Author’s Accepted Manuscript

Biophysical and molecular docking insight into theinteraction of cytosine β-D Arabinofuranoside withHuman serum albumin

Parvez Alam, Sumit Kumar Chaturvedi, TamannaAnwar, Mohammad Khursheed Siddiqui, MohdRehan Ajmal, Gamal Badr, Mohamed H.Mahmoud, Rizwan Hasan Khan

PII: S0022-2313(15)00147-7DOI: http://dx.doi.org/10.1016/j.jlumin.2015.03.011Reference: LUMIN13252

To appear in: Journal of Luminescence

Received date: 4 December 2014Revised date: 27 February 2015Accepted date: 11 March 2015

Cite this article as: Parvez Alam, Sumit Kumar Chaturvedi, Tamanna Anwar,Mohammad Khursheed Siddiqui, Mohd Rehan Ajmal, Gamal Badr, MohamedH. Mahmoud and Rizwan Hasan Khan, Biophysical and molecular dockinginsight into the interaction of cytosine β-D Arabinofuranoside with Human seruma l b u m i n , Journal of Luminescence,http://dx.doi.org/10.1016/j.jlumin.2015.03.011

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1

Biophysical and Molecular Docking Insight into the Interaction of Cytosine β-

D Arabinofuranoside with Human Serum Albumin

Parvez Alam1, Sumit Kumar Chaturvedi

1, Tamanna Anwar

2, Mohammad Khursheed Siddiqui

1,

Mohd Rehan Ajmal1 Gamal Badr

3 and Mohamed H. Mahmoud

4 and Rizwan Hasan Khan

1*

1Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India

2Center of Bioinformatics Research and Technology, Aligarh-202002, India

3Laboratory of Immunology & Molecular Biology, Zoology Department, Faculty of Science,

Assiut University, 71516 Assiut, Egypt

4 Food Science and Nutrition Department, National Research Center, Dokki, Cairo, Egypt

*To whom correspondence should be addressed

Prof. Rizwan Hasan Khan Interdisciplinary Biotechnology Unit,

Aligarh Muslim University,

Aligarh-202002, U.P., India.

E-mail:rizwanhkhan@hotmail.com,

rizwanhkhan1@gmail.com

Phone: 91-571-2720388

Fax: + 91-571-2721776

2

Abstract

Interaction of pharmacologically important anticancer drug Cytosine β-D Arabinofuranoside

with Human serum albumin (HSA) at physiological pH 7.4 has been studied by utilizing various

spectroscopic and molecular docking strategies. Fluorescence results revealed that Cytosine β-D

Arabinofuranoside interacts with HSA through static quenching mechanism with binding affinity

of 2.4⨉103

M-1

. The average binding distance between drug and Trp214

of HSA was found to be

2.23 nm on the basis of the theory of Förster’s energy transfer. Synchronous fluorescence data

indicated that interaction of drug with HSA changed the microenvironment around the

tryptophan residue. UV-visible spectroscopy and circular dichroism results deciphered the

complex formation and conformational alterations in the HSA respectively. Dynamic light

scattering was utilized to understand the topology of protein in absence and presence of drug.

Thermodynamic parameters obtained from Isothermal titration calorimetry (ΔH= -26.01 KJmol-1

and TΔS= -6.5 KJmol-1

) suggested the involvement of van der Waal interaction and hydrogen

bonding. Molecular docking and displacement study with site specific markers suggested that

Cytosine β-D Arabinofuranoside binds to subdomain IB of HSA which is also known as the

hemin binding site. This study will be helpful to understand the binding mechanism of Cytosine

β-D Arabinofuranoside with HSA and associated alterations.

Key words: Serum albumin, Binding, Fluorescence quenching, Molecular docking.

Abbreviations: HSA- human serum albumin, CBDA- cytosine β-D arabinofuranoside, ITC-

isothermal titration calorimetry

3

1. Introduction

Drug-protein interaction is a crucial pharmacological phenomenon that affects structure,

physiological action of carrier protein, distribution and elimination of drugs.[1; 2] Therapeutic

efficacy of drug conjointly depends upon the degree of binding affinity with plasma proteins.

Pharmacokinetic behavior of drug depends on its freely and bound form present in body .[3]

HSA serves as a transport vehicle for several endogenous compounds including fatty acids,

hemin, bilirubin, and tryptophan. Furthermore, distribution, concentration and the metabolism of

various exogenous drugs also depends upon their affinity with serum albumins.[3; 4; 5] Apart

from the roles depicted above, HSA is vital in maintaining osmolarity in plasma and intestinal

fluids and exhibits many enzymatic and antioxidant functions.[6; 7]

Human serum albumin (HSA) is most abundant, single chain, multifunctional, non-glycosylated

and negatively charged protein of molecular weight 66.5 kDa.[8] X ray crystallography revealed

that HSA is globular heart shaped protein consists of three domains viz. domain I, II and III

which are further subdivided into A and B sub domains.[9; 10] Moreover, binding and molecular

docking study depict that HSA binds to large number of small molecules due to diverse binding

sites distributed over the surface of the protein. The two best characterized binding sites for

ligands are located in hydrophobic pocket of sub domain II A and III A which corresponds to

Sudlow site I and Sudlow site II.[11; 12]

Cytosine β-D Arabinofuranoside (CBDA) is also known as Ara-C (Arabinofuranosyl Cytidine)

consists of cytosine base with arabinose sugar as shown in Fig. 1. It is used as chemotherapeutic

agent such as in treatment of acute myeloid leukemia, non-hodgkin lymphoma, acute

lymphocytic leukemia and lymphomas.[13; 14; 15] Antiviral activity of CBDA was utilized for

4

prevention of herpes virus infection [16]. It interferes with DNA synthesis and induces cancerous

cell death. CBDA is among first series of cancer drugs that altered the sugar component albeit

other drugs modify the base. It gets transformed into cytosine arabinoside triphosphate which

causes DNA damage when cells are in S phase of mitosis therefore rapidly dividing cells are

most affected. Moreover, it also inhibits DNA polymerase, RNA polymerase and Nucleotide

reductase needed for DNA synthesis[17]. Previously, our group has reported the binding

mechanism of various detergents, drugs, toxins, pollutants to serum albumins. [18; 19; 20; 21;

22; 23]

In the present study, binding of HSA with CBDA was thoroughly studied by utilizing Isothermal

Titration Calorimetry (ITC) and various spectroscopic techniques. The topology of HSA in

absence and presence of CBDA was studied by using dynamic light scattering measurements.

Moreover, displacement experiment with site specific markers and molecular docking study

have also been performed to reveal the location of CBDA binding site on HSA as well as forces

involved in the binding interaction.

2. Materials and methods

Human serum albumin (A1887), Cytosine β-D Arabinofuranoside (1768), warfarin (A2250),

hemin (H5533) and diazepam (D0406) were purchased from Sigma Aldrich, India. All of the

other reagents were of analytical grade.

2. 1 Sample preparation

HSA (100 µM) stock solution was prepared in 20 mM phosphate buffer (pH 7.4). Protein

concentration was determined spectrophotometrically using of 5.30 at 280 nm by using

Perkin-Elmer Lambda double beam UV-visible spectrophotometer.[24; 25] Drug solution (5

5

mM) was prepared in 20 mM phosphate buffer (pH 7.4) and diluted to various concentrations in

the same buffer. All experiments were carried out in 20 mM phosphate buffer (pH 7.4) at 298K.

2.2 pH determination

pH measurements were carried out on Mettler Toledo pH meter (Seven Easy S20–K) using

Expert “Pro3 in 1” type electrode. The least count of the pH meter was 0.01 pH unit.

2.3 Steady state fluorescence quenching measurement

Fluorescence measurements were performed on a Shimadzu fluorescence spectrophotometer,

model RF-5301. The fluorescence spectra were measured at 25 ± 0.1 °C with a 1 cm path length

cell. Both excitation and emission slits were set at 5 nm. Intrinsic fluorescence was measured by

exciting the protein solution at 295 nm and emission spectra were recorded in the range of 300-

450 nm. The titration of CBDA (0-115 μM) to HSA (5 μM) solution was carried out at 298 K.

The fluorescence intensity at 340 nm (tryptophan) was used to calculate the binding constant

(Kb).

2.4 Synchronous fluorescence spectroscopy

Synchronous fluorescence spectroscopy was carried out by simultaneously scanning the

excitation and emission mono-chromators. Conformational alteration around the tyrosine and

tryptophan residue of HSA can be marked when the wavelength interval (Δλ) was 15 and 60 nm

respectively. The concentration of HSA was 5 μM and CBDA varied in the range (0-115 μM).

All parameters were kept same as discussed in above section

6

2.5 Drug displacement experiment

Different site specific markers, warfarin for (site I), diazepam for (site II) and hemin for

(subdomain IB), were used for performing displacement experiments.[26; 27; 28] Titration of

CBDA was carried out with the solution having protein and site marker in the molar ratio of 1:1.

The fluorescence emission spectra were recorded in a similar way as mentioned in fluorescence

measurements and the Ksv values of drug–protein–marker were evaluated using Stern-Volmer

equation.

2.6 UV-visible absorption measurements

Absorbance measurements were carried out on Perkin-Elmer Lambda 25 double beam UV-

visible spectrophotometer attached with Peltier temperature programmer 1 (PTP-1). Cuvette of

cell length of 10 mm and scanning speed 1000 nm/min was used. The concentration of protein

was kept at 12 μM and 1:0, 1:10 and 1:20 molar ratio of HSA to CBDA were used.

2.7 Circular dichroism measurements

Far-UV CD measurements (190-250 nm) were carried out on JASCO-J 813 spcetro polarimeter

equipped with a Peltier-type temperature controller at 298 K using a quartz cell of path length of

0.1 cm. Three scans were accumulated at a scan speed of 100 nm min-1

. 1:0, 1:10 and 1:20 molar

ratio of HSA-CBDA were taken. The percent α-helical and β-sheet contents of the protein were

calculated by using online K2D3 software [29] using the CD values from 200-250 nm as

extracted from the CD spectra.

7

2.8 Dynamic light scattering (DLS) measurements

DLS measurements were carried out at 830 nm using DynaPro-TC-04 dynamic light scattering

equipment (Protein Solutions, Wyatt Technology, Santa Barbara, CA) equipped with a

temperature- controlled micro sampler. HSA (2 mg/ml) was incubated with CBDA for 12 hours.

The samples were spun at 10,000 rpm for 10 min and were filtered serially through 0.22 and 0.02

µM Whatman syringe filters directly into a 12 µl quartz cuvette. For each experiment, 20

measurements were carried out. Mean hydrodynamic radius (Rh) and polydispersity were

analyzed using Dynamics 6.10.0.10 software at optimized resolution. The Rh was estimated on

the basis of an autocorrelation analysis of scattered light intensity data based on translationa

diffusion coefficient by Stokes- Einstein relationship:

D

kTR

6h (1)

Where Rh is the hydrodynamic radius, k is Boltzmann constant, T is temperature, η is the

viscosity of water and D is diffusion coefficient.

2.9 Isothermal titration calorimetry

Isothermal titration calorimetry allows the measurement of the magnitude of the binding affinity,

and the two contributing thermodynamic terms: enthalpy (ΔH⁰) and entropy (ΔS⁰) changes.

Binding of CBDA to HSA was studied by ITC (VP-ITC (Microcal Inc., Northampton, MA) at 25

°C. Prior to the titration experiment, all solutions were degassed properly on a thermovac. The

sample cell (approximately 1.44 mL) was loaded with HSA (25 µM) and CBDA (1 mM) was

injected into the reaction cell. The titration cell was stirred continuously at 307 rpm, which

ensured rapid mixing but did not cause foaming in the protein solution. Titrations were

8

performed to ensure full occupancy of the binding sites and until the titration signal was

constant. The calorimetric data were analyzed using the MicroCal Origin 7.0 software provided

with the instrument. The enthalpy change for each injection was calculated by integrating the

area under the peaks and then subtracted with control titrations.

The other thermodynamic parameters were calculated according to the equation

∆G⁰ = -RTlnKa = ∆H⁰-T∆S⁰ (2)

where T is the absolute temperature (298 K) and R, is universal gas constant (8.3151 J mol-1

K-1

).

2.10 Molecular docking

The molecular docking study was performed using Autodock 4.2 and Autodock tools (ADT)

using Lamarckian genetic algorithm.[30] The crystal structure of HSA (PDB id: 1AO6) and three

dimensional structure of CBDA (CID: 6253) was obtained from Brookhaven Protein Data Bank

and PubChem respectively. Chain A of the protein was selected water molecules and ions were

removed and all hydrogen atoms were added. Then partial Kollman charges were assigned to

HSA. The protein was set to be rigid and there is no consideration of solvent molecules on

docking. The grid size was set to be 126, 126 and 126 along X, Y and Z axes with 0.564 Å grid

spacing. Autodock parameters used were GA population size: 150 and maximum number of

energy evolutions: 2,50,0000. 10 best solution based on docking score was retained for further

analysis, Discovery studio 3.5 were used for visualization and for the identification of residues

involved in binding. LIGPLOT program was used to analyse amino acid residues involved in the

binding between HSA and CBDA.[31]

9

3. Results and Discussion

3.1 Steady state fluorescence quenching measurement

Fluorescence spectroscopy is a valuable tool to study Protein-ligand interactions. Protein

containing aromatic amino acid residues (tyrosine, phenylalanine and tryptophan) has intrinsic

fluorescence, however among them the contribution of tryptophan is maximum.[22] In

fluorescence quenching experiment titration of drug with protein is carried out to get detailed

information of related binding parameters.[32]

The fluorescence intensity of HSA decreases with gradual addition of CBDA at 298 K as shown

in (Fig. 1A). The maximum emission wavelength of HSA was found at 340 nm and the value

decreases regularly upon increasing concentration of CBDA, indicating that interaction between

drug and HSA occurred. These results are also in accord with the previous reports.[12; 33]

The fluorescence quenching data were analyzed by Stern-Volmer equation (equation 2).[34]

F₀/F = Ksv+1 = kqτ₀ [Q] +1 (3)

where F0 and F are the steady state fluorescence intensities in the absence and presence of

quencher, respectively, KSV is the Stern-Volmer quenching constant and Q is the concentration of

quencher, kq is the bimolecular quenching constant and τ₀ is the life time of tryptophan in the

absence of quencher. Life time of tryptophan for HSA (τ₀) is 5.6 ns.[35] Stern Volmer plot is

shown in Fig. 1B. In order to establish the mechanism of quenching, kq was calculated by using

following equation

(4)

10

In static quenching, quencher physically interact with the excited molecule through chemical

bonds. However, in dynamic quenching, quencher indirectly interact with the molecule. The

bimolecular quenching constant for CBDA calculated to be 1 ⨉ 1011

M-1

s-1

which is greater than

maximum collision quenching constant 2 ⨉ 1010

M-1

s-1

.[36]

This suggests the formation of HSA-CBDA complex occurred through static quenching manner.

Value of binding constant and number of binding sites were obtained from the modified Stern-

Volmer plot as shown in Fig. 1C by using the following equation

Log (F₀/F -1) = logKb + n log [Q] (5)

where F0 and F are the steady state fluorescence intensities in the absence and presence of

quencher, respectively, Kb is binding constant, n is the number of binding sites and Q is the

concentration of quencher. Binding parameters are summarizes in Table 1, the value of binding

constant was found to be in the order of 103

which suggest that binding of CBDA binds to HSA

in static manner. [37]

3.2 Synchronous fluorescence measurements

Synchronous fluorescence spectroscopy provides information about the micro environmental

changes around tyrosine and tryptophan in HSA upon ligand binding. D-value (Δλ) between

excitation and emission wavelength was set at 15 and 60 nm for Tyr and Trp residues

respectively. [38] Change in the polarity around the micro environment of fluorophore is inferred

from the changes in maximum fluorescence emission peak (λ em max). Blue shift and red shift in

λem max signifies enhancement in hydrophobicity and polar environment around the fluorophore

(Tyr and Trp) respectively[39]. As can be inferred from the Fig. 2A, almost no red shift in

emission maximum of HSA was observed in presence of increasing concentration of CBDA

A B

C

A B C

11

when Δλ=15 nm, suggesting that the micro environment around the Tyr residues did not altered

significantly. On the other hand when Δλ=60 nm (Fig. 2B), substantial red shift (~10 nm) was

observed in emission maximum of HSA, inferring that microenvironment around the Trp residue

get significantly altered. CBDA induced conformational changes indicated microenvironment

around the Trp residue (Trp214

) get significantly perturbed and CBDA-HSA complex formation

has occured.[40] This implies that CBDA cause more conformational change near tryptophan

residue than tyrosine. Moreover the fluorescence intensity decreases regularly on addition of

CBDA in both systems which further support the occurrence of fluorescence quenching (Fig 2A

and B)

3.3 Drug displacement experiments

Majority of the drugs bind to HSA at two primary sites known as Sudlow site I and II but few

drugs and fatty acids also binds at subdomain IB of HSA. In order to determine binding site of

CBDA on HSA, competitive binding experiment with site specific markers have been carried

out. Warfarin for site I, Diazepam for site II and Hemin for the subdomain IB were used. To

trace the binding site of CBDA on HSA, KSV values were calculated by using the emitted

fluorescence intensity data in the absence and presence of markers by Stern-Volmer equation.

There was no significant change in the KSV value was observed with warfarin and diazepam

although with hemin KSV value gets markedly reduced (supplementary table 1). This difference in

KSV is sufficient enough to locate binding site on HSA as reported in earlier literature.[41] It is

evident from the displacement experiments that CBDA binds at subdomain IB on HSA.

12

3.4 Fluorescence resonance energy transfer (FRET)

The efficiency of energy transfer can be used to evaluate the distance between the ligand and

fluorophore in the protein. According to Förster’s energy transfer theory the distance between

donor (Trp214

of HSA) and acceptor (CBDA), r and the efficiency of energy transfer, E can be

calculated by following equation.

(6)

where F and F○ are the fluorescence intensities of HSA in the absence and presence of CBDA,

R0 is the Förster critical distance at which the efficiency of energy transfer is 50%, R0 can be

determined by using the following equation.

(7)

where K2

is the factor related to the geometry of donor and acceptor of diploes, n is the refractive

index of the medium, is the fluorescence quantum yield of the donor in the absence of the

acceptor and J is the overlap integral of donor fluorescence emission and the acceptor

absorption, which can be calculated by using following equation.

J =∫

∫

(8)

where F (λ) is the fluorescence intensity of the donor at wavelength λ and (λ) is the molar

extinction coefficient of acceptor at wavelength λ. For HSA, K2, and n were taken as 2/3, 0.118

and 1.33, respectively.[42]

13

The FRET results are summarized in Table 2. The spectral overlap between the absorption

spectrum of acceptor (CBDA) and the fluorescence emission spectrum of donor (HSA) is shown

in Fig. 3. The binding distance (r) for the present system is 2.23 nm. Furthermore, we found that

0.5Ro<r< 1.5Ro, indicating the existence of static quenching due to complex formation between

CBDA and HSA.

3.5 UV-visible absorption spectroscopy

UV-visible spectroscopy is simple and valuable tool to study drug-protein interaction. The

absorption peak around 280 nm because of cumulative absorption of three aromatic amino acid

residues. The formation of CBDA-HSA complex is evident from UV-visible spectra

(supplementary Fig. S1). The progressive increase of absorbance along with blue shift on

increasing drug concentration governs the formation of drug-protein complex. Change in λmax

indicated the change in polarity around the tryptophan residue and hence indicates binding

between CBDA and HSA has led to the change in the conformation of protein. [43]

3.6 Circular dichroism analysis

CD spectroscopy is valuable tool to study secondary structure of proteins and nucleic acids.[44;

45; 46; 47; 48]

The CD experiments were carried out in order to get insight into the conformational change of

HSA induced by CBDA. Binding of ligands to globular protein modulate the intermolecular

forces liable for sustaining the secondary and tertiary structures resulting in conformational

alteration of the protein. The CD spectra of serum albumins exhibited two negative minimal

peaks in UV-region at 208 and 222 nm, characteristics of α-helical structure of the protein.[18;

49]

14

Fig. 4 shows the CD spectra of HSA and HSA-CBDA. HSA exhibits two negative bands at 208

nm and 222 nm characteristic of alpha helical structure[50]. An increase in the negative CD

value without any change in the characteristic pattern of HSA was observed with gradual

increase in CBDA concentration which suggests the increment in alpha helical content in HSA

(55.80 % to 57.92% calculated from K2D3 software). From CD results it can be interpreted that

CBDA leads to stabilize the secondary structure of HSA.

3.7 Dynamic light scattering study

Dynamic light scattering was employed to determine the hydrodynamic radii of native HSA and

CBDA- HSA complex. The hydrodynamic radii of native HSA and in presence of different

molar ratios of CBDA was calculated, data represented in Fig. 5 and Table 3. The lower value of

polydispersity (5-15) indicate the presence of homogenous solution. The Rh of native HSA was

found to be 3.4 nm. [51] The Rh value of HSA starts decreasing on increasing the concentration

of CBDA. This reduction in Rh value may attributed to collapsing of protein as CBDA binds with

HSA. This response may result in a decrease in the molecular volume due to a conformational

change.[49] As CD data suggested, CBDA affects the secondary structure of HSA. This also

might be a reason that conformationally altered secondary structural components tends to

decrease hydrodynamic radius.

3.8 Isothermal titration calorimetry

Isothermal Titration Calorimetry was performed to determine the thermodynamic parameters for

CBDA-HSA interaction. ITC complement the fluorescence studies for the ligands which interact

15

with weak interacting forces with transport proteins as it can be carried out at higher

concentration of both protein and ligand.[52] The final data were best fitted to one binding site

model and the results are presented in Fig. 6 and Table 4. Each peak in the binding isotherms

(Fig. 6 upper panel) represents a single injection of CBDA. The negative deflection from the

baseline on addition of CBDA indicates that process is exothermic. We found that CBDA has

one binding site with affinity of order 104.

The data revealed that the binding of CBDA to HSA

shows a favorable entropical change (0<ΔS). Negative value of ΔH and positive value of ΔS

endorse the involvement of van der Waal interaction and hydrogen bonding between drug and

protein as reported earlier.[33] Moreover negative value of Gibbs’s free energy (ΔG)

demonstrates that the binding process was spontaneous. Overall the binding process was

spontaneous, enthalpy driven and entropically opposed. The differences in fluorescence and ITC

results could be ascertained to the following reason, fluorescence measures local change only

around the Trp residue whereas ITC measures global change.[53; 54]

3.9 Molecular docking study of CBDA-HSA interaction

Molecular docking has been employed to further understand the interaction of CBDA with HSA

and to confirm the binding site of CBDA on HSA. The principle regions of ligand binding on

HSA are located in hydrophobic cavities in subdomain II and III which correspond to site I and

site II respectively. Autodock 4.2 program was used to examine the binding mode of CBDA to

HSA. The docking results showed that CBDA binds within the binding pocket of subdomain IB

(Fig. 7A, B and C). It is apparent from Fig. 7 B that CBDA got insert into the hydrophobic cavity

of the protein, which indicated that the binding location was subdomain IB and this result are

also in consistent with our displacement experiments with site specific markers.

16

The CBDA-HSA complex is stabilized by hydrogen bonds between the drug and the Tyr138

,

Leu812

and Leu185

amino acid residues of the protein with bond lengths of 2.94, 2.83 and 2.92 Å,

respectively. Further, CBDA-HSA complex is also stabilized by hydrophobic interaction

between the Tyr161

, Met123

, Ile142

, and Gly189

amino acid residues of the protein and drug (Table

5). Binding energy calculated by molecular docking was -20.46 KJmol-1

which is very close to

the binding energy obtained from fluorescence and ITC experiments and further support the

spontaneity of reaction.

4. Conclusion

In view of all results it can be concluded that CBDA interacts with HSA through static

quenching mechanism and induce conformation alterations in the protein. The binding process is

spontaneous, exothermic and entropically favoured. Displacement study (with specific site

markers) and molecular docking revealed that CBDA binds at subdomain IB on HSA apart from

major drug binding site (site I and II). Hydrogen bonding and hydrophobic interactions are the

major forces in stabilizing the drug protein complex. Our study provides detailed insight to

understand the mode of interaction of other related drugs that interacts with subdomain IB.

Acknowledgements

Facilities provided by IBU, Aligarh Muslim University, Aligarh are gratefully acknowledged. PA

and SKC are gratify to Council of Scientific and Industrial Research, New Delhi for

providing fellowship in the form of JRF and SRF respectively.

17

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Legend to Figures

Figure 1: (A) CBDA-induced fluorescence quenching of HSA. The concentration of HSA was 5

μM and the concentration of CBDA was varied from 0-115 μM. The intrinsic fluorescence of the

protein was measured in 20 mM sodium phosphate buffer, pH 7.4 at 298 K upon excitation at

295 nm. (B) Stern-Volmer plot for HSA-CBDA interactions (C) Modified Stern-Volmer plot for

the HSA-CBDA interactions at 298 K.

Figure 2: Synchronous fluorescence spectra (A) Δλ 15 (B) Δλ 60, HSA (5 μM) in presence of

increasing concentration of CBDA (0-115 μM).

Figure 3: The spectral overlap of the fluorescence emission spectrum of HSA (curve a) with the

absorption spectrum of CBDA (curve b), CBDA: HSA = 1:1.

Figure 4: Far-UV CD spectra of HSA alone (─) or in complex and in presence of 1:5 (- -) and

1:20 (__

__

) molar ratio of HSA to CBDA.

Figure 5: Hydrodynamic radii pattern of HSA in the absence of CBDA (A) and presence of HSA

+ CBDA (1:5) (C) HSA + CBDA (1:20).

Figure 6: Isothermal titration calorimetry of CBDA-HSA interaction. In the top panel, the heat

released per unit time (μcal/sec) was plotted vs time (min) where each peak corresponds to the

injection of an aliquot of ligand. In the bottom panel, the heat of reaction per injection

(kcal/mole).

Figure 7: (A) Best conformation of CBDA docked to HSA (B) Cartoon representation of

residues involved in binding of CBDA to HSA (C) Ligplot representations of the interactions

between HSA and CBDA.

24

Table 1. Stern-Volmer quenching constants and binding parameters for HSA-CBDA interactions

at 298 K.

Temp

(K)

Ksv

(M-1

)

kq

(M-1

s-1

)

Kb

(M-1

)

n

ΔG⁰

(kJ mol-1

)

R2

298 1.9⨉103 1.9⨉10

11 2.4⨉10

3 1.02 -19.18 0.99

Table 2. (FRET) parameters obtained from HSA-CBDA binding.

J (M-1

cm-1

nm4) R0 (nm) r (nm) EFRET

6.35⨉10-16

1.54 2.23 0.10

25

Table 3. Hydrodynamic radii and polydispersity of HSA in the absence and presence of CBDA.

Condition (Rh) Pd (%)

HSA 3.4±0.03 13.5

HSA + CBDA (1:5) 3.2±0.03 8.1

HSA + CBDA (1:20) 2.6±0.02 5.2

Table 4. Thermodynamic and binding parameters of HSA-CBDA obtained from ITC at 298 K.

n

Kb (M-1

)

ΔH⁰

(kJ mol-1)

TΔS⁰

(kJ mol-1

)

ΔG⁰

(kJ mol-1

)

1.10 1.95⨉104 -26.01 6.5 -19.5

26

Table 5. Molecular docking parameters of HSA-CBDA complex.

.

Figure 1

Binding Site Amino Acids Forces Involved ΔG (kJ mol-1

)

Site IB Tyr138

H-Bonding -20.46

Leu185

H-Bonding

Leu182

H-Bonding

Tyr161

Hydrophobic

Met123

Hydrophobic

Ile142

Hydrophobic

Gly189

Hydrophobic

A B

C

27

Figure 2

Figure 3

a

b

B A

28

Figure 4

Figure 5

A

B C

29

Figure 6

30

Figure 7

Highlights

Comprehensive insight into the interaction of CBDA with HSA.

The interaction process is spontaneous and exothermic.

The main governing forces for stabilising HSA- CBDA complex are van der Waal

interaction and hydrogen bonding.

CBDA binds at subdomain IB on HSA.

A

C

A

B