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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]

Abstract: 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 4.08×103, 3.97×103, 3.45×103, and 3.20×103 Lmol−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 &y& Elsevier]

Abstract: The feasibility of microwave-accelerated derivatization for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was evaluated. The derivatization reaction was performed in a domestic microwave oven. Histidine (His), 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) were selected as test analytes and fluorescein isothiocyanate (FITC) was chosen as a fluorescent derivatizing reagent. Parameters that may affect the derivatization reaction and/or subsequent CE separation were systematically investigated. Under optimized conditions, the microwave-accelerated derivatization reaction was successfully completed within 150s, compared to 4–24h in a conventional water-bath derivatization process. This will remarkably reduce the overall analysis time and increase sample throughput of CE-LIF. The detection limits of this method were found to be 0.023ng/mL for His, 0.023ng/mL for 1-MH, and 0.034ng/mL for 3-MH, respectively, comparable to those obtained using traditional derivatization protocols. The proposed method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of these analytes in human urine. [Copyright &y& Elsevier]