
DASPMI is packed against Pro 384, Leu 387, Ile 388, Phe 395,
Leu 407, Leu 430, Val 433, Ala 449, Leu 453 and also with
hydrocarbon chains of Arg 485 and Glu 450. As there is no
tryptophan residue in the site II, a relatively loose complex is
formed with DASPMI at this site. The strong binding in site I
is due to the larger π-system of the tryptophan residue allowing
a more pronounced π-stacking effect. This loose binding led
us to observe the major component of 70% with a shorter
lifetime of 2.4 ns, which is also further corroborated by the
70% displacement of DASPMI from the BSA-DASPMI
complex by DP.
As binding sites share a common interface, the ligand bound
to site II affects the conformational changes, as well as binding
affinities in site I. Trp 214, conserved in mammalian albumins,
plays an important structural role in the formation of site I by
solvent accessibility, and it participates in an additional hydrophobic packing interaction between the site I and site II interface.
Molecular Docking of the DASPMI-BSA Complex. To
establish which binding site of BSA DASPMI is located in, the
complementary applications of molecular docking computed by
Auto Dock 3.05 have been employed to improve the understanding of the interaction of DASPMI and BSA. For more
accurate docking, we modified the parameters which were 150
for ga_pop_size and 10 000 000 for ga_num_evals. The distance
between the Trp and DASPMI is 6.7 Å (0.67 nm) from
molecular docking, which is close to that from FRET calculation
of experimental data (0.1.2 nm). The DASPMI molecule is
situated in a cavity formed by Trp 214, Lys 195, Tyr 150, Lys
190, Glu 153, Gly 188, Leu 198, His 288, Ser 454, and Arg
197 (Figure 12). The residues close to the probe are mainly
hydrophobic which indicates the existence of hydrophobic
interaction between the residue and probe. The interaction
between the BSA and the ligand is not exclusively hydrophobic
in nature since several ionic and polar residues in the proximity
of the ligand play an important role in stabilizing the probe
molecule via hydrogen bond and electrostatic interaction. The
formation of the hydrogen bond causes a decrease in the
hydrophilicity and an increase in the hydrophobicity which
Albumin Protein with an Ionic Styryl Dye
J. Phys. Chem. B, Vol. 114, No. 32, 2010 10449
TABLE 6: Thermodynamic Parameters of the System
BSA-DASPMI in Reverse Micellar Environment at
Different Temperatures
T
binding constant
(K)
(Kb × 10 )
(kJ mol )
(kJ mol )
(J mol-1 K-1)
298
303
310
315
5.1 ( 0.3
4.7 ( 0.1
4.1 ( 0.3
3.8 ( 0.2
-15.09
-32.67
-33.0
-33.4
-3.8
58
5
∆H
∆G
-1
∆S
-1
stabilize in the DASPMI-BSA system. The calculated binding
free energy (G) for the Trp moiety is (-26.6 kJ mol-1), which
is near the experimental data (-24.81 kJ mol-1). The difference
in Gibbs free energy comes from limitation of the docking
software, which can only handle docking in the single position,
whereas in reality DASPMI binds BSA in two different sites
which has already been proved by site-detecting probes (vide
supra).
Interaction in Reverse Micelle Environment. As the
objective of our study is for a possible application in biological
systems, we investigated all the binding characteristics of
DASPMI with BSA within a model biological system, AOT
reverse micelle. The suitable value of water pool (W0) of
microemulsion for BSA’s stability was found to be by trial
method better for W0 ) 25. Within the water pool, the quenching
rate of BSA with addition of DASPMI is higher than that in
buffer solution (outside the RM). Interestingly, a large change
in spectroscopic parameters like quantum yield, lifetimes, and
radiative and nonradiative decay constant could be observed
inside the reverse micelle compared to that in buffer solution
(Table 1). The binding constant (5.1 × 105) of the BSA-DASPMI
complex is higher in microemulsion than that in buffer solution,
and the change in free energy is -32.67 kJ mol-1 in microemulsion environment. The ∆H0 and ∆S0 values for the binding
reaction between DASPMI and BSA in the microemulsion
environment were found to be -15.09 kJ mol-1 and 58 J mol-1
K-1 (Table 6).
In buffer solution, we used site-selective binding ligand
DNSA and DP in the BSA-DASPMI complex to know the
attachment efficiency; similarly, DNSA was added to the
BSA-DASPMI complex in microemulsion (RM), and we have
found an effective 60% displacement of DASPMI from the
BSA-DASPMI complex (Figure 13a). A similar experiment
with dansylproline (DP) showed that about 40% of DASPMI
has been displaced by DP (Figure 13b). The greater binding
constant in microemulsion than that in buffer solution indicates
greater accessibility of DASPMI toward BSA when BSA is
encapsulated in microemulsion, which possibly is due to the
different nature of water in microemulsion. In AOT microemulsion in the presence of BSA, DASPMI emission shows a
biexponential decay with lifetime values 4.2 ns (60%) and 2.2
ns (40%) compared to the values 4.8 ns (30%) and 2.4 ns (70%)
in buffer. The different attachment efficiency in two sites as
the protein and dye are placed inside RM compared to that in
buffer may be due to one or the combination of the following
reasons: (i) the water activity may change due to the specific
RM milieu, which may have a profound effect on different sites
of protein, as water is considered as an integral part of proteins;
(ii) the dielectric constant of water in RM is different than that
of bulk water (for W0 ) 25, dielectric constant is ∼23); (iii)
the small scale of inner volume of the reverse micelle provides
a “confined space” effect on the cavity of different sites of
protein; (iv) the concentration of ions inside the water core is
higher than that of bulk water which differs the π stacking,
Figure 13. (a) Fluorescence spectra of DASPMI in AOT solution with
the addition of BSA (1) 0 µM, (2) 2 µM, (3) 7 µM, (4) 10 µM, and (5)
15 µM followed by the addition of DNSA (6) 0.01 mM, (7) 0.03 mM,
(8) 0.05 mM, (9) 0.07 mM, (10) 0.08 mM, and (11) 0.09 mM. (b)
Fluorescence spectra of DASPMI in AOT solution with the addition
of BSA (1) 0 µM, (2) 2 µM, (3) 7 µM, (4) 10 µM, and (5) 15 µM
followed by the addition of DP (6) 0.01 mM, (7) 0.03 mM, (8) 0.05
mM, (9) 0.07 mM, (10) 0.08 mM, and (11) 0.09 mM. (c) Percentage
of DASPMI attached in site I and site II of BSA in AOT solution.
hydrophobic interaction, hydrogen bonding, and electrostatic
interaction of protein.
According to Förster’s theory for resonance energy transfer
(FRET) from BSA to DASPMI in microemulsion, the value of
R0, the Förster distance, and rAD, the distance between the donor
and acceptor probe, are found to be 0.94 and 0.99 nm,
respectively. The calculated FRET efficiency in microemulsion
is also seen to be increased to 36% from that of 27% in buffer
solution (Table 4). The greater FRET efficiency and smaller
donor-acceptor distance in RM also indicate that there is a
stronger binding between the BSA-DASPMI complex in RM
than in buffer.
10450
J. Phys. Chem. B, Vol. 114, No. 32, 2010
Conclusions
The most important upshot of the investigation is that we
have demonstrated the site-selective binding of the probe in both
water and microemulsion and reversal of efficiency of interaction
of BSA with DASPMI as BSA moves inside microemulsion, a
realistic biological environment; i.e., subdomain IIA is doubly
active inside RM compared to that in buffer solution, and the
activity of subdomain IIIA in RM is halved compared to that
in buffer solution. In general, binding efficiency also increases
along with a decrease in donor-acceptor distance inside RM.
This in situ knowledge is very important for drug designing
and its delivery, particularly in PDT, and this investigation helps
us to design an efficient drug sensitizer.
Acknowledgment. The authors express thanks to Mr. Subrata
Das, Department of Spectroscopy, I.A.C.S, for taking picosecond
time-resolved data.
Supporting Information Available: Additional experimental details. This material is available free of charge via the
Internet at http://pubs.acs.org.
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