Molecular Interaction Studies of Trimethoxy Flavone with Human Serum Albumin

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 KTMF = 1.060.01610 3 M 21, which corresponds to 25.4 kcal M 21 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 a-helix and an increase in b-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

Binding of Tetracycline and Chlortetracycline to the Enzyme Trypsin: Spectroscopic and Molecular Modeling Investigations

Tetracycline (TC) and chlortetracycline (CTC) are common members of the widely used veterinary drug tetracyclines, the residue of which in the environment can enter human body, being potentially harmful. In this study, we establish a new strategy to probe the binding modes of TC and CTC with trypsin based on spectroscopic and computational modeling methods. Both TC and CTC can interact with trypsin with one binding site to form trypsin-TC (CTC) complex, mainly through van der Waals' interactions and hydrogen bonds with the affinity order: TC>CTC. The bound TC (CTC) can result in inhibition of trypsin activity with the inhibition order: CTC>TC. The secondary structure and the microenvironment of the tryptophan residues of trypsin were also changed. However, the effect of CTC on the secondary structure content of trypsin was contrary to that of TC. Both the molecular docking study and the trypsin activity experiment revealed that TC bound into S1 binding pocket, competitively inhibiting the enzyme activity, and CTC was a non-competitive inhibitor which bound to a non-active site of trypsin, different from TC due to the Cl atom on the benzene ring of CTC which hinders CTC entering into the S1 binding pocket. CTC does not hinder the binding of the enzyme substrate, but the CTC-trypsin-substrate ternary complex can not further decompose into the product. The work provides basic data for clarifying the binding mechanisms of TC (CTC) with trypsin and can help to comprehensively understanding of the enzyme toxicity of different members of tetracyclines in vivo

Differential interactions and structural stability of chitosan oligomers with human serum albumin and α-1-glycoprotein

Chitosan is a naturally occurring deacetylated derivative of chitin with versatile biological activities. Here, we studied the interaction of chitosan oligomers with low degree of polymerization such as chitosan monomer (CM), chitosan dimer (CD), and chitosan trimer (CT) with human serum albumin (HSA) a major blood carrier protein and α-1-glycoprotein (AGP). Since, HSA and AGP are the two important plasma proteins that determine the drug disposition and affect the fate of distribution of drugs. Fluorescence emission spectra indicated that CM, CD, and CT had binding constants of KCM = 6.2 ± .01 × 105 M−1, KCD = 5.0 ± .01 × 104 M−1, and KCT = 1.6 ± .01 × 106 M−1, respectively, suggesting strong binding with HSA. However, binding of chitooligomers with AGP was insignificant. Thermodynamic and molecular docking analysis indicated that hydrogen bonds and also hydrophobic interaction played an important role in stabilizing the HSA-chitooligomer complexes with free energies of −7.87, −6.35, and −8.4 Kcal/mol for CM, CD, and CT, respectively. Further, circular dichroism studies indicated a minor unfolding of HSA secondary structure, upon interaction with chitooligomers, which are supported with fluctuations of root mean square deviation (RMSD) and radius of gyration (Rg) of HSA. Docking analysis revealed that all three chitooligomers were bound to HSA within subdomain IIA (Site I). In addition, RMSD and Rg analysis depicted that HSA-chitooligomer complexes stabilized at around 4.5 ns. These results suggest that HSA might serve as a carrier in delivering chitooligomers to target tissues than AGP which has pharmacological importance

Micro TOF-Q mass spectra.

<p>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.</p

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

<p>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.</p

Secondary structural analysis of the free HSA and its interaction with TMF.

<p>Based on the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008834#pone-0008834-g004" target="_blank">Figure 4</a>, the data analyzed by web based software CDNN 2.1.</p

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

<p>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.</p

Molecular docking of HSA with TMF.

<p>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.</p