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The binding affinity between hydroxyproline (Hyp) and human serum albumin (HSA) was investigated under simulated physiological conditions, using molecular modeling in combination with steady-state fluorescence, synchronous fluorescence, time-resolved fluorescence, UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Molecular modeling studies suggested that the Hyp molecule was situated within subdomain IIA of HSA. The fluorescence quenching analysis indicated that the fluorescence of HSA was quenched by Hyp with a dynamic quenching mechanism. The binding constants were calculated according to Scatchard's equation and implied that Hyp can bind to different binding sites on HSA. The thermodynamic analysis implied that hydrophobic forces were the main interaction in the Hyp-HSA system, which was found to be in line with the results of molecular modeling. Furthermore, the conformational structure of HSA was changed with various amounts of Hyp, which was confirmed by synchronous fluorescence, UV-vis absorption, CD, and FT-IR spectra.

Phthalate esters (PAEs) are globally pervasive contaminants that are considered to be endocrine disruptor chemicals and toxic environmental priority pollutants. In this paper, the interactions between PAEs and human serum albumin (HSA) were examined by molecular modelling, steady state and time-resolved fluorescence, ultraviolet-visible spectroscopy (UV-vis) and circular dichroism spectroscopy (CD). The association constants between PAEs and HSA were determined using the Stern-Volmer and Scatchard equations. The binding of dimethyl phthalate (DMP) to HSA has a single class of binding site and its binding constants (K) are 4.08 × 10(3), 3.97 × 10(3), 3.45 × 10(3), and 3.20 × 10(3)L mol(-1) at 289, 296, 303, and 310K, respectively. The Stern-Volmer and Scatchard plots both had two regression curves for HSA-butylbenzyl phthalate (BBP) and HSA-di-2-ethylhexyl phthalate (DEHP), which indicated that these bindings were via two types of binding sites: the numbers of binding site for the first type were lower than for the second type. The binding constants of the first type binding site were higher than those of the second type binding site at corresponding temperatures, the results suggesting that the first type of binding site had high affinity and the second binding site involved other sites with lower binding affinity and selectivity. The thermodynamic parameters of the binding reactions (ΔG°, ΔH° and ΔS°) were measured, and they indicated the presences of hydrophobic forces and hydrogen interactions in the PAEs-HSA interactions, which agreed well with the results from molecular modelling. The alterations of protein secondary structure in the presence of PAEs were confirmed by UV-vis and CD spectroscopy. The time-resolved fluorescence study showed that the lifetime of Trp residue of HSA decreased after the addition of PAEs, which implied that the Trp residue of HSA was the main binding site., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Abstract: Orange G (OG) was used as a model compound to investigate the binding mechanism between azo dye and human serum albumin (HSA) using a variety of methods. These included nuclear magnetic resonance (NMR), saturation transfer difference (STD)-NMR, steady-state fluorescence, UV–vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR), three-dimensional fluorescence, time-resolved fluorescence spectroscopy, and molecular modeling method under simulated physiological conditions. The data of NMR and STD-NMR indicated that OG was indeed bound to HSA and located in the hydrophobic pocket of HSA. The fluorescence quenching data showed that the binding of OG and HSA quenched the intrinsic fluorescence of HSA, and the dynamic quenching constants were acquired. Thermodynamics analysis and molecular modeling studies suggested that GO bound to the site I on HSA molecule, and indicated the presence of hydrophobic forces. The alterations of protein conformational structures were further examined by synchronous fluorescence, UV–vis absorption, CD, FT-IR, three-dimensional fluorescence, and time-resolved fluorescence spectroscopy. [Copyright &y& Elsevier]

Abstract: In this study, the interaction between C.I. Acid Orange 7 (AO7) and human serum albumin (HSA) was firstly investigated using nuclear magnetic resonance (NMR) spectroscopy in combination with fluorescence quenching spectroscopy, three-dimensional fluorescence spectroscopy, UV–vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling method in vitro. The results of NMR data confirmed that AO7 indeed interacted with HSA, and the hydrophobic portion of AO7 should be embedded to the hydrophobic pocket of HSA. The fluorescence quenching analysis revealed that AO7 can bind to HSA. The conformational change of HSA in the presence of AO7 was confirmed by synchronous fluorescence, three-dimensional fluorescence, UV–vis absorption, FT-IR and CD spectra. The binding distance between AO7 and tryptophan residue of HSA was calculated by the efficiency of fluorescence resonance energy transfer. Molecular modeling showed that hydrophobic force and hydrogen bonds were the major interaction between AO7 and HSA. [Copyright &y& Elsevier]

Abstract: The interaction mechanism of Acid Orange 6 (AO6) with human serum albumin (HSA) was investigated firstly by using fluorescence quenching technique, UV absorbance, circular dichroism (CD), Fourier transform infrared (FT-IR), three-dimensional fluorescence spectroscopy in combination with molecular modeling method under simulative physiological conditions. Fluorescence data indicated that there is a single class of binding sites between AO6 and HSA, and the alterations of HSA secondary structure in the presence of AO6 was confirmed by synchronous fluorescence, UV, CD, FT-IR and three-dimensional fluorescence spectra. The efficiency of fluorescence resonance energy transfer provided the binding distance (r) of 2.83nm for AO6–HSA system. Furthermore, the thermodynamic parameters enthalpy change (ΔH 0) and entropy change (ΔS 0) for the reaction were calculated to be −5.77kJmol−1 and 109.42Jmol−1 K−1, respectively, according to Van''t Hoff equation, these data suggested that both hydrophobic forces and hydrogen bonding play a major role in the binding of AO6 to HSA, which agrees well with the results of molecular modeling study. Experimental results showed that the interaction between AO6 and HSA induced a conformational change of HSA, which was proved by the qualitative and quantitative analysis data of different spectroscopic techniques under simulative physiological conditions. [Copyright &y& Elsevier]