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Deciphering the role of pH in the binding of Ciprofloxacin Hydrochloride to

Bovine Serum Albumin

Article  in  Physical Chemistry Chemical Physics · February 2012

DOI: 10.1039/c2cp00001f · Source: PubMed

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Deciphering the role of pH in the binding of Ciprofloxacin Hydrochloride
to Bovine Serum Albumin
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Uttam Anand, Lisha Kurup and Saptarshi Mukherjee*

Received 1st January 2012, Accepted 6th February 2012
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F

DOI: 10.1039/c2cp00001f

The effect of the added fluoroquinolone, Ciprofloxacin Hydrochloride (CpH), on structural

properties of Bovine Serum Albumin (BSA) was investigated by Circular Dichroism (CD),
steady-state, time-resolved and Dynamic Light Scattering (DLS) spectroscopic approaches. The
intrinsic fluorescence of the Tryptophan (Trp) amino acid residue in the globular protein BSA
was made use of and the effect of pH at two different temperatures was thoroughly investigated.
CD results indicate that CpH induces some structural changes in BSA and this has been well-
supported by steady-state, lifetime and DLS data. The fluorescence intensity of Trp gradually
decreases with the rise in concentration of CpH and we have conclusively proved that at pH 7.4
and 9.2, the mechanism of fluorescence quenching is mostly dynamic in nature, whereas at pH 4.5
mainly static quenching is operational. Thermodynamic parameters have been studied to
rationalize the nature of binding of CpH to BSA, and we have concluded that hydrophobic and
van der Waals forces play an important role in the process of drug–protein interaction at three
different pH values. The lifetime of Trp was found to decrease with the rise in CpH concentration
and the percentage reduction in lifetime was found to be a function of the pH of the medium
under investigation.

Introduction
Fluoroquinolones are a group of broad-spectrum antibacterial
agents with a unique mechanism of action and wider clinical
use. The interaction of such antibiotics with lipid membranes
has been widely studied of late.1–4 The mechanism of action of
such fluoroquinolones has been primarily attributed to be via
both hydrophobic3 and hydrophilic4 pathways, and the latter
is mainly responsible for the route by which they enter the
cytoplasm. In a very seminal work, Bedard and Bryan have
Scheme 1
shown that both ionic and hydrophobic forces are involved in
the binding of a fluoroquinolone, Ciprofloxacin, with liposomes
having negative surface charge.5 Ciprofloxacin (Cp, Scheme 1), a research interests of late and has been extensively studied,10–14
fluoroquinolone anti-bacterial agent (showing activity against yet the mechanism of such interactions is still not clear and
both gram-positive and gram-negative bacteria), is extremely requires further investigations. Among such drug–protein
useful for the treatment of low respiratory tract infections, interactions, the use of Ciprofloxacin (Cp) occupies a seminal
urinary tract infections, acute cystitis, nosocomial pneumonia, position.15,16 Cp is 1-cyclopropyl-6-fluoro-1,4-dihydro-4-
skin and skin structure infections and chronic bacterial infections oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid having an
triggered by Escherichia coli.6–8 The binding of drugs to proteins empirical formula C17H18FN3O3. It is characterized by two
has been recognized as an important factor in drug availability, pK values (pK1 = 6.16 and pK2 = 8.63, as marked in
drug efficacy and drug transport for many years.9 Although Scheme 1) and hence the surface charge of Cp is expected to
the topic of drug–protein interactions has drawn considerable be dependent on the pH of the media. Ciprofloxacin Hydro-
chloride (CpH) is the monohydrochloride monohydrate salt of
Cp and has the empirical formula C17H18FN3O3HClH2O. It
Department of Chemical Sciences, Indian Institute of Science is widely accepted that the overall distribution, metabolism
Education and Research Bhopal, ITI Campus (Gas Rahat) Building,
Govindpura, Bhopal 462 023, Madhya Pradesh, India. and efficacy of many drugs can be altered based on their
E-mail: saptarshi@iiserbhopal.ac.in affinity to bind to serum albumins.17–20 In order to unravel the

4250 Phys. Chem. Chem. Phys., 2012, 14, 4250–4258 This journal is c the Owner Societies 2012

reactivity of chemical and biological systems, spectroscopic
approaches are rather useful as they allow non-intrusive
measurements and estimation of properties of such systems
at low concentrations mimicking physiological conditions.
Among the several proteins studied, Bovine Serum Albumin
(BSA) is perhaps one of the most widely used and best
characterized proteins mainly because of its relatively large
number of charged amino acids on the surface (BSA has a net
charge of 18 at neutral pH).21 The primary structure of BSA
Downloaded by Indian Institute of Science Education and Research – Bhopal on 19 April 2012
consists of nine loops held together by 17 disulfide bonds,
resulting in three domains (I, II and III) each consisting of two
sub-domains (A and B). These disulfide bonds impart rigidity
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F
to the helical, globular structure but at the same time allow
sufficient flexibilty for the protein to undergo conformational
changes based on the experimental conditions it is subjected
to. Its secondary structure consists of about 67% a-helix and
the protein is characterized by having an iso-electric point (pI)
at a pH range of 4.8–5.6.21–23 BSA is synthesized by the liver in
mammals and has a lifetime in the circulatory system of about
19 days. Its concentration varies from 35 up to 55 mg mL1 in
the blood plasma.21 It corresponds to the most abundant
protein in the blood plasma, accounting for B60% of the
total number of globular proteins.21,24 BSA shares 76%
sequence homology with another albumin protein, Human
Serum Albumin (HSA), and houses two tryptophan (Trp)
amino acid residues, one at position 212 and another at
position 134 of the amino acid sequence. In spite of the
complexity in shape and size, the structure and dynamics of
BSA can be easily studied by making use of the intrinsic
fluorescence rendered by the two Trp amino acid residues.
Recent reports reveal that the structural organization and
compactness of the tertiary structure of BSA vary as a
function of pH25,26 and that the protein undergoes reversible
conformational isomerisation, depending on the pH of the
medium.27 It has been reported21,26 that BSA has different pH
dependence in the diluted regime up to a concentration of
3 mg mL1. At a pH range of 4.5 to 7.0, the Normal (N) form
is predominant, whereas, between pH 4.5 and 4.0, a Normal–
Fast (F) transition occurs.25 The N–F transition involves a
decrease in the content of ordered or secondary structure. BSA
in the N form exhibits globular structure, whereas it becomes
partially opened in the F form. Over and above the N and F
forms, BSA is reported to have another conformational
transition between pH 8.0 and 9.0 which is termed as the
Basic (B) transition or N–B transition.25,28 In such cases, BSA
loses some of its structural rigidity which most probably
affects the amino-terminal region of the protein. In this present
report, we try to investigate the effect of the added CpH on the
structural properties of BSA at three different pH values and
monitor how CpH alters the fluorescence properties of the
protein upon binding. The pH induced protein structural
changes may be affected by the protein concentration but
based on fluorescence anisotropy values, it has been reported
that for BSA up to a concentration of 50 mM, there is no
aggregation at various pH values and BSA predominantly
remains in the monomeric form.25b Hence, in the present
report, we may assume that at the BSA concentration used
for our experiments (5 mM), primarily the monomeric form is
present at all the three pH values.
This journal is c the Owner Societies 2012
View Online
Experimental section
Materials
Bovine Serum Albumin (BSA) was purchased from Sigma,
essentially fatty acid free and globulin free. Ciprofloxacin
Hydrochloride (CpH, molecular weight = 385.8) was purchased
from Matrix and was used as received. Protein solutions
were prepared in 25 mM Na-phosphate buffer of different
pH (7.4, 9.2 and 4.5) values. CpH stock solution was prepared
in Milli-Q water. For steady state fluorescence measurements,
the temperature was controlled using a Thermo Electron
Corporation chiller. Temperature during all other experiments
was maintained at 25  0.1 1C, unless or otherwise mentioned.
Steady-state measurements
Steady state absorption measurements were carried out in a
Perkin Elmer UV-Vis Spectrophotometer, Lambda-25. All the
steady state fluorescence measurements were recorded on a
HORIBA JOBIN YVON, FLUOROLOG 3-111. For steady
state experiments, a 5 mM protein (the concentration of BSA
was determined spectrophotometrically) solution in a 25 mM
Na-phosphate buffer solution (having different pH) was taken
having varying concentrations of CpH. The fluorescence spectra
were measured with a 10 mm path length quartz cuvette. BSA
was excited at 295 nm in order to minimize the contribution from
tyrosine. The fluorescence emission was collected from 305 nm to
580 nm with an integration time of 0.1 s. The emission and the
excitation slits were kept at 3 nm and 1.5 nm, respectively.
Time-resolved fluorescence measurements
For lifetime measurements, the samples were excited at 295 nm
using a picosecond diode (IBH-NanoLED source N-295). The
emission was collected at magic angle polarization using a
Hamamatsu MCP Photomultiplier (Model R-3809U-50).
The time-correlated single photon counting (TCSPC) setup
consists of an Ortec 9327 pico-timing amplifier. The data were
collected with a PCI-6602 interface card as a multi-channel
analyzer. The typical Full Width at Half Maximum (FWHM)
of the system response was about 800 ps.
Circular dichroism spectroscopy measurements
Circular Dichroism (CD) measurements were carried out on
an Applied Photophysics (UK) (Model: CIRASCAN) spectro-
polarimeter equipped with a Peltier temperature controller. All
the CD measurements were performed at 298 K and 308 K
with an accuracy of 0.1 K. Spectra were collected at a scan
speed of 200 nm min1 with a spectral bandwidth of 10 nm.
Each spectrum was the average of four scans. Secondary
structure (Far-UV CD) was measured over the wavelength
range of 200–250 nm using a 0.1 cm path length cuvette and
tertiary structure (near-UV CD) was measured over the
wavelength range of 250–300 nm using a 1 cm path length
cuvette. Buffer solutions containing the corresponding concen-
tration of CpH were subtracted from all the measurements.
The results were expressed as mean residue ellipticity (MRE)
in deg cm2 dmol1 which is defined as
yobs ðm degÞM
MRE ¼ ð1Þ
nlC
Phys. Chem. Chem. Phys., 2012, 14, 4250–4258 4251
 View Online

where yobs is the CD in millidegrees, M is the molecular weight

of the protein in g dmol1, n is the number of amino acid
residues (583 in the case of BSA), l is the path length (0.1 cm
for far-UV and 1 cm for near-UV CD) of the cuvette and C is
the concentration of the protein in g L1.

Dynamic light scattering measurements

DLS measurements were done with a Nano ZS (Model:
ZEN3600, Malvern), employing a 5 mW, 633 nm He–Ne laser
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and equipped with a thermostatted sample chamber. All the

DLS experiments were carried out at 298 K. The scattering
intensity data were processed using the instrumental software to
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F

obtain the hydrodynamic diameter (dH) and the size distribution

of the scatterer in each sample. The instrument measures the time
dependent fluctuation in the intensity of light scattered from the
particles in solution at a fixed scattering angle. Hydrodynamic
diameter, dH, is defined as,

dH = kBT/3pZD (2)

where kB = Boltzmann constant, T = absolute temperature,

Z = viscosity coefficient of the solvent and D = translational
diffusion coefficient.

Results and discussion

Circular dichroism spectroscopy
(i) Far-UV CD. The changes in the secondary structure of
BSA induced by the alteration of pH and by the addition of
CpH have been monitored by far-UV (200–250 nm) CD
spectroscopy. BSA shows two minima at 208 and 222 nm,
signifying the presence of a-helical structure. In the native
form, at pH 7.4 and 298 K, the percentage a-helix of BSA has
been reported10 to be about 60% and in the presence of high
concentration of CpH (50 mM), this value reduces to about
52%. A similar percentage reduction of helicity has also been
observed at 308 K for the same buffer solution, exhibiting the
fact that a temperature change of 10 K has almost no effect
on the structure of BSA. Fig. 1(a) shows the representative
far-UV CD spectra of 5 mM BSA in the absence and presence
of 50 mM CpH at both the temperatures at pH 7.4. A similar
trend of reduction in the helical content of BSA was also
observed at pH 4.5 and 9.2. Table 1 summarizes the MRE
values at 222 nm obtained both in the presence and absence of
CpH at three different pH values and two different temperatures.
Khan and co-workers reported that HSA has a helicity of 58.3,
53.9 and 46.3% at pH 7.0, 9.0 and 3.5, respectively, showing that
pH alterations indeed bring in some changes in the secondary
structure of HSA.15 The decrement in helicity in the presence of
CpH can be attributed to the changes in secondary structure of
BSA induced by the added CpH. Very recently, Seedhar and
Agarwal used different antibiotics and have shown that the
added CpH reduces the helical content of the protein.16 It must
be remembered that BSA has a reasonably rigid structure due to
the presence of the seventeen disulfide bonds, and hence even at
high concentrations of CpH, the helical content of the protein
does not change substantially. Such bonds may be ruptured in the
presence of strong denaturing agents (like urea29 and guanidine
hydrochloride30) and this makes the protein to lose almost all its
Fig. 1 (a) Circular Dichroism (far-UV CD) spectra of BSA at pH 7.4
in the absence of CpH at 298 K (solid purple down triangles) and
308 K (solid green up triangles) and in the presence of 50 mM CpH at
298 K (hollow purple down triangles) and 308 K (hollow green up
triangles). The inset shows the zoomed in portion of the spectra having
two isodichroic points. (b) CD (near-UV) spectra of BSA in the
absence of CpH at 298 K at pH 7.4 (green circles) and pH 9.2
(blue circles). The region showing the dips at 262 and 268 nm are
zoomed in for clarity.
Table 1 MRE222 values of BSA in the absence and presence of CpH
at 298 K and 308 K
MRE222/deg cm2 dmol1
pH 7.4 pH 4.5 pH 9.2
[CpH]/mM 298 K 308 K 298 K 308 K 298 K 308 K
0 22 621 22 082 17 578 17 150 18 182 17 600
50 18 490 18 134 15 132 14 731 16 535 16 106
helical content. This aspect of the functional integrity of HSA
(which is very similar to BSA) imparted by the presence of
disulfide bonds has been very recently reported by us.31
Another interesting aspect of Fig. 1(a) is the presence of two
isodichroic points at 201 and 205 nm. An isodichroic point is
the wavelength at which the MRE values are the same for
the two or more protein spectra. For a-helical proteins, an
isodichroic point at 201 nm is observed having very low
absorption of circularly polarized light.32 An isodichroic point
at 205 nm signifies a change in the secondary structure of the

4252 Phys. Chem. Chem. Phys., 2012, 14, 4250–4258 This journal is c the Owner Societies 2012

protein having a substantial absorption of circularly polarized
light.32,33 In our present case, we also observe similar isodichroic
points and the changes in the secondary structure of BSA
induced by the added CpH are also reflected by the presence of
the isodichroic point at 205 nm.
(ii) Near-UV CD. In order to investigate the changes in
tertiary structure of BSA induced by CpH, we carried out
near-UV CD experiments from 250–300 nm. Fig. 1(b) represents
Downloaded by Indian Institute of Science Education and Research – Bhopal on 19 April 2012
the plot of CD (mdeg) against wavelength for BSA in the
absence of CpH at two different pH values. Generally, the
existence of disulfide bonds and aromatic rings present in a
protein are exemplified by the presence of two minima centered
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F
at 262 nm and 268 nm.15,34,35 The structural changes that BSA
undergoes due to alteration of pH can be monitored from the
near-UV CD data. Fig. 1(b) shows that there are two minima at
262 and 268 nm and to have a clearer picture we have zoomed in
the region where such minima appear to exist. From Fig. 1(b),
we can rationalize that there are perturbations in and around the
disulfide and Trp residues.36 This is exemplified by the fact that
upon increasing the pH of the solution from 7.4 to 9.2, the CD
(mdeg) values increase at 262 nm and 268 nm, followed by a
subsequent reduction in the wavelength domain of 280–300 nm.
Khan and co-workers15 has opined that this increase in pH
(from 7.4 to 9.2) may alter the conformation of domain I of BSA
as the protein is isomerising from its Native (N) to the Basic (B)
form. A similar trend is also seen on reduction of pH from 7.4 to
4.5 (figure not shown), where the CD (mdeg) values show a rise
at 262 nm and 268 nm and then the spectra merge in the range of
280–300 nm. This change in CD (mdeg) values (as a result of
change of pH from 7.4 to 4.5) is indicative of the fact that BSA
suffers a loss of tertiary structure due to the unfolding and
separation of domain III from the rest of the molecule whereby
BSA changes from its N form to the Fast (F) form.15 It is
expected that the binding of CpH will also bring in some
structural changes in BSA as encountered with the far-UV CD
data. It was found that up to 5 mM CpH, BSA shows almost no
major alteration in tertiary structure as the near-UV CD spectra
overlap with each other (figure not shown). However, on
increasing the concentration beyond 5 mM, the structural alterations
induced are evident from the changes of the nature of the spectra.
Although, the near-UV CD spectra are not that clean and change
of helicity is not that significant, yet it gives us some indication
that tertiary structure of BSA may be a function of pH and
that CpH also alters the structure of the protein depending on
its concentration. CD data (both far and near-UV) are global
reporters, but the exact changes in the micro-environment are
not that conclusive from such experiments.
CpH induced fluorescence quenching of BSA
Upon increasing CpH concentration in the BSA solutions
under investigation, the absorbance values increase almost
monotonically, and since the absorbance values were rather
high, fluorescence measurements were carried out in front-face
(FF) geometry in order to minimize the primary and secondary
inner filter effects. It must be noted here that both BSA and the
antibiotic CpH have substantial absorption at 295 nm, the
wavelength at which all the samples were excited, hence both
BSA and CpH emit simultaneously but at different wavelengths.
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View Online
Fig. 2 Fluorescence spectra of 5 mM BSA in the absence and presence
of various concentrations of CpH as marked in the figure.
All the emission studies were done at two temperatures
(298 K and 308 K) in order to have a better understanding
of the effect of temperature on the binding of CpH to BSA.
Both at 298 K and at 308 K, the emission maxima of BSA
(in the absence of CpH) were found to be independent of the
pH of the solution and were centred at 348 nm. With the
addition of CpH to the protein solutions at all the temperatures
and pH, the emission maxima of Trp shifted towards the higher
wavelength, and at 50 mM CpH, the emission maxima were
found to be red-shifted by 6–7 nm. As mentioned earlier, BSA
has two tryptophan amino acid residues, Trp 134 and Trp 212,
located in sub-domains IB and IIA, respectively.37,38 Out of
these two Trp amino acid residues, Trp 134 is supposed to be
more exposed to bulk solvent and Trp 212 is more buried.37,38
The added drug molecule binds to site I of domain II of BSA,39
and as we are monitoring the fluorescence quenching of Trp, it
is rather rational to believe that the contribution of fluorescence
intensity that we are monitoring is more from Trp 212. Fig. 2
shows the representative fluorescence spectra of BSA at 298 K
and pH 7.4, both in the absence and presence of varying
concentrations of CpH. As mentioned in the section above that
the addition of CpH alters the structure of BSA, thus it may be
expected that the micro-environment in and around the Trp will
also experience some structural alterations. This CpH induced
structural change of BSA was also exemplified by the red-shift
in the emission spectra. The added CpH binds to BSA and
makes the protein to open its native structure. Consequently,
Trp experiences a more polar environment as compared to its
native state. It must be mentioned here that in the absence of
BSA, CpH exhibits strong fluorescence emission centered at
447 nm (in water) and for all concentrations of CpH (up to 50 mM)
and there was no change in the emission maxima; however the
fluorescence intensity increases with the rise in CpH concen-
tration. This signifies that the CpH experiences the same
environment in water in the absence of BSA. However, when
CpH binds to an organized assembly, here BSA, the emission
peak of CpH shifts to 409 nm in the presence of 5 mM CpH as
a result of the hydrophobic environment it experiences. When
the addition of CpH to the BSA solution is continued, the
peak corresponding to CpH exhibits a red shift by 8 nm,
peaked at 417 nm (for 50 mM CpH, please refer Fig. 2). This
red shift in emission maximum corroborates the fact that upon
binding to BSA, CpH experiences a more hydrophilic (polar)
Phys. Chem. Chem. Phys., 2012, 14, 4250–4258 4253
 View Online

environment as a result of binding to BSA leading to structural Table 2 Quenching constants and binding parameters of BSA +
loss of the protein. The emission peak corresponding to CpH CpH systems
increases (Fig. 2) as a result of an increase in its concentration, KSV  104 K  104
and the peak of Trp of BSA falls in intensity due to the System (L mol1) (L mol1) n
quenching of Trp intensity induced by the added antibiotic.
BSA + CpH; pH 7.4 (298 K) 2.40 1.19 0.93
The concept of fluorescence quenching has vast applications BSA + CpH; pH 7.4 (308 K) 3.15 2.10 0.96
and can be used in understanding the mechanism of drug–protein BSA + CpH; pH 4.5 (298 K) 6.52 10.62 1.06
interaction. The quenching of fluorescence is known to occur BSA + CpH; pH 4.5 (308 K) 3.98 4.14 1.00
BSA + CpH; pH 9.2 (298 K) 3.28 46.60 1.26
by two processes, namely collisional (dynamic) quenching BSA + CpH; pH 9.2 (308 K) 3.93 3.12 0.99
Downloaded by Indian Institute of Science Education and Research – Bhopal on 19 April 2012

and/or formation of a complex between the quencher and

the fluorophore (static quenching).10,40 In the case of static
quenching, with the rise in temperature, the stability of the
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F

complex formed will be reduced and thereby the quenching

constant will be lowered, while in the case of dynamic quenching
(which happens to be diffusion driven), the effective number of
colliding ions increases, enhancing the transfer of energy and
thereby increasing the quenching constant with the rise in
temperature. The fluorescence quenching data were analyzed
according to the Stern–Volmer equation:40

Fo
¼ 1 þ kq t0 ½Q ¼ 1 þ KSV ½Q ð3Þ
F

where F0 and F are the fluorescence intensities of the fluorophore

(Trp in the present case) in the absence and presence of the
quencher Q (CpH in the present case). [Q] is the quencher
concentration, Ksv is the Stern–Volmer quenching constant, kq
is the bimolecular quenching constant and to is the average
lifetime of the protein without the quencher. The value of to is
generally taken to be 108 s. Fig. 3 represents the Stern–Volmer
plots of BSA in the presence of different concentrations of CpH
at pH 7.4. The linear nature of the plots as seen in Fig. 3 does not
necessarily indicate the presence of dynamic quenching as static
quenching can also result in similar linear Stern–Volmer plots.40
Static and dynamic quenching may be best distinguished by their
differing dependence on temperature and more appropriately
by lifetime measurements. Higher temperatures result in
faster diffusion and consequently, there will be a larger
amount of collisional quenching, whereas dissociation of
weakly bound complexes at such temperatures will result in
a lower contribution from static quenching.40 Similar linear
Stern–Volmer plots have also been obtained for the other two
pH values, 4.5 and 9.2, and the respective quenching parameters
have been summarized in Table 2. The magnitudes of KSV give
Fig. 3 Stern–Volmer plots for BSA at pH 7.4 against varying
concentrations of CpH at 298 K and 308 K.
Fig. 4 A plot of log[(F0  F)/F] against log[CpH] at 298 K and 308 K
for BSA and various concentrations of CpH at pH 7.4.
us an estimate of whether dynamic or static quenching is
operational. For pH 7.4 and 9.2, we observe that at 308 K,
the magnitudes of KSV are higher than that observed at 298 K.
However, a reverse trend is encountered at pH 4.5, where the
magnitude of KSV actually reduces on increasing the temperature.
This suggests that at pH 7.4 and 9.2, the contribution of dynamic
quenching is more than what is encountered at pH 4.5, where the
mechanism of static quenching dominates. Besides estimating the
KSV values by eqn (3), the binding constant (K) and binding
affinity (n) have also been estimated by plotting log[(F0  F)/F]
against log[Q] by using the following equation.31,41
log[(F0  F)/F] = log K + n log[Q] (4)
Fig. 4 represents a plot of log[(F0  F)/F] against log[CpH] for
BSA at pH 7.4 under various concentrations of CpH at both
the temperatures. For pH 4.5 and 9.2, similar graphs were
plotted (figures not shown) and the corresponding K and n
values have been summarized in Table 2. It is interesting to
note that the magnitudes of K for both the temperatures at pH
7.4 are of the same order, whereas the values decrease with rise
in temperature by nearly one-fold at pH 4.5 and 9.2. This is
indicative of the fact that static quenching may have some
contribution for those pH at which the magnitudes of K
decrease with increasing temperature and dynamic quenching
is dominant at pH 7.4. Also, the magnitude of n gives us an
estimate of the number of binding sites or the affinity of
binding. For all the cases as mentioned in Table 2, the values
of n are close to unity, which suggests that there is one binding
site in BSA. At pH 9.2, however, the value of n is slightly
higher showing that at this particular condition, BSA has a
stronger binding affinity towards CpH and also this fact is

4254 Phys. Chem. Chem. Phys., 2012, 14, 4250–4258 This journal is c the Owner Societies 2012

Table 3 Thermodynamic parameters of BSA + CpH systems
BSA + CpH pH 7.4 (298 K) pH 7.4 (308 K)
0 4 1
DG  10 (J mol ) 2.32 2.55
DH0  104 (J mol1) 4.36 4.36
DS0 (J K1 mol1) 224.29 224.28
supported by a higher value of binding constant as compared
to the other two pH values of the media.
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Thermodynamic parameters. Thermodynamic parameters
play a pivotal role in controlling and estimating the interactions
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F
associated with drug–protein binding, hence three thermodynamic
parameters, namely, the free energy of binding (DG0Binding),
enthalpy (DH0) and entropy (DS0) have been estimated using
the following equations:
DG0Binding = 2.303RTlog K = DH0  TDS0 (5)
ln K2/K1 = (1/T1  1/T2)DH0/R (6)
Here K1 and K2 are the binding constants at temperatures T1
(298 K) and T2 (308 K), respectively, and R is the universal gas
constant. Table 3 summarizes the thermodynamic parameters
under the various experimental conditions. At pH 7.4, the
DG0Binding for both the temperatures are negative which indicates
that the process of binding of CpH to BSA is spontaneous in
nature and also almost independent of temperature. Both DH0
and DS0 values were positive and on comparing the contribution
of these to DG0Binding it can be concluded that the binding
process is entropy driven at pH 7.4. The major contribution of
the entropy term (TDS0) to the free energy change is indicative
of the fact that the binding of CpH to BSA is driven by
hydrophobic interactions.42 However, at pH 4.5 and 9.2, the
interactions are exothermic in nature (unlike what is encountered
at pH 7.4 where it is endothermic) and the binding is also
spontaneous as indicated by the negative values of DG0Binding.
Again, by comparing the contribution of DH0 and DS0 values
towards the free energy of binding, we conclude that at these
pH values, the process is enthalpy driven, unlike what is
observed at pH 7.4. A large negative DH0 value may be due
to hydrogen bonding interactions, whereas a negative DS0
value is associated with an additional contribution of van der
Waals interactions.43 Ross and Subramanian opined that very
low positive and negative values of DH0 and positive DS0
values are characteristic of electrostatic interactions.44 Whenever
a drug binds to a protein, the solvent molecules are displaced due
to such interaction/binding and a large number of solvent
molecules are responsible for a positive entropy change of the
entire system as a whole.44 Subsequently, forces like hydrogen
bonding, van der Waals and electrostatic interactions play the
predominant role in controlling the overall thermodynamics
of the drug–protein interactions. Once the drug has been
incorporated inside the protein molecule, there exists a competition
between the electrostatic and hydrophobic interactions. In
the present case, BSA has a net negative charge (net charge
of 18 at neutral pH with an iso-electric point at B5.5)21,45
and CpH is ionisable in water. The role played by the different
protonation states of the drug cannot be ruled out as we are
monitoring the structural changes at three different pH values.
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View Online
pH 4.5 (298 K) pH 4.5 (308 K) pH 9.2 (298 K) pH 9.2 (308 K)
2.87 2.72 3.23 2.65
7.10 7.10 20.7 20.7
142.04 142.14 584.41 584.41
Hence, the role of electrostatic interaction needs to be taken
care of. Khan and co-workers suggested that Cp has almost
equal positive and negative charges at pH 7.0 as it exists in the
zwitterionic form.15 At pH 9.0 there is a net negative charge
and at pH 3.5, Cp exhibits a net positive charge.15 On
decreasing the pH of the medium, it has been reported that
BSA undergoes conformational changes and also the absolute
electrical charge of the protein changes.45 At pH 4.5, BSA has
a net positive electrical charge and at pH 9.2, BSA becomes
negatively charged.45 Hence, at pH 4.5, both BSA and CpH
are positively charged and at pH 9.2, both are negatively
charged. So, the interaction between the protein and the drug
molecule at these two pH values is most likely not driven
by electrostatic forces of interaction. Hence, the effect of
hydrophobic interactions plays a more significant role when
the interaction of BSA with CpH is being considered. The
most interesting aspect of the thermodynamic studies is that
the binding of CpH to BSA is pH dependent and the pH value
of the solution governs whether the binding is entropy or
enthalpy driven, as discussed above.
Time resolved fluorescence studies of BSA–CpH interaction
The investigation of intrinsic fluorescence from proteins
has been regarded as an effective method to study protein
conformational dynamics. In spite of the fact that the structures
of BSA are rather complicated, they have been widely studied
by making use of the Trp residue which is used as an intrinsic
fluorophore. In aqueous solutions at neutral pH, Trp exhibits
multiple exponential decays and this has been attributed to
the existence of rotational conformational isomers, called
rotamers.10,31,40 By using the concept of rotamers, the lifetime
and conformational changes around the Trp residue can easily
be correlated and explained in organized media.31 The process
of binding of drugs/quenchers to the protein also leads to
distortion of the indole ring (of Trp) planarity and thus
changes the local environment around the Trp causing the
decrement in lifetime and sometimes changes the contribution
of rotameric lifetimes also. In the present work, we have
investigated the effect of binding of CpH on the lifetime of
BSA at three different pH values. Fig. 5 shows representative
decays (log plot) of BSA at pH 7.4 in buffer and in the
presence of 50 mM CpH. Upon increasing the concentration
of CpH, the lifetime of Trp has been observed to decrease and
such a decrement is a function of the pH of the solution.
Similar plots as depicted by Fig. 5 were obtained for pH 4.5
and 9.2 (figures not shown) and the most interesting observation
was that even in the absence of CpH, the lifetime of Trp was
lower at pH 4.5 and 9.2 than that estimated at pH 7.4. The inset
of Fig. 5 represents the changes in the average lifetime values of
Trp in the presence of various concentrations of CpH at three
different pH values. The lifetime decays for all the measurements
Phys. Chem. Chem. Phys., 2012, 14, 4250–4258 4255
 View Online

It is thought that conformer C is rather stable and conversion

from C to either A or B is difficult in a ns time scale.47 Since Trp
exhibits multi-exponential decays in aqueous solutions, the faster
component can be attributed to the existence of the conformer C,
whereas the longer lifetime mainly arises from the rapid inter-
converting A and B conformers. The contribution of these
lifetimes has been ascribed to the relative populations of the
various conformers present in the system.31 From Table 4, we
can observe that at pH 7.4, the contribution from conformer C
Downloaded by Indian Institute of Science Education and Research – Bhopal on 19 April 2012

is around (16  3)% whereas that from conformers A and/or B

is around (84  3)%. The shorter lifetime has been attributed to
conformer C whereas the longer lifetime was thought to be from
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F

the rapid inter-converting A and B conformers.31,46 When the

Fig. 5 Fluorescence lifetime decays of BSA at pH 7.4 (i) in the
absence of CpH and (ii) in the presence of 50 mM CpH. The scattered
points represent the actual decay profile while the solid blue line
represents a bi-exponential fit to that decay. The inset represents the
fall of the average lifetime (hti) with increasing concentration of CpH
at three different pH values, as marked in the figure.
were found to be bi-exponential having very good w2 values
(which indicates the goodness of the fitting) and the lifetime
parameters have been tabulated in Table 4.
To ascribe the individual contributions of lifetime in a
multi-exponential decay as observed in the present case is
rather difficult; however, we will make an effort to explain the
relative amplitudes of the lifetimes as depicted in Table 4. The
contribution of Trp lifetime has been mainly ascribed to
the relative populations of the various conformers present in
the excited state as mentioned below.30,31,46
pH changes from 7.4 to 4.5 and 9.2, the contributions of the
relative amplitudes suddenly change and the contribution from
conformer C rises to around (27  3)% whereas that from
conformers A and/or B decreases to about (73  3)%. The
planarity and rotation of indole is more restricted at pH 7.4,
and this makes the lifetime higher as compared to the other two
pH values. When the pH changes, the inter-conversion from
conformer A and/or B to C becomes easier as the protein loses
some of its secondary structure. Although a change in pH of the
medium is more instrumental in bringing about changes in
lifetimes, however, the addition of CpH does not have a major
contribution in controlling the relative amplitudes of lifetimes.
This may be due to the fact that the addition of the drug
may not induce the inter-conversion of the various rotameric
forms in BSA. Out of the two lifetimes (t1 and t2) observed
(please refer Table 4), t1 is more sensitive towards the addition
of CpH and hence the reduction of t1 is rather more as

Table 4 Fluorescence lifetime parameters of BSA + CpH systems

System a1 t1/ns a2 t2/ns htia,b/ns w2

BSA (5 mM) pH 7.4 +CpH (0 mM) 18.82 3.48 81.18 6.75 6.14 1.08
+CpH (10 mM) 17.45 2.86 82.55 6.67 6.00 1.04
+CpH (25 mM) 13.50 2.03 86.50 6.43 5.84 1.08
+CpH (50 mM) 16.15 1.71 83.85 6.31 5.57 1.06
BSA (5 mM) pH 4.5 +CpH (0 mM) 26.75 2.90 73.25 5.96 5.14 1.05
+CpH (10 mM) 28.00 2.84 72.00 5.89 5.04 1.12
+CpH (25 mM) 29.55 2.90 70.45 5.87 4.98 1.13
+CpH (50 mM) 24.68 2.51 75.32 5.70 4.90 1.03
BSA (5 mM) pH 9.2 +CpH (0 mM) 25.01 2.59 74.99 6.12 5.23 1.07
+CpH (10 mM) 24.66 2.34 75.34 5.96 5.07 1.05
+CpH (25 mM) 27.45 2.25 72.55 5.88 4.88 1.10
+CpH (50 mM) 30.18 2.11 69.82 5.81 4.69 1.10
a b
hti = (a1t1 + a2t2)/(a1 + a2). 5%.

4256 Phys. Chem. Chem. Phys., 2012, 14, 4250–4258 This journal is c the Owner Societies 2012

compared to the longer lifetime (t2). As stated above in this
section, the addition of CpH does not bring a major change in
lifetime at pH 4.5 and 9.2, the change of t1 at these two pH values
is also smaller as compared to what is observed at pH 7.4.
In order to have a clearer understanding of the nature of
quenching at the three different pH values, we have also used
the average lifetime values as an indicator. At pH 7.4, the
average lifetime of Trp decreases from 6.14 ns (in the absence
of CpH) to 5.57 ns (in the presence of 50 mM CpH). However,
Downloaded by Indian Institute of Science Education and Research – Bhopal on 19 April 2012
the average lifetime decreases from 5.14 ns (in the absence of
CpH) to 4.9 ns (in the presence of 50 mM CpH) at pH 4.5 and
from 5.23 ns (in the absence of CpH) to 4.69 ns (in the
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F
presence of 50 mM CpH) at pH 9.2 (please refer Table 4 and
the inset of Fig. 5). The average lifetime values of Trp in the
absence of CpH suggest that pH alterations do bring in some
structural/conformational changes and this was indicated by CD
measurements as discussed earlier. The percentage reduction
values of the average lifetimes in the presence of 50 mM CpH
Fig. 6 Dynamic Light Scattering spectra of BSA in the absence and
presence of 50 mM CpH, as marked in the figures at (a) pH 7.4, (b) pH 4.5
and (c) pH 9.2.
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are about 10% at pH 7.4 and 9.2 and about 4.6% at pH 4.5
which is clearly visualized from the inset of Fig. 5. The values
of percentage reduction in lifetimes suggest that the mechanism
of quenching induced by CpH is dynamic in nature at pH 7.4
and 9.2, whereas it is static in nature at pH 4.5. The time-
resolved parameters agree very well with those calculated
by CD and steady-state measurements. Thus, the dynamic
quenching mechanism which could not be clearly deciphered
by steady-state measurements has been clarified by time-resolved
spectroscopy.
Dynamic light scattering studies of BSA–CpH interaction
For the further confirmation of structural changes incorporated
in BSA due to addition of CpH, we performed Dynamic Light
Scattering (DLS) experiments at three different pH values with
different concentrations of CpH (ranging from 0–50 mM).
Fig. 6(a)–(c) represents the normalized hydrodynamic diameter
(dH) distribution plot of BSA in the absence and presence of
50 mM CpH at three different pH values. The hydrodynamic
diameter of BSA alone in buffer was found to be around
7.3 nm (i.e. the hydrodynamic radius = 3.65 nm) at pH 7.4
which is close to the reported values.48–50 The dH values
change to 9.6 and 7.5 nm at pH 4.5 and 9.2, respectively,
showing a similar kind of trend that has been reported for
HSA at acidic and basic pH.50 It was observed that at all the
three pH values, the dH values for BSA increase in the presence
of 50 mM CpH. At pH 7.4, the value increases from 7.3 to 9.8 nm;
and from 9.6 to 10.5 nm at pH 4.5 and from 7.5 to 8.3 nm at pH
9.2 respectively. The increase in dH is due to binding of CpH to
BSA as well as the structural changes induced in BSA. Hence
CpH induced structural deformation can easily be justified from
these data as the increase in dH is not equal in all the cases. Also,
our DLS data are in excellent agreement with our CD data as by
the latter technique we have shown that in the absence of CpH,
BSA suffers a maximum loss of structure at pH 4.5, where dH is
maximum in the absence of CpH. Although DLS data give us an
estimate regarding the average size of the total protein, yet the fact
that both changes in pH of the medium as well as the addition of
CpH do bring in structural changes in BSA and this has also been
supported by other experimental techniques mentioned above.
Conclusion
The main objective of the present study was to decipher the
role of pH in the binding of the antibiotic CpH to the protein
BSA. At all pH values, the added CpH bring in some
structural changes in BSA and such changes were thoroughly
investigated by both far and near-UV CD spectroscopy. The
concept of isodichroic points as observed in the CD spectra of
BSA in the presence of CpH suggests the changes of secondary
structure induced by CpH. The fall in Trp fluorescence
intensity with the rise of CpH concentration was mainly due
to quenching of Trp and the rise of CpH intensity was a result
of the increase in its own concentration. With the rise in
concentration of CpH, both Trp and CpH undergo a red-shift
in their emission maxima, which further supports the fact that
BSA opens up its native-like structure. Such CD data are in
excellent agreement with our reported DLS data as well.
Steady-state data suggest that the mechanism of quenching
Phys. Chem. Chem. Phys., 2012, 14, 4250–4258 4257

of the Trp emission induced by various concentrations of
CpH is dependent on the pH of the media and by lifetime
measurements we have conclusively proved that at pH 7.4 and
9.2, the mechanism of quenching is mostly dynamic in nature,
whereas, at pH 4.5, it is mainly static quenching that is
operational. By the rational use of Stern–Volmer and modified
Stern–Volmer equations, we have estimated the binding
properties of the drug to the protein. Thermodynamic parameters
reveal that this binding is spontaneous in nature at both the
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temperatures and hydrophobic and van der Waals forces play an
important role in determining such interactions. We have also
concluded that electrostatic forces of interactions do not have any
Published on 06 February 2012 on http://pubs.rsc.org | doi:10.1039/C2CP00001F
significant role in the binding process. The percentage reduction of
lifetimes has also been shown to be a function of the pH and a
time-resolved approach has been used to differentiate between the
two mechanisms of quenching that are prevalent in this present
investigation. The concept of rotamers of Trp has been used to
ascribe the existence of the two independent lifetimes as observed
in the present report. The data reported in this present work can
be used in understanding protein conformational dynamics and
will pave the way to unravel the effect of pH on binding of drugs
to proteins.
Acknowledgements
We sincerely thank Professor Vinod Kumar Singh, Director
IISER Bhopal, for his constant encouragement and support and
the Advanced Instrumentation Research Facility, Jawaharlal
Nehru University (JNU), New Delhi, for helping us to carry out
the CD spectroscopy experiment. We also thank Professor Kankan
Bhattacharyya, IACS Kolkata, for allowing us to use the DLS
instrument and Mr. Dibyendu Kumar Sasmal and Mr. Tridib
Mondal for helping us with the DLS measurements. SM thanks Dr
Sunando Datta and Dr Rajesh Kumar Murarka, IISER Bhopal,
for many stimulating discussions. UA thanks IISER Bhopal and
LK thanks CSIR, Govt. of India, for providing fellowships.
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