Ology Science
Research Article
Journal of Research Analytica
Spectroscopic Study of the Interaction between
Dipicolinic Acid And Human Serum Albumin
Abstract
This study investigates the interaction between human serum albumin (HSA) and dipicolinic acid (DPA) in two
different buffers (tris-HCl and phosphate) using UV/visible absorption and fluorescence spectroscopy. Both
absorption and fluorescence spectra of the mixture indicate that there is moderate interaction between HSA
and DPA. The Stern-Volmer quenching constant (Ksv) and binding constant (Ka) were measured at three different
temperatures. The quenching mechanism is believed to be static by forming a complex between the two since
Ka and Ksv are essentially equal to each other, and both decrease with a temperature increase. Through the
temperature dependent measurements it is found that in tris buffer the nature of the binding interaction is
mainly by electrostatic force, but does not exclude van der Waals forces and Hydrogen bonding. On the other
hand, in phosphate buffer the dominant intermolecular forces between the two are van der Waals forces and
Hydrogen bonding. Our study also demonstrates that tris buffer is a better choice than phosphate for the study
of HSA since the latter also interacts with HSA.
Matthew Feliciano1, Marisa Kroger1, James Irizarry1, Sophia Prentzas1, Jianwei Fan1* and Enju Wang2*
Department of Chemistry and Biochemistry, Manhattan College, Riverdale, NY, 10471, USA
2
Department of Chemistry, St. John’s University, Jamaica, NY, 11439, USA
1
Corresponding author: Jianwei Fan and Enju Wang
*
Received : September 15, 2016; Accepted: October 10, 2016; Published: October 14, 2016
E-mail: [email protected] (J.F) and [email protected] (E.W)
Copyright: ©2016 OLOGY Group.
J Res Anal. (2016) Volume 2 • Issue 4
ISSN : 2473-2230
1
Citation: Feliciano M, Kroger M, Irizarry J. Spectroscopic Study of the Interaction between Dipicolinic Acid and Human Serum Albumin. J Res Anal.
2016; 2(4): 102-107.
Introduction
Instrumentation
Human serum albumin (HSA) is the most abundant plasma
protein in human blood. It is a single chain globular protein
that contains three structurally homologous domains, I, II and
III, and two subdomains within each. It has many positive and
negative centers, and its own charge is negative at physiological
condition [1,2]. Domains II and III contain two primary drug
binding sites (Sudlow’s site I in subdomain IIA and Sudlow’s
site II in subdomain IIIA.) The bulky heterocyclic anions bind to
Sudlow’s site I whereas aromatic carboxylates bind to Sudlow’s
site II [1,2]. When binding to HSA, most drugs are transported
in the circulatory system with albumin. Due to the bound form
being inactive, strong binding to HSA causes a decrease of the
concentration of active drugs in the body. Thus, the study of
the drug binding and binding strength with HSA is an active
research area [3-10]. There are two types of fluorophores in HSA:
one tryptophan (W214) in subdomain IIA, which dominates the
fluorescence of albumin, and several tyrosyl residues in different
subdomains. The fluorescence intensity of HSA is highly
susceptible to the local chemical environment. Solvent and
ligands in the solution could induce the protein conformational
change and reduce the emission through dynamic and static
quenching. Thus, the ligand-albumin binding information is
often acquired through fluorescence quenching measurements
[3-10].
The fluorescence spectra of all solutions were measured with
a Photon Technology International (PTI) spectrofluorometer
equipped with a 1.0 cm quartz cell connected to a thermostat
bath. The excitation wavelength for HSA was set at 280 nm, and
the emission spectra were taken in the range of 290-500 nm.
The slit width of excitation and emission monochromators were
both set at 5 nm. The absorption spectrum was recorded with an
Agilent 8453 UV/visible photodiode array spectrophotometer.
Dipicolinic acid (DPA), 2,6-pyridinedicarboxylic acid, is a
chemical component of bacterial spores (endospores) [11-14].
When the appropriate conditions are present, endospores can
germinate into active cells and release DPA. Thus chemical
detection of spores is usually done through the detection
of DPA. Besides, DPA is hazardous in case of ingestion or
of inhalation. Since the binding between HSA and many
ligands usually change the distribution, metabolism and
excretion properties of the ligands, determining the binding
and thermodynamic parameters with HSA is essential
to understanding the toxicological profile and other
pharmacodynamics of the DPA [12-16]. Our research aims to
study the binding mechanism of HSA to DPA by using UV/
visible and fluorescence spectroscopy. Through the steadystate fluorescence measurements, it was found that DPA
quenches the fluorescence intensity of HSA statically since the
Stern-Volmer quenching constant and the binding constant
are essentially the same. The thermodynamic parameters of
the binding were also determined at three temperatures in
phosphate buffer and tris-HCl buffer, respectively. Although
there are many reports on the study of the interaction between
HSA and different drugs, few were carried out in parallel in
different buffers to compare the effect of buffers. From our study
we found that the buffers do affect the binding constants and the
thermodynamic parameters, as well as the type of the binding
forces between DPA and HSA.
Experimental
Materials
HSA and DPA were purchased from Sigma Aldrich. 0.05 M
tris-HCl buffer (pH = 7.2) and 0.05 M phosphate buffer (pH =
7.2) were prepared using analytical reagent grade. All solutions
were prepared with ultrapure deionized water (Figure 1).
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ISSN : 2473-2230
Fluorescence titration
200 µL of the stock HSA (1.00 × 10-5 M) were mixed with
1800 µL of 0.05 M tris-HCl buffer (pH = 7.2) in a 1-cm cuvette
which was placed in a constant temperature cell holder for 10
minutes to reach thermal equilibrium. The titration of HSA by
DPA was done by adding 1.00 × 10-4 M DPA to the HSA at 2 µL
intervals and recording the fluorescence spectrum (290-500 nm)
of the mixture after each addition. The titration stopped when
the fluorescence quenching of HSA by DPA was saturated at
about 24 µL of added DPA. The titration was carried out at room
temperature and two elevated temperatures below physiological
conditions (295, 305 and 308 K). The UV/visible absorption
spectra of all solutions were recorded at 295 K.
The titration of HSA in phosphate buffer was done slightly
differently: 200 µL of the stock HSA (1.00 × 10-5 M) were mixed
with 1800 µL of phosphate buffer (pH = 7.2), and incubated
overnight (for convenience). Then, 1.00 × 10-4 M DPA were
added to the HSA at 2 µL intervals until the fluorescence
quenching was saturated. The titration was also carried out at the
three temperatures mentioned above.
Results and Discussion
UV/visible absorption spectra
Figure 2 is an overlay of the UV/visible spectra of the
solutions of 1.00 × 10-5 M HSA, 1.00 × 10-4 M DPA, 50/50
mixture of HSA and DPA, and the calculated spectrum for the
50/50 mixture. Although the spectrum of the mixture (curve b)
displays a broad band with two overlapping peaks resulting from
DPA (273 nm) and HSA (280 nm), the measured absorbance of
the mixture is lower than the calculated sum of HSA and DPA
absorbance at the same concentration (curve c). This indicates
that there is a complex formation between HSA and DPA in the
ground state (Figure 2).
It can also be seen from Figure 2 that due to the closeness of
the absorption maxima of HSA and DPA, there is an inner filter
effect by DPA if the excitation wavelength of HSA is set at 280
nm. Inner filter effect is the competition for exciting photons
between HSA and DPA which results in the apparent decrease
of fluorescence intensity of HSA [17]. To compensate for the
co-absorption of excitation photons by DPA, the corrected
fluorescence intensity of HSA was calculated.
Fluorescence quenching spectra
The emission spectra of 1.00 µM of HSA in tris-HCl in the
absence and presence of DPA are displayed in Figure 3. The
emission spectrum of DPA was first run with the excitation at
J Res Anal. (2016) Volume 2 • Issue 4
Ology Science
Citation: Feliciano M, Kroger M, Irizarry J. Spectroscopic Study of the Interaction between Dipicolinic Acid and Human Serum Albumin. J Res Anal.
2016; 2(4): 102-107.
280 nm, and no fluorescence intensity was detected for DPA.
However, the deionized water displays a small Raman scattering
peak at 310 nm. To correct for this artificial peak, the emission
spectra of HSA were subtracted by the emission spectrum of the
buffer solution using the instrument software. As an increasing
amount of DPA (1.00 × 10-4 M) was added to 2 mL of 1.00 µM
of HSA, the emission intensity of HSA decreased, but the extent
of the quenching gradually slowed down. The largest quenching
was achieved when 24 µL of DPA were added, corresponding to
1.19 µM added DPA, or when the molar concentration ratio of
HSA to DPA reaches around 1 to 1 (Figure 3).
Figure 3 also shows that DPA not only quenches but also
shifts the emission maximum, λem, of HSA to a shorter wavelength
(from 337 nm to 333 nm). Although this shift is small it is
consistent from trial to trial. So this could be an indication that
DPA changes HSA’s tryptophan micro-environment, making it
more hydrophobic [3-10,17].
The emission quenching of HSA in phosphate buffer by the
addition of DPA shows a similar trend but to a lesser extent
compared with HSA in tris-buffer.
O
O
N
HO
OH
Absorbance
Figure 1: Structure of DPA.
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
c
b
a
260
280
300
320
340
Wavelength (nm)
Figure 2: Overlay of the UV-visible spectra. (a) 1.00 × 10-5 M HSA,
(b) 50/50 mixture of HSA and DPA, (c) Calculated 50/50 mixture of HSA and
DPA and (d) 1.00 × 10-4 M DPA.
Intensity (a.u. x 106)
2
A
DPA Added
1.5
J
1
Fcorr
= Fobs × e A/2 320
340
360
380
400
420
440
460
Wavelength (nm)
Figure 3: Emission spectra of 1.00 µM of HSA in tris buffer in the absence
and presence of DPA at 295 K. λex = 280 nm. The DPA (1.00 × 10-4 M) added
(A-J): 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 24 µL, corresponding to DPA
concentrations: 0, 0.0999, 0.200, 0.299, 0.398, 0.498, 0.596, 0.695, 0.794,
0.892 and 1.19 µM.
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(1)
where Fcorr and Fobs are the fluorescence intensities corrected
and observed, respectively. A is the absorbance of DPA at the
excitation wavelength (280 nm) at the same concentration as it
is in the mixture. The equation holds true when the absorbance
is less than 0.3.
The corrected intensity was then fitted with the Stern-Volmer
equation [17]:
F0 / Fcorr= 1 + K sv [Q ] (2)
where Ksv is the Stern-Volmer quenching constant, and [Q]
is the concentration of the quencher. F0 is the emission intensity
in the absence of the quencher, and Fcorr is the corrected emission
intensity in the presence of the quencher. Figure 4 displays F0/Fcorr
of HSA against the concentration of DPA in two different buffers
and at three different temperatures. The slope of the line gives Ksv
values which are summarized in Table 1. It can been seen from
Figure 4 that the Ksv values decrease as the temperature increases
in both buffers, and the Ksv values are smaller in phosphate buffer
than those in tris-buffer at the same temperature (Figure 4)
(Table 1).
It is well known that for pure dynamic quenching the SternVolmer constant is Ksv = 𝑘𝑞 × 𝜏, where 𝑘𝑞 is the bimolecular
quenching rate constant, and 𝜏 is the lifetime of the excited state
of HSA, which equals 1 × 10-8 s [10, 17]. The maximum value
of kq is kd, the diffusion rate constant in aqueous solution, which
is 1010 M-1 s-1, or kq ≤ kd = 1010 M-1 s-1 [5]. However, the value
of kq calculated from our measured Ksv value and the 𝜏 of HSA
is much larger than kd, i.e., kq = Ksv / 𝜏 = 1 × 105/ 1 × 10-8 = 1013
>>1010. This indicates that the dynamic quenching mechanism is
unlikely the dominate process [17-20].
Furthermore, the dynamic quenching is a nonradiative
decay based on the collision between DPA molecules and the
excited state of HSA, which competes with the fluorescence of
HSA. As temperature increases, more collisions occur, therefore
more quenching occurs leading to an increase of Ksv. Our result
shows that Ksv decreases with temperature increases. This is more
consistent with a static quenching mechanism by forming a nonemissive complex between DPA and HSA which dissociates at
elevated temperatures.
Binding parameters
functions
0.5
0
The observed emission intensity of fluorescence was corrected
for inner filter effect using the following equation [17,18]:
Quenching mechanism
d
240
Stern-Volmer quenching constant
and
thermal
dynamic
For pure static quenching, the Stern-Volmer constant Ksv
is the same as Ka, the association or binding constant of DPA
with HSA and Ka can also be derived from the following binding
process [21].
P + nD  Dn P (3)
where P is the free protein, D is the drug or quencher, n is the
number of binding sites on the protein, and DnP is the proteindrug complex.
J Res Anal. (2016) Volume 2 • Issue 4
ISSN : 2473-2230
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Citation: Feliciano M, Kroger M, Irizarry J. Spectroscopic Study of the Interaction between Dipicolinic Acid and Human Serum Albumin. J Res Anal.
2016; 2(4): 102-107.
1.4
1.6
F0/F corr.
F0/F corr.
1.4
1.2
1
0.8
a
0
0.5
1
1.5
1.2
1
0.8
b
0
0.5
1
[DPA] (µM)
[DPA] (µM)
1.5
Figure 4: Stern-Volmer plots of HSA at three temperatures: (a) in tris buffer and (b) in phosphate
buffer. (■) 295K, (▲) 303K (●) 308K.
Using the mass action law, the binding constant (Ka) is
obtained through the following equation:
[ D P]
[ P][ D]
4)
Since the free protein is the only fluorescent species,
[ P] Fcorr
=
Fo
[ P ]o
(5)
where [P]0 is the initial concentration of free protein, and
Fcorr and F0 represent the emission intensities of protein with and
without the presence of the drug. Substituting Eq. (5) into Eq.
(4), the following double log equation can be derived:
log ( F0 − Fcorr / Fcorr ) =
log( K a ) + n log[ D] (6)
Eq. (6) was used to determine the binding constant (Ka) and
the number of binding sites (n) on protein. Figure 5 gives the
plots of log (𝐹0−𝐹𝑐𝑜𝑟r/𝐹𝑐𝑜𝑟r) vs. log [DPA] at three temperatures
and in two buffer solutions. The slope of the line gives the
number of binding site, and the antilog of the intercept gives the
binding constant. These values are listed in Table 1 (Figure 5).
The data in Table 1 indicate that Ksv and Ka are essentially
the same at three temperatures, and they both decreases as
temperature increases in both buffer solutions. These data further
prove that the quenching mechanism is static by forming a
complex between DPA and HSA in both buffer media.
The number of binding sites on HSA (n) was found as
one (1), i.e., there is one binding site in subdomain IIA of
HSA to combine with DPA. This is consistent with our other
measurements, i.e., the quenching is saturated when the molar
ratio of HSA to DPA in the solution reaches around 1:1 (Figure
3). It is also comparable with the values reported in other papers
[3-10,17-19].
Thermodynamic parameters
To obtain the thermodynamic parameters of the binding
interaction, the Vant Hoff equation [22] is used:
log( K a ) =
S o
− H 0 1
+
2.303 R T 2.303R
(7)
where Ka is the binding constant, and ∆H and ∆S are enthalpy
and entropy changes of the binding reaction, respectively. Figure
6 displays the Vant Hoff’s plots in tris-buffer and phosphate
buffer, and ∆H and ∆S are obtained from the slope and the
intercept of the graph. Gibbs free energy is obtained from ΔG =
∆H - T∆S. All calculated thermodynamic parameters are listed
in Table 2 (Figure 6).
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Force contributing to binding
The force between HSA and the ligands involves hydrophobic
force between two hydrophobic groups such as benzene rings, van
der Waals, Hydrogen bonding and electrostatic force. From the
sign of thermodynamic functions, the type of binding force can be
deduced [23-25]. It is suggested by Ross and Subramanian [23],
that if ∆𝐻 > 0 and ∆𝑆 > 0, hydrophobic interaction occurs since
the process involves the reorganization of the solvent structures
around the protein and the ligand species; if ∆𝐻 < 0, ∆𝑆 > 0, the
force is electrostatic; and if ∆𝐻 < 0, ∆𝑆 < 0, van der Waals force
and hydrogen bonding dominate. The more H-bonding form in
the process, the more negative the ∆𝐻 is. Since DPA contains a
heterocyclic ring and carboxylic groups, and is negatively charged
at pH = 7.2, it could have all these types of interactive forces
with HSA which contains many positive and negative centers. In
tris buffer, since the calculated ∆𝐻 <0, and ∆𝑆 > 0, the binding
interaction between HSA and DPA is attributed to electrostatic
attraction between negative DPA and positive center of HSA.
However, due to the small value of ΔS in tris buffer, the binding
interaction cannot exclude van der Waals forces and Hydrogen
bonding. On the other hand, in phosphate buffer, since both
∆𝐻 and ∆𝑆 are negative, the binding interaction between HSA
and DPA is mainly van der Waals forces and H-bonding. The
large negative values of ∆𝐻 are indicative of the presence of
H-bonding, especially in the phosphate buffer.
The effect of buffers on the binding interaction
between HSA and DPA
Parallel investigations on the interaction of HSA and
DPA were carried out in both tris-HCl buffer and phosphate
buffer at pH = 7.2, respectively. It was found that tris buffer
Table 1: Ksv, Ka and n at three temperatures in tris buffer and in phosphate
buffer, respectively.
Tris buffer
Phosphate buffer
Temp
(K) Ksv × 105 (M-1) Ka × 105 (M-1) n Ksv × 105 (M-1) Ka × 105 (M-1) n
295
4.8 ± 0.7
4.6 ± 0.8 0.98 1.7 ± 0.8
2.0 ± 0.5 0.94
1.1 ± 0.03 0.95
303
3.3 ± 0.3
3.3 ± 0.3
1.1
1.1 ± 0.01
2.8 ± 0.1
1.1 0.75 ± 0.04 0.79 ± 0.01 0.75
308
2.8 ± 0.3
Table 2: Thermodynamic parameters in tris buffer and in phosphate buffer.
Temp (K)
295
303
308
∆H (kJ/
mol)
- 29.2
Tris buffer
∆S (J/
∆G (kJ/
Kmol)
mol)
-32.0
+ 9.83
-32.1
-32.1
J Res Anal. (2016) Volume 2 • Issue 4
∆H (kJ/
mol)
-52.9
Phosphate buffer
∆S (J/
∆G (kJ/mol)
Kmol)
-29.9
-78.0
-29.3
-28.9
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Citation: Feliciano M, Kroger M, Irizarry J. Spectroscopic Study of the Interaction between Dipicolinic Acid and Human Serum Albumin. J Res Anal.
2016; 2(4): 102-107.
0
-0.4
-0.6
-0.4
-0.8
Log (F0-Fcorr./Fcorr.)
Log (F0-Fcorr./Fcorr.)
-0.2
-0.6
-0.8
-1
-1.2
-1
-1.4
a
-1.2
-1.4
-0.8
-0.6
-0.4
-0.2
Log [DPA]
0
b
-1.6
-1.8
0.2
-0.8
-0.6
-0.4
-0.2
Log [DPA]
0
0.2
Figure 5: log (𝐹0−𝐹𝑐𝑜𝑟r/𝐹𝑐𝑜𝑟r) vs. log [DPA] at three temperatures. (a) in tris-buffer and (b) in phosphate, (■) 295K, (▲) 303K, (●) 308K.
5.4
5.7
5.3
5.2
Log (Ka)
Log (Ka)
5.6
5.5
5.4
327
332
337
5
4.9
a
322
5.1
b
4.8
342
322
327
332
337
342
1/T x 105 (K-1)
1/T x 105 (K-1)
Figure 6: log (Ka) vs. 1/T (a) in tris buffer and (b) in phosphate buffer.
has little interaction with HSA because the emission intensity
of HSA is very stable in tris buffer until the DPA is added. On
the other hand, the emission intensity of HSA decreased slowly
in phosphate buffer even in the absence of DPA. When DPA
was added to HSA in phosphate buffer, the emission intensity
of HSA decreased substantially, but hardly stopped decreasing
even after a long period of waiting (more than 30 min). It
seems that phosphate buffer itself has some weak interaction
with HSA, and may compete with DPA since its concentration
is much larger than DPA. The interaction between phosphate
and HSA is likely due to an electrostatic attraction considering
the highly negative charges carried by the phosphate ions at the
experimental condition. At pH = 7.2, for phosphoric acid (pKa1 =
2.148, pKa2 = 7.198 and pKa3 = 12.375) the first hydroxyl group
is 100% deprotonated and the second one is 50% deprotonated.
For DPA (pKa1 = 2.16 and pKa2 = 4.76), both –COOH groups
are deprotonated and negatively charged at pH = 7.2. Therefore,
phosphate ions can compete with DPA for the same positive
centers of HSA by electrostatic attraction. On the other hand,
the tris buffer (pKa = 8.072) is partially neutral and partially
positively charged (+1) at pH = 7.2, and will not compete with
DPA for binding to the positive centers of HSA.
To overcome the instability problem of HSA in phosphate
buffer, 1.00 µM HSA and phosphate buffer were mixed and
incubated overnight (for convenience) before DPA was added.
The incubation allows the interaction between HSA and
phosphate to be saturated/stabilized in order to minimize the
interference by phosphate ions when DPA is added to HSA.
This procedure seems to work well since the measurements of
the quenching by DPA are more reproducible and take less time
to stabilize in incubated HSA-phosphate solution. However,
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the interaction between HSA and phosphate could weaken
the attraction between HSA and DPA. This is consistent with
the experimental results that both Stern-Volmer quenching
constant, Ksv, and binding constant, Ka, in phosphate buffer are
smaller compared to these obtained in the tris buffer (Table 1).
Besides, since the positive centers on HSA are partially shielded
by negatively charged phosphate ions, the interaction between
HSA and DPA is mainly by van der Waals force and hydrogen
bonding (Table 2). On the other hand, in tris buffer, the main
forces between HSA and DPA are electrostatic force as well as
van der Waals and hydrogen bonding.
Conclusion
This study found that DPA does quench the fluorescence
of HSA and affects the microenvironment of the tryptophan
on HSA. The quenching mechanism is believed to be static
by forming a complex between them since both Ka and Ksv
are essentially equal to each other, and both decrease with a
temperature increase. The binding site for DPA is one (1) in
subdomain IIA of HSA.
This work also compared the effect of buffers on the interaction
between HSA and DPA. In tris buffer, the thermodynamic
parameters obtained from temperature-dependent measurements
indicate that the binding interaction is mainly electrostatic
forces, but does not exclude van der Waals forces and Hydrogenbonding. In phosphate buffer, the intermolecular forces between
the two are van der Waals interaction and Hydrogen bonding.
Our study also found that HSA is more stable in tris buffer than
in phosphate buffer, which may contribute to the selection of
buffers in future studies.
J Res Anal. (2016) Volume 2 • Issue 4
ISSN : 2473-2230
5
Citation: Feliciano M, Kroger M, Irizarry J. Spectroscopic Study of the Interaction between Dipicolinic Acid and Human Serum Albumin. J Res Anal.
2016; 2(4): 102-107.
Acknowledgment
This work was supported by Manhattan College and St. John’s
University. J. Fan thanks Dr. Constantine Theodosiou for many
helpful suggestions, and Dr. Alexander Santulli for some graphics
work.
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