Molecular interaction studies of trimethoxy flavone with human serum albumin

Abstract

Background: Human serum albumin (HSA) is the most abundant protein in blood plasma, having high affinity binding sites for several endogenous and exogenous compounds. Trimethoxy flavone (TMF) is a naturally occurring flavone isolated from Andrographis viscosula and used in the treatment of dyspepsia, influenza, malaria, respiratory functions and as an astringent and antidote for poisonous stings of some insects.

Methodology/principal findings: The main aim of the experiment was to examine the interaction between TMF and HSA at physiological conditions. Upon addition of TMF to HSA, the fluorescence emission was quenched and the binding constant of TMF with HSA was found to be K(TMF) = 1.0+/-0.01x10(3) M(-1), which corresponds to -5.4 kcal M(-1) of free energy. Micro-TOF Q mass spectrometry results showed a mass increase of from 66,513 Da (free HSA) to 66,823 Da (HAS +Drug), indicating the strong binding of TMF with HSA resulting in decrease of fluorescence. The HSA conformation was altered upon binding of TMF to HSA with decrease in alpha-helix and an increase in beta-sheets and random coils suggesting partial unfolding of protein secondary structure. Molecular docking experiments found that TMF binds strongly with HSA at IIIA domain of hydrophobic pocket with hydrogen bond and hydrophobic interactions. Among which two hydrogen bonds are formed between O (19) of TMF to Arg 410, Tyr 411 and another one from O (7) of TMF to Asn 391, with bond distance of 2.1 A, 3.6 A and 2.6 A, respectively.

Conclusions/significance: In view of the evidence presented, it is imperative to assign a greater role of HSA's as a carrier molecule for many drugs to understand the interactions of HSA with TMF will be pivotal in the design of new TMF-inspired drugs.

Figure 1. Chemical structure of Tri-methoxy flavone.

The molecular mass is 312.31 Da and its molecular formula is C₁₈H₁₆O_5.

Figure 2. Fluorescence emission spectra of HSA–TMF in 0.1 M phosphate buffer pH 7.2, λ_ex = 285 nm, temperature = 25±1°C.

A) Free HSA (0.025 mM) and free HSA with different concentrations of TMF (0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 mM). B) Plot of log (dF/F) against log [Q]. λ_ex = 285 nm λ_em = 362 nm.

Figure 3. Micro TOF-Q mass spectra.

A) free HSA, and B) HSA along with TMF. The concentration of free HSA and TMF were 0.15 µM and 0.2 µM, respectively.

Figure 4. Circular dichroism of the free HSA and HSA+TMF complexes.

The free HSA and HSA+TMF complexes in aqueous solution with a protein concentration of 0.025 mM and TMF concentrations were 0.01, 0.025 and 0.08 mM.

Figure 5. Temperature-dependent CD-Spectra of free HSA and HSA+TMF complexes.

A) HSA alone and B) CD spectra of HSA+0.08 mM TMF complexes. C) Secondary structure composition calculated from Figure 5A, HSA alone and Figure 5B, HSA-TMF complexes with temperature dependent. The temperature dependance for both free HSA and HAS+TMF complexes from 25 to 85 with an interval of 10°C.

Figure 6. Molecular docking of HSA with TMF.

A) Schematic representation of HSA molecule. Each subdomain is marked with a different colour (Red for subdomain IA; yellow, IIA; purple IIIA; blue, IIB; orange, IB; green, IIIB) Asn391, Arg410 and Tyr411 involved in Binding of TMF, Trp-214 are coloured white. B) Graphical representation of HSA-TMF complex (prepared by using SILVERv1.1.1 visualizer), TMF Complex represented as capped sticks, and the residues as ellipsoid model. Three H-bonds (as highlighted by the dashed lines in green colour) were formed between TMF and HSA. The hydrogen bond lengths were represented in green colour. C) Graphical representation of HSA showing TMF docked in the binding pocket of HSA using GOLDv3.2.TMF, depicted in stick model (light green), and HSA, represented in solid (better) with ray model. The image was visualised by using PyMol.