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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

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Front. Biol.    2017, Vol. 12 Issue (3) : 199-209    https://doi.org/10.1007/s11515-017-1449-z
RESEARCH ARTICLE
Analysis of curcumin interaction with human serum albumin using spectroscopic studies with molecular simulation
Turban Kar1, Pijush Basak2, Srikanta Sen3, Rittik Kumar Ghosh1, Maitree Bhattacharyya1,2()
1. Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata-700019, West Bengal, India
2. Jagadis Bose National Science Talent Search, Kolkata-700107, India
3. 229A/230, Mira Tower, Lake Town, Block-A, Kolkata-700089, India
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Abstract

BACKGROUND: Curcumin has emerged to be utilized as a superb beneficial agent, due to its naturally occurring anti-oxidant, anti-inflammatory and anti-carcinogenic property.

METHODS: The interaction of curcumin with human serum albumin, the main in vivo transporter of exogenous substances, was investigated using absorption spectroscopy, steady-state fluorescence, excited state life-time studies and circular dichroism spectroscopy.

RESULTS: Isothermal titration calorimetry techniques inferred one class of binding site with binding constant ～1.74×105 M−1 revealing a strong interaction. The binding profile was analyzed through the evaluation of the thermodynamic parameters, which indicated the involvement of hydrophobic interactions (burial of non-polar group). Fluorescence lifetime of tryptophan residue was observed to decrease to 1.94 ns from 2.84 ns in presence of Curcumin. Percentage of α helicity of human serum albumin was also reduced significantly upon binding with curcumin as evidenced by circular dichroism measurement leading to conformational modification of the protein molecule.

CONCLUSIONS: On the basis of such complementary results, it may be concluded that curcumin shows strong binding affinity for human serum albumin, probably at the hydrophobic cavities of the protein and at or around the tryptophan residue. Molecular Docking analysis of HSA and curcumin provided light on the number of binding sites at an atomic level, which were already determined at a molecular level in spectroscopic measurements. Our study unfolds the modes of interaction of curcumin with human serum albumin in the light of different biophysical techniques and molecular modeling analysis.
Keywords curcumin      human serum albumin      fluorescence quenching      conformational change      thermodynamic parameters     
Corresponding Author(s): Maitree Bhattacharyya   
Online First Date: 26 May 2017    Issue Date: 19 June 2017
 Cite this article:   
Turban Kar,Pijush Basak,Srikanta Sen, et al. Analysis of curcumin interaction with human serum albumin using spectroscopic studies with molecular simulation[J]. Front. Biol., 2017, 12(3): 199-209.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-017-1449-z
https://academic.hep.com.cn/fib/EN/Y2017/V12/I3/199
Fig.1  Fluorescence spectra of human serum albumin (15 µM) in presence of increasing amounts of curcumin; concentration corresponds from 0 µM to 650 µM. Axis acronyms –X axis (a.u. – arbitrary units); Y axis (nm – nanometers).
Fig.2  Panel (A) and (B) represent Stern–Volmer and modified Stern–Volmer plots of F0/F and log [(F0−F)/F] vs. concentration of curcumin where the concentration of human serum albumin remains fixed at 15 µM.
Protein Ligand SV equation R2 Ksv (103mol−1) Modified SV log [( F0−F)/F)] = logKa + n log [Q] R2 Binding
constant
Ka(103mol−1)		 No. of binding
site
Human serum albumin Curcumin y = 375.36x−0.3012 0.9554 0.35 y= 1.4024x + 3.1522 0.992 1.419 1.402
Tab.1  Quenching constant and curcumin binding parameters to HSA
Fig.3  The fluorescence lifetime decay profiles of the (A) protein-curcumin complex and (B) native HSA; Axis acronyms –X axis (ns – nanoseconds).
Protein t1 t2 t3 Average ( t) c2
Human serum albumin Native
Curcumin-HSA complex		 0.30
0.80		 1.37
1.67		 6.85
3.34		 2.84
1.93		 1.21
1.40
Tab.2  Fluorescence life time decay profile of Tryptophan in human serum albumin in absence and presence of curcumin
Fig.4  Isothermal titration calorimetric profiles for the binding of curcumin to Human serum albumin. The top panel (A) represents raw data for the sequential injection of curcumin solution (80 µM) into HSA (3 µM) solution. The lower panels (B) show the integrated heat results after correction of heat of dilution against the mole ratio of Human serum albumin /curcumin. The points (closed squares) were fitted to a one-site model and the solid lines represent the best-fit results.
Protein Ligand Binding constant
(103mol−1)		 Enthalpy change
(kcal/mol)		 Entropy change
(kcal/mol)		 Free energy change (Δ G)
( kcal/mol)		 Process
Human serum albumin Curcumin 1.11 −37.55 ×10 6 −12.68 × 10 6 −6.06 van Der Waal’s interaction and hydrogen bonding
Tab.3  Thermodynamic parameters for HSA-Curcumin interaction derived by ITC method
Fig.5  Circular dichroism spectra of HSA observed at 25°C in presence of increasing concentration of curcumin (0 µM to 650 µM). Axis acronyms –X axis (nm – nanometers); Y axis (mdeg – millidegrees).
Concentration of curcumin (M) α-helix
(%)
0
4.343 ×10−4
4.886 ×10−4
5.429 ×10−4
5.972 ×10−4
6.515 ×10−4 		51
47.6
47.4
46.5
45.3
43.3
Tab.4  Estimation of secondary structure content of human serum albumin with gradually varying concentration of curcumin
Fig.6  The docked structures of (A) the keto form and (B) the enol form are shown. The keto-curcumin is shown in cyan ball and stick model and the enol-curcumin is shown in yellow ball and stick model. The Human serum albumin structure is represented in line-ribbon style.
Fig.7  The docked structures of (A) the keto form and (B) the enol form with its protein neighborhood are shown. The curcumin variants are shown as ball and stick model. The Human serum albumin neighborhood is represented in wireframe style.
Fig.8  Comparison of the binding modes of the two forms of curcumin. The keto-form is represented as ball and stick while the enol-form as shown in stick form.
Molecule name Δ Gbinding(kcal/mol) Ki
(μM)		 Ki (μM)
experimental
Curcumin
(keto form)
Curcumin
(enol form)		 −7.32
−6.92		 4.3
8.4		 1.79
Tab.5  The best binding energies and the computed binding constants obtained by the docking runs for two different forms of curcumin. The values have been obtained using molecular docking platform (Autodock 4.0)
HSA-Curcumin (keto) HSA-Curcumin (enol)
HSA Curcumin HAS Curcumin
1
2		 ΔSASA (Å 2)
ΔG (kcal/mol)		 −387.0
−2.33		 −539.4
−3.89		 −370.4
−2.26		 −539.4
−3.77
Tab.6  Change in SASA values for HSA and curcumin molecule due to complex formation and the free energy change due to complex formation
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[1] Pijush Basak,Tanay Debnath,Rajat Banerjee,Maitree Bhattacharyya. Selective binding of divalent cations toward heme proteins[J]. Front. Biol., 2016, 11(1): 32-42.
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