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Comprehensive Studies on the Interaction of Pyrimidine Derivatives and Bovine Serum
Albumin by Multi-spectroscopic Approach: A Comparative Study.
Anita J. Bodake*a ,Sunil D. Kumbhara, Anil H. Goreb, Prafulla B. Choudharic, Nilotpal Barooahd, Prashant V. Anbhuleb, Yogesh S Sonavanee, Govind B. Kolekarb, and
aDepartment of Chemistry, Rajaram college, Vidyanagar, Kolhapur-416004, India. bFluorescence Spectroscopy Research Laboratory, Department of Chemistry,Shivaji University,
Kolhapur-416004, India.
cDepartment of Pharmaceutical Chemistry BharatiVidyapeeth College of Pharmacy, Kolhapur-
416013, India.
dRadiation& Photochemistry Division, Bhabha Atomic Research Centre, Mumbai-400085, India.
eDepartment of Chemistry, University of Split, 21000, Split, Croatia.
*Corresponding author: Email: [email protected]; Fax: +91 0231 2531989
Abstract
The binding of quinolone derivative NBPT/BPT/MBPT to bovine serum albumin (BSA)
was investigated by various spectroscopic methods and molecular docking analysis. The
fluorescence quenching spectroscopic results showed that NBPT/BPT/MBPT bind to the protein
BSA. The thermodynamic parameter of the system shows increase in temperature with gradual
decrease in Stern-Volmer quenching constant thereby indicating Static quenching mode.
Negative entropy and positive enthalpy indicates that the hydrogen bonding interaction. The CD
spectral study indicates reduction of α-helical structure in BSA and small changes in the tertiary
structure of the protein. NBPT/BPT/MBPT interacts strongly with BSA and small changes in
protein morphology was advised by molecular docking results. Moreover docking results show
that the ETMTMHQC binds to BSA at ASN390 residue
Keyword: Bovine serum albumin; Site selective binding; Förster resonance energy transfer.
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1 Introduction:
Serum albumin is the most abundant protein, having 67,000 molecular weight and occurs
mainly in single polypeptide chain. It is made up of 583 amino acids which comprises of two
tryptophanyl, one methionyl, and four methionyl residues. Serum albumin binds to number of
biomolecules and ligands such as penicillin, thyroxine, ascorbic acid, sulfonamides, and
barbiturates. It’s basic roll is to transport some lipid classes, with free fatty acids [1-3].
In-vitro study of some heterocyclic compounds and their interaction with bovine serum
albumin at various conditions is carried out. This information will lead to binding and
transportation of the biomolecules in the drug delivery system. The pharmacokinetics (counting
distribution, metabolism, and elimination) and pharmacodynamics study of heterocyclic
compounds MBPT, NBPT and BPT is focused in the present chapter.[4] The effect of interaction
of BSA with heterocyclic compounds containing electron donating MBPT, electron withdrawing
NBPT and BPT is studied. This in-vitro study will provide helpful information for the synthesis
of the new biological compounds and specific information for drug delivery system. With this
aspect, the interaction of BSA and the synthesized biomolecules is planned and carried out [5-9].
5-(benzylidene)-pyrimidine-2,4,6,-(1H,3H,5H)-trione (BPT), 5-(4-nitrobenzylidene) -
pyrimidine-2,4,6,-(1H,3H,5H)-trione (NBPT), 5-(4-methoxy benzylidene)-pyrimidine-2,4,6,-
(1H,3H,5H)-trione (MBPT) [10].
2. Material and Methods:
2.1 Materials and Apparatus:
Bovine serum albumin, Barbituric acid and aldehyde were purchased from Sigma-
Aldrich analytical reagent grade and used without purification. Bovine serum albumin is used to
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prepare the stock solution in deionized water and the final concentration of this solution is
1.0×10-5 M. BSA stock solution was preserved in the dark at 273-277 K. The phosphate-buffer
solution was prepared in (0.1M) of the pH=7.4. The stock solution of biomolecules samples
MBPT, NBPT and BPT with 1x10-6mol dm-3 concentration were prepared in deionized water and
DMSO in (95:5) proportion. The UV-visible absorption spectra was measured on UV-Vis-NIR
spectrophotometer (Shimadzu UV-3600) and fluorescent emission spectra was monitored by PC-
based spectrofluorometer (JASCO Japan FP-750) equipped with Xenon lamp. A 1.0 cm quartz
cell was used for measurements. Slit width was fixed to 10 nm for excitation and emission
spectra measurement. A circular dichroism (CD) spectrum is measured with a Jasco J-815
Spectropolarimeter (Jasco, Tokyo, Japan) at wavelength range 200-300nm at room temperature,
using a 1 cm quartz cell. The docking analysis using Biopredicta module of V life MDS 4.4.
2.2 Measurements of Spectrum:
In the present study, ideal concentrations of protein and MBPT, NBPT and BPT were
selected. On the basis of experiments under several physiological conditions described above,
1.0 mL BSA solution and 2.0 mL phosphate-buffer solution, a known volume of standard
MBPT, NBPT and BPT solutions were added to 0.5 to 6 mL standard flask and diluted up to the
mark with deionized water. UV-Vis absorption spectra were measured at room temperature from
200-600nm using a quartz cuvette with 1.0 cm path length. The fluorescence quenching spectra
of BSA were produced at the excitation wavelength (λex = 280 nm) and emission wavelength
(λem = 290-600 nm). All measurements were noted after 10 minutes of equilibrium at the
suitable temperatures 298K, 308K, and 318K. The 3D fluorescence spectra recorded the
excitation wavelength range of 250-320 nm and the emission wavelength range of 260-550 nm
for all biomolecule-BSA system.
3. Results and Discussion:
3.1 Fluorescence Interaction Studies:
Fluorescence is a helpful and elusive method used to explore the change in the
fluorophore micro environment upon binding with the biomolecule [11, 12]. The result shows
more useful information in the binding mechanism of biomolecule with BSA. The fluorescence
intensity was observed in BSA with MBPT, NBPT and BPT systems and the fluorescence
spectra were observed at 290nm to 600nm with excitation at 280nm [13]. These results indicate
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decrease in the fluorescence intensity of BSA with the addition of NBPT, BPT and MBPT in
figure 1(A, B, and C)
Figure1: Fluorescence quenching spectra of BSA in the presence of
NBPT(A)/BPT(B)/MBPT(C)(CBSA= 1.0×10-6mol dm-3; CNBPT 10-6mol dm-3) (a) 0.0, (b) 0.5, (c)
1.0, (d) 1.5, 3.0,(g) 4.0,(h) 5.0,(i) 6.0. Figure 2 The Stern-Volmer plot of F0/F against
concentrations of the quencher in figure 2(NBPT(D)/BPT(E)/MBPT(F)).
B
C
E
F
A D
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The intensities of NBPT-BSA, MBPT-BSA and BPT-BSA systems spectra were recorded
NBPT-BSA > MBPT-BSA > BPT-BSA and arranged according to the decreasing order of
intensities.These spectra decreases the fluorescence intensity of a variety of molecular reactions
viz., ground-state complex formation, energy transfer, excited state reactions, collisional
quenching and molecular changes in terms of quenching. These surprising results are due to
static type of quenching spectra. The results are determined using the following Stern-Volmer
equation (1 and 2)
��� = 1 + ���� = 1 + ����…������������������������������������������(1)
���� = ���…�������������������������������������������������������������������(2) ��in the above equation stands for fluorescence intensity of pure BSA whereas � represents the fluorescence intensity of BSA in presence of biomolecule. ���is the Stern-Volmer quenching constant, �� is the quenching rate constant of BSA and �is the normal fluorescence lifetime of BSA (10-8s)[14-16]. at different temperature 298K, 308K 318K are taken and the values of Ksv
and Kq are calculated. It is observed that with increase in temperature the stability of complex
decreases. The overall study of different MBPT-BSA, NBPT-BSA, BPT-BSA systems shows
static type of quenching.
3.2. Binding Constant and Binding Sites:
The fluorescence spectra at different temperatures is used to calculate the binding
constant (k) and number of binding sites(n) with protein molecule, using the subsequent
equations (3.3)
��� ��� − �� � = ���� + �����…������(�)
Where, �� and � are the fluorescence concentration deprived with the medicine, correspondingly, � is binding constant. �is the number of binding sites.[17]. The value of the k is calculated from slope and the value of (n) is calculated from intercept. This slope and intercept
is the double logarithm regression curve of log ((F0−F)/F) versus log [Q]. The binding constant
and binding sites at different temperatures (298K, 308K and 318K) are represented in table
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1.1(BSA-NBPT), 1.2(BSA-BPT) and 1.3(BSA-MBPT). These results indicates that on
increasing the temperature of the system consequently the stability of complex decreases. The
results shows that the MBPT-BSA, NBPT-BSA, BPT-BSA systems and the binding constant (k)
values are different as shown table no 2.1(BSA-NBPT), 2.2(BSA-BPT) and 2.3(BSA-MBPT).
The binding constants (k) of the systems are arranged in the decreasing order as NBPT-BSA >
MBPT-BSA > BPT-BSA [18-20]
3.3 Analysis of Thermodynamic Parameters and Binding Modes:
The thermodynamic analysis at different temperatures exhibit more information in the
formation of complex. The values of ∆H, ∆G and ∆Swere calculated [21]. Here, the(∆�)value produces the possibility of forming the protein-biomolecule complex, (∆�) indicate the changes in enthalpy (the formation of complex in binding mode) and (∆�)indicate the entropy change (a disorder in the system, including ordering and disordering of solvent).
��� = �−� ������������������������������������������������������������(4) Where �� is the free energy change, � is the gas constant at room temperature,
and � is the binding constant which is gained after the fluorescence data.The (∆�)and (∆�)are the important factors for evaluating the interaction forces, hydrophobic binding, hydrogen bond, electrostatic and van der Waal’s interactions . The thermodynamic parameters of
BSA-MBPT, NBPT and BPT system are determined from the given Van’t Hoff equation (5) [22,
23].
"��� = �−�(���/� ) �+ �(���/�)���������������������������������������(5) The enthalpy change (∆�) and entropy change (∆�)are calculated from the graph. The
graph of the lnK versus 1/T is depicted in figure 3(A, B and C). Moreover, the slope is the
enthalpy change (∆�)and intercept is the entropy change(∆�). Thus (∆�) is calculated using following Gibb’s equation (3.6). Ross and Subramanian have summarized the thermodynamic
parameters and their correlation to define the principle interaction force between a biomolecule
and BSA. On the basis of the data shown in table 3.1(BSA-NBPT), 3.2(BSA-BPT) and 3.3(BSA-
MBPT) for MBPT, NBPT, and BPT with BSA,the negative (∆�) value observed cannot be mainly attributed to electrostatic interactions since for electrostatic interactions (∆�)is very
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small, almost zero. The negative
(∆�) and entropy change (∆�)bonding and hydrophobic interactions played a role in the b
BSA. In addition, the negative value of
MBPT, NBPT and BPT and BSA is spontaneous [1, 9].
Figure 3: Van’t Hoff plot for the interaction between of BSA and
in1.0mMphosphate buffer pH=7.4
absorption withBSA emission;T=298K, C
A
B
C
small, almost zero. The negative (∆�)and positive (∆�)values, in other words enthalpy change ) obtained in this case therefore indicates that the hydrogen
bonding and hydrophobic interactions played a role in the binding of NBPT, BPT and MBPT to
In addition, the negative value of (∆�) specifies that the interaction process between MBPT, NBPT and BPT and BSA is spontaneous [1, 9].
.
Hoff plot for the interaction between of BSA and NBPT(A)/BPT
phosphate buffer pH=7.4Figure 4: Spectral overlap of NBPT(D)/BPT(E)/MBPT(F)
BSA emission;T=298K, CBSA = CNBPT= 1.0x10-6moldm-3
values, in other words enthalpy change
obtained in this case therefore indicates that the hydrogen
inding of NBPT, BPT and MBPT to
specifies that the interaction process between
/BPT(B)/ MBPT(c)
/BPT(E)/MBPT(F)
D
E
F
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Table 1. Stern-Volmer quenching constants and bimolecular quenching rate constants
for the interaction of NBPT/BPT/MBPT with BSA at varioustemperatures.
pH T(K) 10-5
Ksv dm3
mol-1
10-13
kq/(dm3
mol-1
s-1
)
R2
7.40 298 7.9765 7.9765 0.9726
NBPT 308 6.9887 6.9887 0.9733
318 6.6099 6.6099 0.9875
pH T(K) 10-5
Ksv dm3
mol-1
10-13
kq/(dm3
mol-1
s-1
)
R2
7.40 298 4.2086 4.2086 0.9072
BPT 308 2.4076 2.4076 0.9399
318 1.7809 1.7809 0.9608
pH T(K) 10-5
Ksv dm3
mol-1
10-13
kq/(dm3
mol-1
s-1
)
R2
7.40 298 3.5819 3.5819 0.9896
MBPT 308 3.1140 3.1140 0.9871 318 2.7798 2.7798 0.9783
3.4. FRET Study:
Forster resonance energy transfer theory (FRET) is used to determine the distance
between the biomolecule and BSA. The energy transfer (E) can be calculated by the given
equation (7, 8 and 9) [24, 25].
E = 1 − F�F =R()
R() + *)…����������������������������������������������(7)
Where, F and F� are the fluorescence concentrations, E is the efficiency of energy transfer between donor and acceptor, ‘r’ is the portion of the distance between the NBPT/BPT/MBPT
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and Tyr moiety of BSA.R�is the critical distance at 50% efficiency of energy transfer R�can be found from the succeeding equation: 8 [26].
R�) = 8.8 × 10123K2N16∅J…����������������������������������������(8) Where spatial issue of alignment is �K2 = 2 3; and N = 1.336 is the refractive index of the medium, ∅ = 0.15, R� = 2.19�nm, E = 0.29. The production spectrum of the donor and absorbance spectrum of the acceptor is intended byJ = 3.93 × 10-15cm3�L�mol-1,� is the overlap essential spectra of the BSA-NBPT/BPT/MBPT system figure 4(A, B and C), and which can be
attained by the succeeding equation: 9.
J = ΣF(λ)ε(λ)λ6∆λ
ΣF(λ)∆λ …���������������������������������������������������(9)
Where F(λ) the fluorescence intensity of the fluorescent donor of wavelength isλ, ε(λ) is the molar immersion coefficient of the acceptor of wavelength λ.The fluorescence quenching of BSA after interacting with MBPT, NBPT and BPT shows that energy transfer takes place amidst
MBPT, NBPT and BPT and BSA. The calculated value of E, R0 and r present in table 4. The
present study implies that the energy transfer from BSA to MBPT, NBPT and BPT takes place
with great possibility show in figure 4(A, B and C) [26, 27].
3.5 Three-dimensional Fluorescence Spectroscopy:
Three-dimensional fluorescence spectroscopy is the most emerging technique that
exhibits conformational changes and shows exorbitant information in biomolecule and protein
interaction. It shows the morphology in biomolecule-BSA interaction and also gives information
about binding [28]. In this case, BSA spectra with and without addition of MBPT, NBPT, and
BPT, shows excitation wavelength and emission wavelength as well as fluorescence
intensity.This extensive emission wavelength and beneficial fluorescence intensity shows that,
there is strong binding of the MBPT, NBPT and BPT with BSA in figure 5(c, e, g). The contour
figures of the BSA-MBPT, BSA-NBPT, and BPT-BSA system in figure 5(d, f, h) shows bird’s
eye view, as well as different spectra, which deliver some binding information in BSA-MBPT,
BSA-NBPT, and BPT-BSA system. Peak first is the Rayleigh scattering peak and peak second is
BSA 5(A) with representation of (λex = λem) [29]. In this system, excitation and emission
wavelength is detected at 280nm and 350nm respectively. The addition of small amount MBPT,
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NBPT and BPT in BSA changes the environment of the system totally. It is also observed that
there is a slump in the fluorescent intensity of BSA in presence of the MBPT, NBPT, and BPT.
All this information confirms the binding of the BSA
Figure 5: Three-dimensional projection and corresponding excitation emission matrix
fluorescence diagrams(a) BSA and (b) Contour plot BSA and
plot MBPT-BSA complex CBSA=C
NBPT-BSA complex CBSA=C NBPT
BPT-BSA complexCBSA=C BPT = 1×10
NBPT and BPT in BSA changes the environment of the system totally. It is also observed that
there is a slump in the fluorescent intensity of BSA in presence of the MBPT, NBPT, and BPT.
formation confirms the binding of the BSA-MBPT, BSA-NBPT and BSA
dimensional projection and corresponding excitation emission matrix
(a) BSA and (b) Contour plot BSA and (c) MBPT-BSA and (d) Contour
=C MBPT = 1×10-6mol dm-3. (e) NBPT-BSA and (f) Contour plot
NBPT =1×10-6mol dm-3 and (g) BPT-BSA and (h) Contour plot
= 1×10-6mol dm-3
NBPT and BPT in BSA changes the environment of the system totally. It is also observed that
there is a slump in the fluorescent intensity of BSA in presence of the MBPT, NBPT, and BPT.
NBPT and BSA-BPT [30].
dimensional projection and corresponding excitation emission matrix
BSA and (d) Contour
BSA and (f) Contour plot
BSA and (h) Contour plot
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3.6 Circular Dichroism Spectroscopy:
The circular dichroism spectroscopy provides the secondary structure and conformational
changes in protein. This spectroscopy plays an important role in interaction system. It shows
excellent information in secondary structure, folding and binding properties of proteins and
secondary structure of the protein-biomolecules system which is important in pharmacokinetic
and pharmacodynamics [22]. The spectrum is shown in ultraviolet (UV) region with n-π* and π-
π* transitions. The addition of small amount of MBPT, NBPT and BPT with BSA, the secondary
structure is rapidly formed and these spectra are recorded at 200-250nm in UV CD region shoes
in figure 6. This spectra shows two bands at 208nm and 222nm, which is a typical signals of
BSA. On addition of MBPT, NBPT, and BPT to BSA, the peak intensity decreases and amount
of helicity in BSA is reduced. This reduced percentages of the ∝-helicity in BSA calculated from the given equation (10 and 11).
MRE = ObservedCD(mdeg)Cp × h × l × 10 …���������������������������������������(10)
Where, experimental CD is the detected ellipticity at 208 nm, n is the quantity of amino acid residue, l is path length of the cell, and Cp is the molar attentiveness of the protein. The standards of n are taken as 583 for BSA .The α-helical contented is then designed from the MRE values at 208 nm.
∝ −helix(%) = (−MRE2�X − 4000) × 10033000 − 4000 …����������������������(11)
Where MRE2�X the experimental MRE assessment of proteins at 208nm, 4000 is the MRE value of the β procedure and random coil conformation at 208nm and 33,000 is the MRE assessment of a pure α-helix at 208 nm.For pure BSA, the α-helicity was about 46% which
decreased to∼43% 42% 40% BPT, MBPT, and NBPT respectively. After the addition of BPT, MBPT, and NBPT (1x10-5M). The decrease in intensity indicates shrinkage in α-helical content,
which is due to the binding of BPT, MBPT and NBPT with amino acid residues. The CD spectra
indicates the basic peak and the shape of position in protein does not change even after binding
with BPT, MBPT and NBPT [31,32].
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Figure 6: Circular dichroism (CD) spectra of free BSA and its NBPT-BSA,BPT-BSA, and
MBPT-BSA complex. CBSA=1×10-6mol dm-3, C(NBPT, BPT andMBPT)=1.5×10-6mol dm-3, pH-7.4,
T=301K.
3.7 Comparative Analysis of the Three Derivatives:
Figure 7: Binding of NBPT/ BPT/ MBPT
with BSA (Green color is hydrogen bond
interaction).
NBPT
BPT
MBPT
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We have performed the silico analysis of the all the three selected derivatives on
the bovine serum albumin. All the three molecules showed excellent binding affinity with
BSA. The binding energy of all the three molecules in the range (-70.43to -64.35
kcal/mol) indicated that the stability of the biomolecule-BSA complex is very good.
NBPT is having least binding energy of -70.43 kcal/mol and showed hydrogen bond
interaction with ASN390 (2.3 Ǻ), LYS413 (1.9 Ǻ), THR448( 2.5 Ǻ) and SER488(1.4Ǻ)
in figure 7. Hydrogen bond interaction has binding energy of 1 and 40 kcal/mol, which
makes it stronger and stable form of interaction compared to the other interaction.NBPT
was found to be interacting with the BSA via formation of four hydrogen bond
interaction which makes it as strong binder. MBPT showed similar binding potential to
that of nitro via formation of hydrogen bond interaction with ASN390 (2.3 Ǻ), ARG409
(1.8 Ǻ), TYR410 ( 2.5 Ǻ ) in figure 7 at bindingenergy -68.42 kcal/moland LYS413
(1.4Ǻ). In case of BPT, the hydrogen bond interactions are limited so it was found to be
weaker binder to BSA in figure 7 at binding energy -64.35 kcal/mol [33-36].
Table 2.1 Binding constants (K) and number of binding site (n) of competitive experiment of NBPT-BSA system.
pH T(K) 10-6
K (dm3
mol-1
) N R2
7.40 298 8.1477 1.4167 0.9934
308 7.4136 1.292 0.9838
318 6.9757 1.2125 0.9809
Table 2.2 Binding constants (K) and number of binding site (n) of competitiveexperiment of BPT-BSA system.
pH T(K) 10-6
K (dm3
mol-1
) N R2
7.40 298 7.1793 1.4823 0.9885
308 5.6305 1.2364 0.9799
318 4.4885 1.0421 0.9657
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Table 2.3 Binding constants (K) and number of binding site (n) of competitiveexperiment of MBPT-BSA system.
pH T(K) 10-6
K (dm3
mol-1
) N R2
7.40 298 6.4391 1.1648 0.9939
308 6.9549 1.2699 0.9835
318 7.5902 1.3984 0.9926
Table 3.1. Thermodynamic parameters of NBPT–BSA In interaction at pH 7.40.
T(K) ∆H(kJ mol-1
) G(kJ mol-1
) ∆S(J mol-1
K-
1)
R2
298 -106.567 -60.429 202.424 0.9842
308 -62.453
318 -64.477
Table 3.2. Thermodynamic parameters of BPT–BSA in interaction at pH 7.40.
T(K) ∆H(kJ mol-1
) G(kJ mol-1
) ∆S(J mol-1
K-1
) R2
298 -244.405 -203.97 683.67 0.9953
308 -210.81
318 -217.65
Table 3.3. Thermodynamic parameters of MBPT–BSA in interaction at pH 7.40.
T(K) ∆H(kJ mol-1
) G(kJ mol-1
) ∆S(J mol-1
K-
1)
R2
298 -104.264 -140.960 472.671 0.9938
308 -145.686
318 -150.413
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Table 4. Forster resonance energy transfer theory study with BPT, NBPT and MBPT.
Derivative Name
K2 N Φ E R0 nm \= 101]3^_L_��1]
R nm
NBPT-BSA 2/3 1.33 .015 0.5062 2.387 6.621 2.4
BPT-BSA 2/3 1.33 0.15 0.0760 1.989 2.021 3.0
MBPT-BSA 2/3 1.33 0.15 0.3746 2.057 2.742 2.2
4 Conclusion:
Advanced multi-spectroscopic techniques and excellent docking analysis methods were
used for the investigation of protein biomolecule interaction study. These methods shows more
specific information in biomolecule interaction with protein. The interaction of different
biomolecule depends on the molecular properties like conjugation, lone pair and heterocyclic
groups. The biomolecules and its derivatives were synthesized and their interaction with protein
(BSA) was studied.
Fluorescent study revealed that the binding site for all the derivatives is approx. 1 and all
derivative shows static type of quenching. The increase in temperature of the system decreases
the Ksv value for all the derivatives. FRET study elucidate the distance between donor and
acceptor. The distance between NBPT-BSA, MBPT-BSA, BPT-BSA and BPT-BSA was found
to be 4.1, 5.1, and 6.5 respectively. Thus it shows that bonding between donor and acceptor is
less than 7 nm where complex can be found.
The thermodynamic study shows that negative (∆�)and positive (∆�)values, in other words enthalpy change (∆�) and entropy change (∆�) obtained in this case therefore indicates that the hydrogen bonding and hydrophobic interactions played a role in the binding of NBPT,
BPT and MBPT to BSA. The reaction is spontaneous which is due to its negative ∆� value.
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The three-dimensional study of BSA as well as NBPT-BSA, MBPT-BSA and BPT-BSA
complex spectra are found different from each other which explains that the complex is formed
for all these systems. The CD spectral study of these system shows that the spectra of BSA is
strongly reduced by NBPT compared to other systems which explains further that α-helicity of
the biomolecule is strongly reduced by NBPT and hence a strong bonding between NBPT-BSA
is compared to other systems. The binding energy of all three molecules is in the range between -
70.43 to -64.35 kcal/mol which indicates that the stability of ligand BSA complex is excellent.
6.5 References:
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[2]. A. Jahanban-Esfahlan, V. Panahi-Azar, S. Sajedi, Food Chem., (2016), 202, 426-431.
[3]. X. Zhao, F. Sheng, J. Zheng, R. Liu, J. Agric. Food Chem. (2011), 59, 7902-7909.
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