chapter-4: photophysical properties of...
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Chapter-4: Photophysical Properties of Luminol
4.1. Introduction Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, LH2) is a versatile chemical that
shows striking blue chemiluminescence in presence of certain metal ions when treated with
appropriate oxidizing agent like hydrogen peroxide. This unique feature of LH2 is often exploited
by forensic investigators to detect trace amount of blood left in the crime scene. LH2 is also used
by the biologists as a cellular assay to detect copper, iron and cyanides etc [103-107]. Further,
LH2 enhanced chemiluminescent probes have been used to characterize and quantify the
secretion of oxygen by phagocytozing cells [108]. The use of LH2 chemiluminescence has also
been reported recently for facile detection of proteins [109], cancer biomarkers [110], as well as
for reactive oxygen species produced by human neutrophils [111]. The ultra-high sensitivity of
time-resolved chemiluminescence behavior of LH2 can be used to measure OH/O2- radical
species concentration as low as 2×10-7 mol dm-3 in water [112]. An important aspect of LH2
chemiluminescence is its different degrees of sensitivity from one substance to another. LH2
shows higher sensitivity to animal or human blood, organic tissues and fluids than to other
compounds containing metal ions, such as paints, metallic surfaces, household products, or
vegetable enzymes. Therefore, the light emitted by LH2 has different intensities and time
duration, depending on the material of contact making it an efficient forensic marker.
4.2. Luminol Fluorescence in Homogeneous Media 4.2.1. General spectral behavior: Importance of hydrogen bonding
Solution phase spectroscopic properties of LH2 have drawn enormous interest in recent
times due to the biochemical relevance of its photoactivity. Photophysical properties of LH2 in
different solvents and solvent mixtures as well as its interaction with several biological
molecules were reported in the literature [113-117]. LH2 exhibits two principle absorption bands
in 300 and 350 nm region, whereas, a single broad fluorescence emission appears at 400 nm
region. Interestingly, the fluorescence emission peak shows large spectral shift towards longer
wavelength in hydrogen bonding solvents. This shift is believed to be due to the stabilization of
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the charge transfer excited state of the intermolecular hydrogen bonded complex of LH2 with
solvent [113].
Figure 4.1 Possible tautomeric structures of luminol (LH2). Numbering scheme used in calculation is also shown.
Intermolecular hydrogen bonding and solubility of organic systems is known to play a
crucial role in determining the biological activity as well as its application in forensic science [79,
118-120]. In general, formation of intermolecular hydrogen bond between solute and solvent
results in a decrease in Gibb’s free energy and thus promotes mixing. Hydrogen bonding can
occur in different modes, depending on the structure of the solute and solvent. The situation
becomes more complicated when a solute molecule possesses multiple hydrogen bonding site
and the solvent molecule can act both as a proton donor as well as a proton acceptor. Under
this condition, the competition among different molecular species resulting from hydrogen
bonding interaction between the solute and the solvent molecules becomes inevitable. LH2
provides a unique example to study the hydrogen bonding effect because the molecule itself
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can exist in more than one prototropic species (figure 4.1) having multiple hydrogen bonding
sites. The keto-amine (I) structure can go to the enol-imine (III) form in single step or through
the intermediate structures IIa and/or IIb, respectively. These inter-conversion and associated
spectroscopic properties will depend strongly on the relative abundance of several species, as
well as their hydrogen bonding mode with the solvent. Furthermore, the efficacy of hydrogen
bond formation in the excited state may change due to charge redistribution after excitation.
Also, relatively strong and unstructured fluorescence of LH2 is an additional advantage to use
the spectroscopic ruler for quantitative estimation of the effect of different solvent parameters.
The chemiluminescence and the fluorescence band of LH2 in water appear in the same
wavelength region (425 430 nm) [115]; so quantitative characterization of this band on
different solvent parameters is indispensable.
In the following sections, we use steady state spectral properties of LH2 in a series of
pure solvents with varying polarity as well as hydrogen bond donor and acceptor abilities to find
quantitative information about their relative contribution. The list of all the solvents and some
of the important physical parameters are collected in table 4.1. Furthermore, the effects of
specific LH2 – water hydrogen bonding on the solution phase spectral properties were
theoretically modeled by using density functional approach.
Figure 4.2 Absorption (a), Fluorescence emission (b and c, at exc = 350 and 290 nm, respectively) and excitation (d, mon = 425 nm) spectra of 1.210-5 mol dm-3 aqueous solution of LH2.
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Table 4.1 Solvent parameters
No. Solvent f(,n)a ET(30)b c c *c
1. Acetonitrile 0.30 45.6 0.19 0.40 0.75
2. Ethyl acetate 0.19 38.1 0.0 0.45 0.55
3. Benzene 0.0 34.3 0.0 0.10 0.59
4. Tetrahydrofuran 0.21 37.4 0.0 0.55 0.58
5. 1,4-dioxane 0.03 36.0 0.0 0.37 0.55
6. Toluene 0.02 33.9 0.0 0.11 0.54
7. DMSO 0.26 45.1 0.0 0.76 0.1
8. DMF
9. Dichloromethane
10. 1-Butanol
0.27
0.22
0.26
43.2
40.7
49.7
0.0
0.0
0.84
0.69
0.1
0.84
0.88
0.81
0.47
11. Methanol 0.31 55.4 0.98 0.66 0.60
12. 1-Propanol 0.27 50.7 0.84 0.90 0.52
13. 1-Pentyl alcohol 0.25 49.1 - - -
14. Water 0.32 63.1 1.17 0.47 1.09
15. Acetone 0.28 42.2 0.08 0.48 0.71
16. Isopropanol 0.27 48.4 0.76 0.95 0.48
a Polarity parameter; b Reichardt solvent parameter; c Kamlet-Taft solvent parameters.
4.2.2. Steady state spectral properties in pure solvents Figure 4.2 shows some representative absorption and emission spectra of LH2 in
aqueous medium and table 4.2 summarizes the steady state spectral behavior of LH2 in solvents
with varying polarity and hydrogen bonding parameters. In homogeneous solvents, LH2 shows
two absorption maxima. One relatively structured high energy peak appear at 280320 nm
region; whereas the other unstructured, broad low energy absorption in the 330380 nm
region. However, the emission obtained by exciting at both these absorptions show strongly
intense, unstructured and broad spectra ranging from 375 to520 nm. The excitation spectra
corresponding to this emission again show a broad peak at 350 nm. In a recent report [115],
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Vasilescu et. al. proposed an acid-base type of equilibrium to explain the two absorption bands
of LH2 in highly alkaline (pH 12.0) DMSO solution for the formation of corresponding dianion
of structure III. This was further confirmed by the concomitant quenching of LH2 fluorescence
and appearance of new broad band at 475 480 nm with increasing alkali concentration.
However, the formation of the dianionic species in the present experimental condition of
neutral aqueous LH2 solution (pH 6.4) can be ruled out. The absence of any new fluorescence
band further supports this hypothesis. The origin of the broad absorption in 330380 nm region
can be assigned as S1(π) ← S0(π) transition; whereas, the origin of the high energy absorption at
300 nm may be due to S2 ← S0 excitation. From the relatively large absorption coefficient (max
22275 dm3 mol-1 cm-1) of this band, which is comparable to that of 350 nm absorption (max
23300 dm3 mol-1 cm-1), it can be concluded that this transition is also π*←π in nature. The
assignment of these absorptions as well as further verification for the long wavelength
absorption at 475480 nm to be originated from the anionic species, is confirmed from
theoretical calculations described in the following sections.
Although, the results in table 4.2 do not show any regular variation of steady state
spectral properties, careful observation reveal several interesting trends. For example,
fluorescence maxima (νem) shows appreciable shift in protic solvents along with almost two fold
increase in fluorescence quantum yield (φf), when compared with their aprotic counterpart.
Furthermore, full width at half maxima (FWHM) for both the absorption and emission peaks are
much higher in water compared to the other solvents. All these results indicate that
consideration of hydrogen bonding interaction is very important to describe LH2 photophysics,
more particularly in aqueous medium.
4.2.3. Solvatochromism of luminol photophysics: Estimation of
contributions from solvent parameters To verify the effect of solvent polarity, several steady state spectral parameters of LH2 in
a variety of solvents mentioned in table 4.1 were plotted against the solvent polarity parameter
∆f(, n). From the results given in figure 4.3, it is clear that the spectroscopic properties of LH2 do
not show any regular solvatochromism behavior on the solvent polarity parameter. This
observation points to the existence of specific solute-solvent interactions. As a trial, the
empirical solvent polarity scale, ET(30), built with a betaine dye, was used, as it is the most
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popular choice to correlate several solvent dependent spectral properties. The uni-parametric
scale depends on both the solvent dielectric properties and hydrogen bonding acidity, but it
does not take care of solvent hydrogen bonding acceptor basicity [76]. The specificity of Lewis
acid base interactions in ET(30) parameter arise from the negative charge localized on the
phenolic oxygen of betaine molecule. As it is seen in figure 4.4 again, there is no linear
correlation of either LH2 absorption/emission energies or Stokes-shift even with this parameter.
A break point, mostly influenced by LH2 emission properties like fluorescence maxima (νem) and
Stokes shift (∆νss), is obtained around ET(30) = 38 kcal mol-1. This clearly indicates that apart from
solvent polarity, LH2 solvatochromism is strongly modulated by both solvent hydrogen bond
donor acidity and solvent hydrogen bond acceptor basicity parameters also.
Figure 4.3 Variation of absorption (a), emission (em) energies and Stokes-shift (ss) with solvent polarity parameter, f(,n).
In-view of this situation, one must look at multi-parametric approach, as devised by
Kamlet and Taft and mentioned in equation (3.3), to assess the contribution of different solvent
parameters on LH2 solvatochromism. The s, a and b coefficients in equation (3.3) were all
obtained by multiple linear regression analysis and the results are given in tables 4.3 and 4.4.
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Table 4.2 Steady state spectral properties of LH2 in homogeneous solventsa
Solventb abs /cm-1 em /cm-1 exc /cm-1 ss /cm-1 f em /cm-1 exc /cm-1
1 28169 25126 28490 3043 0.26 3053 3333
2 28172 25253 28490 2919 0.21 2902 3333
3 28170 24876 28249 3294 0.20 3053 3099
4 28165 25316 28410 2849 0.24 2902 3263
5 28170 25126 28490 3044 0.25 2853 3712
6 28169 24876 28410 3293 0.21 3158 4010
7 27933 24876 28090 3057 0.33 2902 3263
8 28090 25063 28249 3027 0.26 2902 3310
9 28011 24876 28490 3135 0.29 3106 3358
10 28410 24331 28011 4079 0.52 3004 3590
11 28169 24450 28329 3719 0.42 3057 4201
12 28090 24390 28090 3700 0.58 3106 3768
13 27933 24450 28090 3483 0.58 3057 3793
14 28572 23474 28410 5098 0.78 3211 4388
15 28328 25063 28090 3265 0.31 2902 3001
16 28011 24510 28169 3501 0.50 3057 3846
aAbbreviations used: = absorption, emission and excitation energy; ss = Stokes shift; f = fluorescence quantum yield; = corresponding full width at half maxima (FWHM), bThe name of the solvents are listed in table 4.1.
Few representative correlation diagrams of the experimental values with those
calculated from multiple regression analysis using equation (3.3), are shown in figure 4.5. A
close look into the tables reveals several interesting feature for LH2 solvatochromism: (i) in
general, the contributions from a as well as b parameters are significant relative to the s
parameter, indicating the importance of solvent hydrogen bonding in LH2 spectroscopy; (ii) the
excited state spectral properties like fluorescence maxima, Stokes-shift, quantum yield etc. are
mostly controlled by solvent hydrogen bond acidity function (a parameter), whereas, both a and
b contributes almost equally in the absorption property. This indicates an efficient charge
localization in LH2 upon excitation (see DFT calculation results in the following section); (iii) the
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charge localization in the excited state is further confirmed by the negative values of both a and
s [121]; (iv) almost two fold increase in fluorescence quantum yield in protic medium (table 4.2)
is mainly due to the hydrogen bond acidity of the solvents with a/s 1.5. However, solvents like
DMSO with higher hydrogen bonding acceptor (HBA) tendency has very little effect (8%) on φf.
For example, the φf value of LH2 in water is about 0.79 compared to that in 1,4-dioxane (0.25)
and DMSO (0.34). This observation is also in line with our finding that the yield of LH2
fluorescence decreases substantially in presence water soluble proteins like bovine and human
serum albumins and discussed more in detail in the next section. The decreased fluorescence
intensity of LH2 on binding to albumins most likely reflects reduced water access to the
chromophore in the bound state. Finally, (v) the large spectral shift in water and other
hydroxylic solvents, as observed in table 4.2, is due to the negative value of a parameter and its
corresponding larger contribution (table 4.4). In summary, the solvatochromic analysis reveals
that in polar protic solvents, like water for example, several spectroscopic species may be
present due to the hydrogen bonded donor and acceptor properties of the solvent in the ground
state; however, in the excited sate, the main fluorescing species is originated due to the
hydrogen bonded complex formation of LH2 through the solvent hydrogen bonding acidity
behavior.
Figure 4.4 Variation of absorption (a), emission (em) energies and Stokes-shift (ss) with solvent ET(30) parameter. The solid line guides the eye along the variation of em and ss with the solvent parameters.
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Table 4.3 Regression fit to solvatochromic parameters towards the steady state spectral properties of LH2.a
Property (P) P0 s a b R2 SD
abs /cm-1 28183.14 70.06 260.90 -267.79 0.90 140.3
em /cm-1 25362.49 -652.76 -1448.0 -391.26 0.94 166.7
exc /cm-1 28492.90 25.60 26.92 -424.06 0.92 147.2
ss /cm-1 2820.65 722.80 1347.85 -657.84 0.88 223.3
f 0.074 0.225 0.33 0.05 0.89 0.06
em /cm-1 3061.69 28.84 233.77 -277.26 0.85 71.7
exc /cm-1 3566.69 -30.11 851.47 -491.86 0.89 281.3
a The regression analysis was done using equation (3.3); R2 and SD indicate correlation coefficient and standard deviation in the regression analysis, respectively.
Figure 4.5 Correlation diagram of LH2 emission energy (em), Stokes-shift (ss) and fluorescence quantum yield (f) with predicted values from Kamlet-Taft equation.
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Table 4.4 Relative values (in percentage) of the solvatochromic parameters towards LH2 steady state spectral properties.
Property (P) Ps(%) Pa(%) Pb(%)
abs 11.70 43.57 44.72
em 30.65 51.03 18.31
exc 5.37 5.64 88.89
ss 26.49 49.34 24.11
f 37.19 54.54 8.26
em 5.34 43.30 51.36
exc 2.19 61.99 35.82
4.3. Calculation Using Density Functional Theory
4.3.1. Energetic of different conformers in the ground state
Full geometry optimization of different conformers of LH2 in isolated condition
(structures given in figure 4.1 along with the numbering scheme) as well as with different
degree of hydration, was done using B3LYP/6-311++G(d,p) methodology. The fully optimized
structures are shown in figure 4.6(a-d). The energy parameters, relative to the most stable
structure, are given in table 4.5. It is clear that the conformer IIb is the most stable structure in
the isolated, monohydrated as well as in the dihydrated configuration. However, comparison of
relative energies indicates that LH2 most likely exists in dihydrated complex structure
represented by IIb-S3. The structure represented by I-S3 is about 7.6 kJ mol-1 higher in energy
than IIb-S3. It may still be possible that this high energy structure will have relatively less
abundance in the solution along with the structure IIb-S3 in the ground state; and more so, in
the excited state (see below). However, the existence of all other conformers represented by IIa
and III can be neglected to discuss the spectroscopic behavior of LH2 in water. This is because of
their relatively higher energy; they are unlikely to be present in solution mixture. It is to be
noted here that only primary hydrogen bonded complex with water was considered to give
different complexes like S1, S2, and S3 (figure 4.6). It is obvious that additional solvent
molecules will combine to give secondary water cluster around LH2 and the actual hydrated
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structure is far more complex than what is considered for these calculations. However, we
restrict ourselves only in the first hydration layer, as the effect of internal hydrogen bonding
(IHB) is expected to diminish very fast from the center of origin. Consequently, it is reasonable
to believe that any further addition of water structure will have insignificant contribution
toward the relative energy parameters.
Table 4.5 Relative energy (kJ mol-1) of fully optimized structures of different conformers of LH2 in gas phase calculated at B3LYP/6-311++G(d,p) level.
Structurea I IIa IIb III
Isolated 95.504 99.626 81.421 141.022
Monohydrated (S1) 52.952 58.062 41.553 90.538
Monohydrated (S2) 50.450 57.507 41.715 93.064
Dihydrated (S3) 7.635 14.544 0.0 44.182
a See figures 4.6(a-d) for the optimized structures of different conformers with varying degree of hydration.
Figure 4.6 (a) Fully optimized structures of I conformer of LH2 in isolated, monohydrated I-S1 and I-S2, and dihydrated I-S3 states. The geometry optimization was done at B3LYP/6-311++G(d,p) level of calculation. Bond lengths and angles are given in Å and degrees, respectively.
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Figure 4.6 (b) Fully optimized structures of IIa conformer of LH2. The other details are similar to figure 4.6(a).
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Figure 4.6 (c) Fully optimized structures of IIb conformer of LH2 in isolated (i), monohydrated IIb-S1 and IIb-S2 (ii & iii, respectively), and dihydrated IIb-S3 (iv) states. The geometry optimization was done at B3LYP/6-311++G(d,p) level of calculation. Bond lengths and angles are given in Å and degrees, respectively.
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Figure 4.6 (d) Fully optimized structures of III conformer of LH2. The other details are similar to figure 4.6(a).
4.3.2. TD-DFT calculation on the excited state The excited states of the two most stable ground state structures of dihydrated LH2
discussed above, i.e. for I-S3 and IIb-S3, were calculated using TD-DFT procedure. The calculated
transition energies and the corresponding oscillator strengths for several singlet excitations
within the experimentally observed absorption wavelength range of 260400 nm region for
both the structures are given in table 4.6. It is noted that the nature of transition as well as its
energy and oscillator strength is comparable for both the structures. The calculated S1←S0
transition wavelength of 335 nm is in close agreement with the experimentally observed value
of 350 nm and the gas phase absorption energy of 354 nm (tables 4.2 and 4.3). The nature of
the second lowest energy transition in the experiment cannot be assigned unambiguously
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because of the close energy separation of the next two calculated values (294 and 281 nm,
respectively) and their similar oscillator strengths. However, all the associated orbitals those
might be involved in excitation with significant contribution in this wavelength range, viz.
HOMO-1, HOMO, LUMO and LUMO+1 (table 4.6), are shown to be of π type in nature (figure
4.7). Further, to confirm that no proton dissociated anionic species contributes in this
wavelength region, TD-DFT calculation was performed on fully optimized anionic species of
conformer IIb. The lowest energy absorption appears at 450 nm region that is in close
agreement with the experimentally obtained value of 475480 nm reported by Vasilescu et. al.
[115]. These authors proposed the existence of proton dissociated dinionic structure of
conformer III responsible for this absorption. However, from the energy parameters given in
table 4.5, it is clear that the formation of this conformation itself is very unlikely. So, the anionic
species responsible for the long wavelength absorption is believed to be originated from
conformer IIb. Furthermore, comparison of the nature of HOMO and LUMO in figure 4.7 reveals
that on excitation, the electron density is more localized on carbonyl oxygen and imino nitrogen
atoms (O12 and N10, respectively in figure 4.1), thereby increasing the basicity at these points
significantly. This confirms the importance of hydrogen bond donating ability (acidity) of the
solvent to discuss the spectroscopic behavior of LH2, more particularly in the excited state, as
indeed observed from LSER analysis discussed above.
Table 4.6 Calculated singlet excited state transitions, associated energies and oscillator strength (f) of I-S3 and IIb-S3.a
I-S3 IIb-S3
Singlet
State
Transition Energy /nm f Transition Energy /nm f
1 HL 335 0.13 HL 332 0.14
2 H-1L 294 0.01 HL+1 294 0.07
3 H-1L+1
HL+1
282 0.07 H-1L
HL+1
281 0.05
a The frontier orbitals HOMO and LUMO are designated as H and L, respectively.
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Figure 4.7 Frontier orbital diagram for the most stable ground state conformer of LH2 (structure IIb). The arrow mark in LUMO indicates charge localization on the oxygen and nitrogen atoms on electronic excitation.
The calculated energy difference between I and IIb in the ground state is 14.1 kJ mol-1.
The potential energy surface (PES) (figure 4.8), constructed by intrinsic reaction coordinate (IRC)
calculation from the transition state (TS), indicates that the water assisted conversion of I to IIb
is associated with a large activation barrier of 55 kJ mol-1. So, in ground state, relative
abundance of the high energy structure (I-S3) will be very less, both from kinetic and
thermodynamic point of view. However, TD-DFT calculation results show that, the relative
energy difference between I-S3 and IIb-S3 is very small in the first excited state (3.4 kJ mol-1).
Hence, simple Boltzmann distribution predicts approximately 25% population of excited state to
exist as I-S3 in solution at room temperature. Therefore, the photochemistry of LH2 can be
considered as an average property of both these structures. As shown in table 4.6, the transition
energy, nature of excitation as well as the corresponding oscillator strength of both these
structures is similar to each other. Naturally, it is expected for them to show similar
spectroscopic behavior, particularly in non-interacting solvents. However, as these conformers
differ considerably in their mode and extent of hydrogen bonding, it is possible to form different
hydrogen bonded clusters in protic solvents with little difference in energy. The broad nature of
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the emission spectra of LH2 in protic solvents, as discussed before, may be due to the ensemble
averaged spectral properties of all these microstructures.
Figure 4.8 Ground state potential energy surface for water-mediated conversion of I to IIb obtained from IRC calculation using B3LYP/6-311++G(d,p) methodology. The energy of the transition state (TS) structure (given in the right hand panel, bond lengths and angles are in Å and degrees, respectively) and six points on both the sides are shown.
4.4. Luminol Fluorescence in Aqueous Buffer and Mixed Solvents
4.4.1. LH2 fluorescence in aqueous buffer: Presence of more than one
species In homogeneous buffer medium LH2 shows two absorption bands, one at 295 nm and
the other at 350 nm. The emission obtained by exciting at both these absorptions show
strongly intense, unstructured and broad spectra ranging from 375520 nm with the maximum
at 420 nm. The excitation spectra corresponding to this emission resembles very closely with
the absorption spectrum [119].
Picosecond time-resolved fluorescence decay measurement of LH2 emission in
homogeneous buffer solution indicates a bi-exponential decay function as confirmed by visual
inspection of the fitting data as well by the statistical parameters shown in figure 4.9. It is
observed that about 24% of the total excited fluorophore decays at about 2.4 ns, whereas, the
remaining 76% population has an average fluorescence decay time of 9.8 ns. The measured
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TCSPC data is in good agreement with the decay times of LH2 reported previously in aqueous
medium [77]. As discussed in the previous section, out of several possible isomers, only
structures I and IIb (figure 4.1) are responsible for the fluorescence behavior of LH2 in aqueous
solution [119]. Density functional theory calculation using B3LYP/6-311++G(d,p) formalism
predicts that although the calculated spectroscopic properties like vertical transition energy and
associated oscillator strength etc. are similar for both the isomers, structure IIb is more stable by
14.1 kJ mol-1 energy compared to structure I in the ground state; whereas, in the excited state
the energy difference is only 3.4 kJ mol-1. From this excited state energy difference, a simple
Boltzmann distribution calculation predicts about 25% of the fluorescence is contributed from
hydrated I; whereas, the rest is due to the contribution from structure IIb. In analogy with this
observation and the pre-exponential factors associated with LH2 fluorescence decay time
discussed above, the short and long decay time component of LH2 fluorescence can be assigned
to structures I and IIb, respectively. These two conformers differ considerably in their mode and
extent of hydrogen bonding in aqueous medium. Very small energy difference among different
hydrogen bonded clusters and a possible dynamic interconversion among them in the excited
state may contribute towards the broad nature of LH2 fluorescence spectra, particularly in protic
solvent like water. Although, time-resolved data and theoretical calculation results give a
definite indication of the presence of more than one fluorescing species, these conformers
could not be resolved spectrally.
Figure 4.9 Time-resolved fluorescence decay profile (open squares) and simulated data (solid line) with one and two exponential decay functions (represented by 1-exp and 2-exp, respectively) for LH2 in aqueous buffer (pH = 7.4). exc = 375 nm and mon = 430 nm. IRF indicates the instrument response function. Upper panel shows the distribution of weighted residuals for 2-exp fitting. Value of reduced chi-square (2) in different fitting model is also indicated.
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4.4.2. Steady state spectral properties in mixed solvents Before going into the detail of the fluorescence properties in hydrophobic environment,
it is worth discussing the behavior of the probe in the homogeneous mixture of water and 1,4 –
dioxane, as these mixtures are known to mimic the micellar environment very closely [122].
With increasing the volume fraction of water in 1,4-dioxane, both the fluorescence spectral
position and intensity shift regularly and finally shows about 30 nm red shift along with almost
three times increase in fluorescence quantum yield (f) in pure aqueous medium.
Representative fluorescence emission spectral profiles along with the corresponding data are
shown in figure 4.10a. However, the fluorescence excitation spectra (figure 4.10b) do not show
any major change while going from 1.4-dioxane to water; only exception being the loss of
vibrational structure in the high energy band accompanying by little broadening of the 350 nm
peak. This difference in fluorescence properties in aqueous media is due to the stabilization of
the excited state through formation of specific hydrogen bond of LH2 with water [123]. The
hydrogen bond formation occurs through the solvent hydrogen bond donation property
towards the electron rich charge localized centers like imine nitrogen and carbonyl oxygen of
the 2- and 4-positions of the phthalhydrazide ring system of LH2.
Figure 4.10 Steady state fluorescence emission (a) and excitation (b) spectra of LH2 in varying water/1,4-dioxane content. (a) Volume percent of water = 0 (1), 10 (2), 50 (3), 75 (4) and 100 (5). Inset shows the fluorescence emission maxima (max
em) and quantum yield (f) in different systems. (b) 1,4-dioxane (1) and water (2).
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4.5. Interaction of Luminol with Surfactants & -Cyclodextrin
4.5.1. Steady state spectral properties in micellar media The absorption maximum for the aqueous solution of LH2 is practically unaffected by the
presence of added surfactant, indicating very low absorbance sensitivity to the changes in
surfactant concentration. However, the intensity of the fluorescence spectrum changes with the
amount of surfactant in solution. Gradual addition of all the surfactants (CTAB, TX-100 and SDS)
is associated with an initial increase within very small surfactant concentration followed by
steady decrease in LH2 fluorescence intensity. Figure 4.11 shows some representative spectral
profile along with the pattern of intensity variation in each case.
Figure 4.11 Change in LH2 fluorescence emission profile (exc = 360 nm) with increasing concentration of CTAB (a), TX-100 (b) and SDS (c). The concentration of the surfactants are (a) [CTAB]/ mM = 0.0 (i), 0.5 (ii), 1.4 (iii), 2.7 (iv), 4.7 (v), 7.8 (vi), 12.5 (vii), 18.7 (viii); (b) [TX-100]/ mM = 0.0 (i), 0.25 (ii), 0.30 (iii), 0.5 (iv) and (c) [SDS]/ mM = 0.0 (i), 2.7 (ii), 8.0 (iii), 11.9 (iv), 12.5 (v), 21.9 (vi). Inset shows the variation in fluorescence intensity at 425 nm in each case.
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The increase in fluorescence intensity at very low surfactant concentration is due to the
increased solubility of the organic fluorophore; however, it is clear that the fluorescence
intensity decreases continuously after a certain concentration of surfactant in the solution,
which is quite close to the critical micelle concentration (cmc) of the individual surfactant
system. Once the micellar structure is formed, the fluorophore is partitioned in the hydrophobic
micellar pseudo-phase from the bulk aqueous medium. As a result, the average steady state
fluorescence intensity of the solution would decrease, which is in consistent with the
observations made above for homogeneous solvents like water buffer and 1,4-dioxane.
Interestingly, it is evident from the inset of figures 4.11(a) and 4.11(c) that the rate at which
intensity decreases after the micelle formation is relatively higher in case of cationic micellar
system CTAB in comparison with anionic SDS. However, it is rather difficult to quantitatively
compare the TX-100 data given in inset of figure 4.11(b) as the concentration range used in this
case are at least an order of magnitude lower than the other two. Apparently, the fluorescence
peak position is not too sensitive towards the micellar medium. However, careful analyses of the
data presented in table 4.7 as well as figure 4.11 reveal that fluorescence spectral blue shift is
somewhat greater in case of interaction with CTAB (3 nm) in comparison with the other
surfactant systems. In analogy with the results discussed above for water/1,4-dioxane solvent
mixture, a blue shift of the fluorescence maximum suggests that the polarity of the micellar
environments are less than the polarity of the bulk water. However, the fluorescence maxima of
LH2 in fully micellized condition (table 4.7) indicate that the probe is not incorporated into the
core, rather, it stays more or less in the interfacial region of the micelle. Nevertheless, the data
presented in table 4.7 indicates stronger interaction of LH2 with CTAB. The decrease in LH2
fluorescence intensity in micellar medium can be rationalized on the basis of the passage of the
relatively non-polar fluorophore (structure IIb, figure 4.1) towards more hydrophobic interfacial
region. However, due to extensive charge localization in the excited state [123 (a)], LH2 interacts
more strongly with cationic micelle like CTAB. The presence of this dipole-dipole type of
interaction in addition to the hydrophobic force causes much stronger interaction with CTAB in
comparison with other surfactant systems as evidenced by (a) rapid fall in fluorescence
intensity, and also (b) detectable blue shift in fluorescence spectral position. This point is further
confirmed from the time resolved studies as well as thermodynamics of ligand binding in BSA
discussed below. Similar results were reported recently for the fluorescence behavior of 8-
hydroxypyrene-1,3,6-trisulfonate, trisodium salt (HPTS) [124]. It was shown that the extent of
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excited state deprotonation and quenching of neutral and/or anionic fluorescence of HPTS is
drastically different and depends strongly on the nature (cationic, anionic or neutral) of the
surfactant. It is to be noted here that while discussing the principle driving force and the stability
of LH2-micelle complex in different surfactant systems, neither the dimension of the micelle nor
the aggregation number was taken into consideration. However, a rigorous description of the
forces responsible for binding the chromophore in the micellar sub-domain should involve the
size of the respective micelles and its change with increasing surfactant concentration as well as
the spatial distribution of the fluorophore in the interfacial region. Nevertheless, a qualitative
description based on the averaged parameters of the probe as well as the micellar system is
reasonable, given the tendency of the hydrophobic environments to quench LH2 fluorescence
and also the time-dependent fluorescence decay behavior in different media discussed in the
following section [123 (b)].
Table 4.7 Fluorescence spectral behavior of LH2 (5 M) in homogeneous buffer solution and in different heterogeneous medium.a
Medium maxem
(nm)
f Decay parameters 2
1 /ps (1) 2 /ns (3) 3 /ns (3)
Buffer (pH=7.4)
426 0.78 - 2.4 (0.24) 9.8 (0.76) 1.19
CTAB 0.5 mM
12.5 mM
426
423
0.84
0.61
-
-
2.5 (0.24)
2.4 (0.61)
9.9 (0.76)
9.5 (0.39)
1.12
1.14
TX-100 0.15mM
0.6 mM
426
426
0.89
0.77
-
-
2.3 (0.25)
2.4 (0.36)
9.7 (0.75)
9.6 (0.64)
1.20
1.15
SDS 2.4 mM
20 mM
426
426
0.81
0.59
-
-
2.4 (0.24)
2.5 (0.34)
9.9 (0.76)
9.5 (0.66)
1.15
1.20
BSA 6 M
30 M
423
420
0.30
0.11
<100 (0.65)
<100 (0.60)
1.8 (0.12)
1.0 (0.27)
8.9 (0.23)
8.8 (0.13)
1.02
1.2
HSA 6 M
30 M
426
426
0.62
0.53
<100 (0.71)
<100 (0.73)
2.0 (0.08)
1.6 (0.09)
9.0 (0.21)
9.0 (0.18)
1.10
1.12
aThe pre-exponential factor and the decay time is represented by and , respectively; the relative error in measuring values is within 0.1 ns.
90
4.5.2. Time-resolved fluorescence properties The fluorescence decay behavior was also monitored in presence of added surfactants.
All the experimental decay curves could be well reproduced again with two-exponential decay
functions as evidenced by the statistical parameters like reduced chi-square (χ2) and distribution
of weighted residuals. Figure 4.12 shows some representative decay profile in varying
concentration of CTAB along with the fitting data and table 4.7 collects all the relevant data for
different surfactants. From the table, it is clear that the fluorescence decay time of the two
components remains practically unaffected in presence of all the surfactants. The relative
contribution of the first decay component increases in presence of all the surfactant
concentration above cmc. However, in case of CTAB, this increase is almost two and half times
in comparison with that of the bulk aqueous medium. This observation further supports the idea
of relatively stronger interaction in CTAB micelle as discussed before.
Figure 4.12 Time resolved fluorescence decay profile (open circles) of aqueous LH2 in presence of varying concentration of CTAB along with the simulated data (solid lines). [CTAB] /mM = 0.5 (1) and 12.5 (2), respectively. The upper panels show the distribution of weighted residuals for 2-exponential fitting in each case. IRF indicates the instrument response function.
91
4.5.3. Fluorescence behavior in β-cyclodextrin (CD) and mixed CD-
surfactant system
The unique property of cyclodextrin (CD) to encapsulate organic compounds inside its
hydrophobic central cavity make them potential candidate as extremely efficient molecular
vehicles for drug delivery [125]. Furthermore, the reduced polarity and restricted geometry of
the interior cyclodextrin cavities gives an opportunity to study different photophysical
properties in tailored environmental conditions. The inclusion of the organic probe is primarily
controlled by the size fitting of the guest toward the host cavity [126]. The LH2 fluorescence
intensity seems to increase moderately on addition of CD till it reaches a plateau along with a
spectral blue shift of about 5 nm (figure 4.13a). As seen in the previous section about the LH2
fluorescence quenching in micellar medium, it is rather surprising to observe an enhancement in
fluorescence intensity on binding with the hydrophobic CD cavity. In fact, little increase in
molecular fluorescence of LH2 derivatives in presence of CD was reported as early as in mid-
eighties; however, no possible explanation was given for that [127]. Recently, Maeztu et. al. also
reported the enhancement of chemiluminescence intensity of LH2 and its derivatives at alkaline
pH in presence of natural cyclodextrins [128,129]. In addition to the size requirement for the
entire or partial inclusion of the guest molecule inside the CD cavity, additional hydrophobic
forces are also important in determining the geometry of the complex [130,131]. Nevertheless,
an increase in LH2 fluorescence intensity in presence of CD can stem from the fact that considers
the restricted non-radiative motion, and thereby reducing the vibrational deactivation of the
caged analyte.
The apparent binding constant and stoichiometric ratio of the inclusion complex can be
determined from the modified Benesi-Hilderbrand (BH) equations described in the previous
chapter [equations (3.19) and (3.20)] using the fluorescence data. Figure 4.13b shows the
double reciprocal plot for LH2-CD system. The linearity for 1:2 case indicates that LH2 is bound
with two CD molecules and the binding constant is calculated to be 4.2 × 106 M-2 from the slope
and intercept of the simulated data. Interestingly, the fluorescence intensity is also found to
increase upon gradual addition of CD in LH2 solution containing a fixed amount (12.5 mM) of
CTAB (figure 4.14). However, BH plot in this case indicates a complex with 1:1 stoichiometry
with an apparent binding constant of 101.4 M-1 (inset, figure 4.14). This difference in
stoichiometry may be due to the fact that the nature and intensity of the feeble forces
92
responsible for the formation of the complex changes in presence of an ionic micellar medium
like CTAB.
Figure 4.13 (a) Variation of LH2 fluorescence spectral profile on addition of -cyclodextrin (CD). [CD] /mM = 0 (i), 0.3 (ii), 0.7 (iii) and 3.8 (iv). Inset shows the intensity variation at 425 nm. (b) Double reciprocal plot for 1:1 (solid line) and 2:1 (dotted line) of the CD-LH2 complex obtained from equations (3.19) and (3.20).
Figure 4.14 Variation in intensity of fluorescence for LH2 (12 µM) in fixed concentration of CTAB (17 mM) with increasing concentration of -cyclodextrin. [CD] /mM = 0 (i), 0.7 (ii), 1.2 (iii), 1.8 (iv), 3.2 (v) and 4.0 (vi). Inset shows the double reciprocal plots.
93
4.6. Binding of Luminol with Serum Albumins
4.6.1. Interaction of luminol with human serum albumin (HSA) (i) Fluorescence quenching in presence of HSA
Figure 4.15a shows the emission spectra of LH2 in the presence of various concentration
of HSA. It is observed that the fluorescence intensity of LH2 decreases regularly with the
increasing concentration of HSA without any significant shift in the emission maximum. The
rapid quenching of LH2 fluorescence indicates a strong interaction of LH2 with HSA. The Stern-
Volmer (SV) plot for the LH2 fluorescence quenching (shown in the inset of figure 4.15a) shows a
downward curvature. This kind of deviation from linear SV plot is a typical characteristic feature
of fluorescence quenching involving two types of fluorophore populations; one of which is not
accessible to the quencher [4(a)]. A modified form of SV equation, given in equation (4.1), can
be used to analyze this type of fluorescence quenching data assuming the total fluorescence to
be the contribution from both the accessible and inaccessible fluorophores.
∆
=[ ]
+ 4.1)
where, ∆F (= F0 – F) is the difference of fluorescence intensity in absence and presence of
quencher (Q) concentration; Ka is SV quenching constant of the accessible fraction (fa).
Figure 4.15 (a) Variation of LH2 fluorescence with increasing concentration of HSA. [HSA] /M = 0 (1), 3 (2), 6 (3), 15 (4), 21 (5), 30 (6). The excitation wavelength is 360 nm. Inset shows the downward deviation of SV plot. (b) Modified Stern-Volmer plot for LH2 fluorescence quenching by HSA at 308 K. Correlation coefficient (R) and standard deviation (SD) are given for the linear fitting. The numbers in parenthesis indicate the relative error in slope and intercept.
94
Apparent values of Ka and the fraction of fluorophore accessible to the quencher (fa) can
be obtained from the slope and intercept of the plot of F0/∆F vs [Q]-1. Linear variation of the
modified SV plot was obtained at all the temperatures with acceptable statistical parameters. A
representative plot at 308 K is shown in figure 4.15b in case of LH2/HSA system. Apparently, the
magnitude of the SV quenching constant (2.0±0.2 x 105 M-1) and fraction (4047 %) of the
accessible fluorophore for quenching does not vary significantly within the temperature range.
Figure 4.16 (a) Fluorescence spectra of aqueous LH2 in presence of 0.6 mM TX-100 (2) and 12.5 mM CTAB (3) relative to the normalized spectrum (1) at cmc (0.3 and 0.9 mM, respectively). (b) Increase in fluorescence intensity of the LH2/HSA solution in presence of bilirubin. [HSA] = 30 M and [BIL] /M = 0 (1), 0.5 (2) and 1.0 (3).
The idea of fractional accessibility of LH2 towards binding with protein and consequent
fluorescence quenching is further supported from the steady state and time resolved
fluorescence study in presence of different micellar medium discussed in the previous sections.
The fluorescence intensity of LH2 is found to decrease (figure 4.16a) in presence of different
surfactants like SDS, CTAB and also TX-100, when the concentration of surfactants in solution is
above critical micelle concentration (cmc). Moreover, time-resolved fluorescence decay analysis
reveals that although the decay time on the first component (I) remains almost same (2.4 ns),
the long component (IIb) decays relatively faster in presence of micellar environment when
compared with that in aqueous buffer solution (table 4.7). Organic fluorophore are known to
penetrate into the micellar core or can reside in the micelle-water interface when the surfactant
concentration in solution is above cmc and therefore, experiences a relatively non-polar
environment surrounding it [132]. In case of LH2, the decrease in fluorescence intensity can also
be ascribed due to the passage of component IIb in the micellar environment, thereby reducing
95
both the intensity as well as fluorescence decay time of this species. However, component I
remains in the homogeneous (aqueous) phase of the micellar solution and the decay time
remains unaltered. The calculated dipole moment of species I and IIb (2.12 and 1.92 D,
respectively) is not too different to conclude about the preferential encapsulation of species IIb
in the relatively hydrophobic micellar core with certainty; however, this definitely gives an
indication towards this prediction necessary to explain the observed experimental results.
It is known that HSA contains a hydrophobic portion on its surface (sub-domain IIA),
which is normally interior to the protein and forms the primary binding region for a large
number of hydrophobic ligands like fatty acids, bilirubin as well as several indole derivatives like
tryptophan [22(b), 133]. It seems that out of two species of LH2 (I and IIb) present in solution,
the more hydrophobic IIb is accessible to bind to the albumin and consequently, the overall
fluorescence intensity is quenched. The importance of sub-domain IIA in binding LH2 is further
reinforced by ligand replacement process in presence of bilirubin (BIL). On subsequent addition
of BIL to an LH2/HSA solution, the fluorescence intensity of LH2 increases continuously (figure
4.16b). BIL is known to have very strong affinity towards HSA sub-domain IIA with apparent
binding constant varying within the range of approximately 106 108 dm3 mol-1 [134]. This value
is higher by almost four order in magnitude compared to that in case of LH2 (see below). With
the preferential binding of BIL into the ligand binding site, the bound fraction of LH2 is expelled
in the more aqueous phase; consequently the fluorescence intensity increases.
Figure 4.17 (a) Fluorescence decay profile (open circles) and simulated data (solid line) with three exponential decay function of LH2 in presence of 30 M HSA. IRF indicates the instrument response function. Inset shows the same data in shorter time scale.
96
The fluorescence decay behavior of LH2 in presence of albumin is markedly different
from what is observed in homogeneous buffer and also the micellar medium (table 4.7).
Although the decay time corresponding to species I (inaccessible for quenching) and IIb
(accessible for quenching and therefore, bound to ligand binding domain of protein) still found
to be 2 ns and 9 ns respectively, a major portion (ca. 70%) of the excited fluorophore decay
with very short instrument limited life time (<100 ps). The presence of the first decay
component in presence of HSA is obvious from figure 4.17 and also observed in presence of BSA
(see below). A comparison of the statistical parameters reveals that two exponential fitting of
the experimental data is insufficient to produce acceptable statistics and a minimum of three-
exponential decay function is necessary for good fitting. However, no such fast decay was
observed for LH2 in presence of other heterogeneous micellar medium (table 4.7). As a first
guess, a ground state complex formation may be approximated for the rapid fluorescence
quenching along with a very fast LH2 decay component in presence of albumins. However,
relatively weak binding constant values (see below) and also the absence of any additional
absorption peak for the LH2-HSA complex suggests that they do not really form a ground state
complex. Instead, there may be a closely spaced fluorophore-quencher pair; where, the
fluorescence is quenched rapidly in presence of the quencher and appears to be dark under this
experimental condition. This rapid transient quenching is associated with the additional very fast
decay time which is followed by the normal (slower) diffusion of the fluorophore and the
quencher.
(ii) Analysis of the nature and thermodynamics of binding equilibrium
The equilibrium between the free and bound ligands for the binding of small molecules
to a set of equivalent sites in a macromolecule is given by the following equation [135]:
푙표푔 ∆ = 푙표푔퐾 + 푛. log [푄] (4.2)
where, K is apparent binding constant to a site and n is the number of binding site per
macromolecule. ∆F (=F0-F) is the difference in fluorescence intensity in absence and presence of
quencher Q, respectively.
The dependence of log(∆F/F) on log[Q] is linear at all the temperatures studied here.
Some representative plots are shown in figure 4.18. The correlation coefficient (R) is 0.98±0.01
and the standard deviation (SD) values obtained in each case are within 0.05 indicates that the
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assumptions made in deriving equation (4.2) is valid under the present experimental condition.
The binding constant values obtained from the intercepts of figure 4.18 are further used to
calculate the thermodynamic parameters of LH2-HSA interaction.
Figure 4.18 Calculation of binding constant in LH2/HSA system using equation (4.2) at different temperatures.
If the enthalpy change (∆H) does not vary significantly in the temperature range studied,
van’t Hoff relation [equation (4.3)] can be used to evaluate the enthalpy and entropy change for
LH2 binding to HSA [136]; whereas the corresponding Gibb’s free energy parameter is calculated
from the relationship given in equation (4.4).
푙표푔퐾 = − ∆.
+ ∆.
(4.3)
∆퐺 = ∆퐻 − 푇∆푆 (4.4)
All the parameters are listed in table 4.8 and negative values of ∆G assert that the binding
process is spontaneous in the whole temperature range. Both the enthalpy and entropy change
for LH2 binding to HSA are also found to be negative. The interaction forces between drug and
biomolecules may include electrostatic as well as van der Waals interaction, multiple hydrogen
bond formation, hydrophobic and steric contact within the cavity etc. Ross and Subramanian
[137] studied a series of protein association reaction with variety of ligands and proposed three
possible reasons for the negative values of ∆H and ∆S. Those include non-bonded (van der
Waals) interactions, hydrogen bond formation in low dielectric media and protonation due to
the association process. The possibility of protonation during the LH2-HSA association at the
experimental pH seems unlikely. Moreover, our previous report suggests that LH2 possesses
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several potential hydrogen bonding sites and even the hydrogen bonded conformers are
thermodynamically far more stable (80 kJ mol-1 for species IIb) than the corresponding non-
hydrogen bonded structures [123]. So, the possibility of hydrogen bond formation even inside
the protein binding domain cannot be ruled out. Overall, it is not possible to account for the
thermodynamic parameters of LH2-HSA binding based exclusively on a single intermolecular
force model. A combination of van der Waals interaction as well as possible hydrogen formation
may contribute in the whole process. Meanwhile, it is clear from table 4.8, that major
contribution for the ∆G term comes from ∆H rather than ∆S; so the binding process is believed
to be enthalpy driven.
Table 4.8 Thermodynamic parameters corresponding to binding of LH2 with HSA.a
Temperature /K Binding constant /dm3 mol-1
∆H /kJ mol-1 ∆S /J K-1 mol-1 ∆G /kJ mol-1
298 50.121.78 -31.7 -74.9 -9.38 303 28.861.23 -9.00 308 32.481.35 -8.63 313 22.171.54 -8.26 318 20.991.38 -7.88
a The binding constant values were calculated from the slope of equation (4.2). The other thermodynamic parameters were obtained using equations (4.3) and (4.4) with an error limit of 10%.
Figure 4.19 Variation of LH2 fluorescence spectral profile on addition of bovine serum albumin (BSA). [BSA] /M = 0 (i), 1.7 (ii), 3.5 (iii), 5.1 (iv), 6.9 (v), 8.6 (vi), 10.3 (vii) and 17.2 (viii). Inset shows the intensity variation at 425 nm.
99
4.6.2. Interaction of luminol with bovine serum albumin (BSA) (i) Fluorescence quenching in presence of BSA
The fluorescence intensity of LH2 decreases regularly with the increasing concentration
of BSA accompanied with 10 nm blue shift in the emission maximum. Figure 4.19 shows the
representative emission spectral profile in presence of various concentrations of BSA. The rapid
quenching of LH2 fluorescence indicates a strong interaction of LH2 with BSA. In inset of figure
4.19, the fluorescence intensity was plotted against the quencher concentration. Interestingly,
the decrease in intensity shows a clear sigmoidal shape. It is to be noted here that the LH2
fluorescence intensity was also found to decrease with HSA concentration [138]; however, the
nature of fluorescence intensity decrease is non-sigmoidal (inset of figure 4.20). The decrease in
LH2 fluorescence intensity can be assumed again as the passage of the probe molecule from the
aqueous bulk phase and binding towards a more hydrophobic region in the ligand binding
domain of the proteins.
Figure 4.20 Variation of LH2 fluorescence with increasing concentration of HSA. [HSA] /M = 0 (1), 3 (2), 6 (3), 15 (4), 21 (5), 30 (6). The excitation wavelength is 360 nm. Inset shows the intensity variation at 425 nm.
Binding of LH2 in albumin is further reinforced by ligand replacement process in
presence of bilirubin (BIL). On subsequent addition of BIL to an LH2/BSA solution, the
fluorescence intensity of LH2 is recovered (figure 4.21). The binding constant value (106 108M-1)
of BIL to BSA is higher by almost three order in magnitude compared to that in case of LH2 in
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BSA (see below). With the preferential incorporation of BIL into the ligand binding site, the
bound LH2 is expelled in the more aqueous phase; consequently the fluorescence intensity
increases. Interestingly, this recovery of fluorescence in presence of BIL can be explored in
further detail and used as an assay for efficient application of LH2 for analytical and/or forensic
purposes.
Figure 4.21 Increase in fluorescence intensity of the LH2/BSA solution in presence of bilirubin (BIL). [HSA] = 26 M and [BIL] /M = 0 (i), 0.3 (ii), 0.5 (iii) and 1.0 (iv). The excitation wavelength is 360 nm in all the cases.
Like HSA, the fluorescence decay behavior of LH2 in presence of BSA is also markedly
different from what is observed in homogeneous buffer and also the micellar medium (table
4.7). The presence of the first decay component in presence of BSA is also obvious from figure
4.22. Furthermore, a comparison of the statistical parameters like reduced chi-square values as
well as visual inspection of the weighted residuals confirm that two exponential fitting of the
experimental data is insufficient and a minimum of three-exponential decay function is
necessary.
(ii) Stern-Volmer analysis of the quenching data
The fluorescence quenching data was analyzed by Stern-Volmer (SV) relation [equation
(1.20)]. As can be seen in figure 4.23, a plot of F0/F of LH2 versus [BSA] exhibit a good linearity (R
= 0.994) and affords KSV value 2.42±0.01 × 105 M-1 at 298 K. The amplitude weighted average
lifetime in absence of quencher, <휏>0, can be calculated from the bi-exponential decay data of
101
LH2 in homogeneous buffer given in table 4.7 and found to be about 8.0 ns. From this data, the
bi-molecular quenching rate constant, 휅q, can be calculated as 3×1013 M-1 s-1. This value is
almost three orders of magnitude greater than the maximum diffusion limited quenching rate
constant, which is known to be of the order of 2×1010 M-1 s-1 [139]. Therefore, the quenching
process is assumed mainly to be controlled by static quenching mechanism rather than
dynamically controlled process.
Figure 4.22 Fluorescence decay profile (open circles) and simulated data (solid line) with three exponential decay function of LH2 in presence of 30 M BSA. exc = 375 nm and mon = 430 nm. IRF indicates the instrument response function. Inset shows the same data in shorter time scale. Upper panels show the distribution of weighted residuals and 2 values obtained from the fitting of the experimental data using three exponential (3-exp) and two exponential (2-exp) decay models, respectively.
For a static quenching process, KSV can be regarded as the association constant (Ka) for
the formation of the fluorophore-quencher complex in the ground state [4(a)]. The idea of static
quenching can further be supported by doing temperature variation experiment. The calculated
Ka values from the linear SV plots given in figure 4.23 are collected in table 4.9. It is seen that the
magnitude of Ka decreases with increase in temperature, which further supports the formation
102
of a protein-LH2 complex responsible for the fluorescence quenching. Interestingly, the Ka value
in this case is about four orders of magnitude higher than that of LH2-HSA binding [138], which
indicates far stronger interaction of LH2 with BSA.
Figure 4.23 Stern-Volmer (SV) plots of LH2 fluorescence quenching in presence of BSA at 298 K (a), 303 K (b), 313 K (c) and 318 K (d). The parameters obtained from linear fitting of the data using equation (1.20) are also shown in each case.
(iii) Thermodynamics of ligand binding
The binding constant values given in table 4.9 at different temperature are further used
to calculate the thermodynamic parameters of LH2-BSA interaction. All the parameters obtained
from the linear van’t Hoff plot (not shown) are also listed in table 4.9. The negative values of ∆G
assert that the binding process is spontaneous in the whole temperature range. Further, the ∆G
value for LH2-BSA complex formation is about three times more than the LH2-HSA interaction,
which is consistent with the assumption of more favorable binding in the former. The high
negative value of ∆H in this case indicates that the binding is an enthalpy driven process;
although the positive entropy change points to the fact that the structural distortion also
contributes a major part in the binding process. In contrast, the binding of LH2 in HSA was
reported to be associated with negative ∆S value [138]. Overall, the present study reveals that
103
although BSA and HSA are structurally similar, the mechanism and consequently the
thermodynamics of binding of LH2 in these carrier proteins are distinctly different (see below).
Table 4.9 Thermodynamic parameters corresponding to binding of LH2 with BSA.a
Temperature /K Binding constant / 105 M-1
∆H /kJ mol-1 ∆S /J K-1 mol-1 ∆G /kJ mol-1
298 2.4230.108 -13.529 57.997 -30.812
303 2.2920.111 -31.102
308 2.2740.166 -31.392
313 1.9430.103 -31.689
318 1.7090.089 -31.971
a The binding constant values were taken from the slope of the Stern-Volmer plot. The other thermodynamic parameters were obtained using equations (4.3) and (4.4) with an error limit of 10%.
It is to be noted here that LH2 fluorescence quenching in presence of HSA showed a
downward curvature in SV plot and was explained on the basis of fractional accessibility of the
fluorophore to bind within the protein cavity [138]. However, no such curvature was found with
BSA in the present study. This difference can be explained on the basis of the location of the
binding sites of the albumins. Although BSA is structurally homologous to HSA, the former
contains an extra binding pocket (sub-domain IA) which is relatively more exposed to the
aqueous medium in addition to a more hydrophobic binding region in sub-domain IIA for both
the proteins [140(a-d),141]. In a recent communication, we have shown that the relatively non-
polar fraction of LH2 binds in the hydrophobic binding region in sub-domain IIA of HSA [138].
However, in case of BSA, the exposed binding region in sub-domain IA is also available for the
relatively more polar fraction of LH2 in addition to the hydrophobic binding in sub-domain IIA for
the non-polar fraction similar to the HSA binding. As a result, the overall fluorescence quenching
follows simple SV relation.
These two binding sites in BSA can be assumed to be cooperative in nature and results a
sigmoidal decrease in fluorescence intensity with increasing quencher concentration. The strong
nature of LH2 binding is also confirmed by fluorescence spectral blue shift of ca. 6 nm in BSA
compared to the bulk aqueous medium (figure 4.19). On the other hand, in HSA apparently no
104
spectral shift is observed (figure 4.20). Furthermore, the relative quenching of LH2 fluorescence
intensity is almost five times in BSA compared to HSA under similar quencher concentration.
An important equation involved in quantitative description of cooperative binding
process and analysis of the LH2 fluorescence quenching in presence of BSA is given by the
modified Hill equation [142, 143]:
∆퐹 = 퐹 × [ ]
. [ ] (4.5)
Where, ∆F (= F0 – F) is the change in fluorescence intensity in presence of substrate
concentration [S] and ∆Fmax is the maximum change in fluorescence intensity, K0.5 is the
concentration of substrate that gives half maximal fluorescence change and nH represents the
Hill coefficient. The value of nH is usually a whole number and if it is greater than one, the
situation is represented by positive cooperativity; whereas, the value of nH equal to one
represents a situation without any cooperativity.
Figure 4.24 Hill plot for LH2-BSA system at 298 K (a), 303 K (b), 313 K(c) and 318 K (d). The solid line indicates the simulation of experimental data points using equation (4.5). The corresponding parameters obtained from the fitting result are also shown.
105
Figure 4.24 represents the simulation of LH2-BSA data using equation (4.5) at different
temperature. It is evident that the experimental data points are nicely correlated and the
statistical parameters are fairly acceptable with correlation coefficient greater than 0.99 in all
the cases. Interestingly, the magnitude of nH is very close to two, indicating the positive
cooperativity with involvement of both the binding sites in BSA. However, similar analysis in
case of HSA (figure 4.25) results the value of nH to be very close to one, indicating the absence of
any cooperative mechanism in this case. Initial binding of LH2 in the more exposed ligand
binding site at the surface of BSA causes a massive structural change in a cooperative manner so
that the hydrophobic binding site in sub-domain IIA also becomes easily accessible for LH2
binding. This reorganization in the protein structure on ligand binding is also manifested from
the overall positive change in ∆S value calculated above. Further, the cooperativity is manifested
in almost four orders of magnitude higher value of binding constant and also about three times
greater negative free energy change in case of BSA binding while comparing with HAS [123
(b),138].
Figure 4.25 Hill plot for LH2-HSA system at 298 K (a), 303 K (b), 308 K(c) and 318 K (d). The solid line indicates the simulation of experimental data points using equation (4.5). The corresponding parameters obtained from the fitting result are also shown.