Spectroscopic Investigations on the Interaction between Carbon Nanotubes and Catalase on Molecular Level

The interactions between well‐dispersed multiwalled carbon nanotubes (MWCNTs) and catalase (CAT) were investigated. The activity of CAT was inhibited with the addition of MWCNTs. After deducting the inner filter effect, the fluorescence spectra revealed that the tryptophan (Trp) residues were exposed and the fluorescence intensities of CAT increased with the increase in the MWCNTs concentration. At the same time, the environment of the Trp residues became more hydrophobic. The results of UV–vis absorption spectroscopy and CD spectra indicated that the secondary structure of CAT had been changed, and the amino acid residues were located in a more hydrophobic environment. Meanwhile, the UV–vis spectra indicated that the conformation of the heme porphyrin rings was changed. The microenvironment of CAT activity sites may be interfered by MWCNTs. This research showed that MWCNTs could not only contribute to the conformational changes of protein but also change the enzyme function.     

Probing the Interaction of Mutiwalled Carbon Nanotubes and Catalase: Mutispectroscopic Approach

In this study, the mechanism of the interaction between multiwalled carbon nanotubes (MWCNTs) and catalase was investigated by fluorescence, UV–vis, and circular dichroism (CD) spectroscopy under physiological conditions. The fluorescence quenching mechanism of catalase by MWCNTs was shown to be a static quenching procedure and was a result of the formation of a catalase–MWCNT complex. The secondary structure and conformation of the catalase adsorbed on MWCNTs was determined by CD and UV‐vis spectroscopy, and the results indicate that the catalase in this complex is partially unfolded with its lost in α‐helical content and obtainment in β‐sheet content. Moreover, binding of MWCNTs to catalase inhibited the enzymatic activity, which may trigger some toxic effects and undesirable physiological consequences. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:493‐498, 2012;Viewthis article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21454     

Study on the interaction of β‐carotene and astaxanthin with trypsin and pepsin by spectroscopic techniques

β‐Carotene and astaxanthin are two carotenoids with powerful antioxidant properties, but the binding mechanisms of β‐carotene/astaxanthin to proteases remain unclear. In this study, the interaction of these two carotenoids with trypsin and pepsin was investigated using steady‐state and time‐resolved fluorescence measurements, synchronous fluorescence spectroscopy, UV–vis absorption spectroscopy and circular dichroism (CD) spectroscopy. The experimental results indicated that the quenching mechanisms of trypsin/pepsin by the two carotenoids are static processes. The binding constants of trypsin and pepsin with these two carotenoids are in the following order: astaxanthin–trypsin > astaxanthin–pepsin > β‐carotene–trypsin > β‐carotene–pepsin, respectively. Thermodynamic investigations revealed that the interaction between the two carotenoids and trypsin/pepsin is synergistically driven by enthalpy and entropy, and hydrophobic forces and electrostatic attraction have a significant role in the reactions. In addition, as shown by synchronous fluorescence spectroscopy, UV–vis absorption spectroscopy and CD, the two carotenoids may induce conformational and microenvironmental changes in trypsin/pepsin. The study provides an accurate and full basic data for clarifying the binding mechanisms of the two carotenoids with trypsin/pepsin and is helpful in understanding their effect on protein function and their biological activity in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Spectroscopic and molecular simulation studies on the interaction of di‐(2‐ethylhexyl) phthalate and human serum albumin

Di‐(2‐ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in industrial production, but may have a potential health risk. In this study, the binding characteristics of DEHP with human serum albumin (HSA) in aqueous solution at pH 7.4 were determined using UV/vis absorption, fluorescence, Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD), along with a molecular simulation technique. Analysis of the fluorescence titration data at different temperatures suggested that the fluorescence quenching mechanism of HSA by DEHP was static. The calculated thermodynamic parameters indicated that hydrophobic forces played a predominant role in formation of the DEHP–HSA complex, but hydrogen bonds could not be omitted. Site marker competitive experiments and denaturation studies showed that the binding of DEHP to HSA primarily took place in subdomain IIA of HSA, and molecular docking results further corroborated the binding sites. The synchronous fluorescence, UV/vis absorption, FTIR and CD spectra revealed that the addition of DEHP induced changes in the secondary structure of HSA. Protein surface hydrophobicity (PSH) tests indicated that DEHP binding to HSA caused an increase in the PSH. Moreover, the effects of some metal ions on the binding constant of DEHP − HSA interaction were also investigated. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation on the interaction of nanoAg with Cu–Zn SOD

Silver nanoparticles (nanoAg) are used more and more widely, particularly because of their antimicrobial properties. The effect of exposure to nanoAg on the structure of superoxide dismutase (SOD) was thoroughly investigated using fluorescence measurements, synchronous fluorescence spectroscopy, steady‐state and time‐resolved fluorescence quenching measurements, UV/Vis absorption spectroscopy, resonance light scattering (RLS), circular dichroism (CD), isothermal titration calorimetry (ITC) and high‐resolution transmission electron microscopy (HRTEM). Through van der Waal's force, nanoAg interacted with Cu–Zn SOD and influenced the active site by inducing structural changes, which influenced the function of SOD. The fluorescence studies show that both static and dynamic quenching processes occur. This paper provides reference data for toxicological studies of nanoAg, which are important in the future development of nanotechnology. Copyright © 2015 John Wiley & Sons, Ltd.     

Study on the mechanism of the interaction between acteoside and pepsin using spectroscopic techniques

The interaction of acteoside with pepsin has been investigated using fluorescence spectra, UV/vis absorption spectra, three‐dimensional (3D) fluorescence spectra and synchronous fluorescence spectra, along with a molecular docking method. The fluorescence experiments indicate that acteoside can quench the intrinsic fluorescence of pepsin through combined quenching at a low concentration of acteoside, and static quenching at high concentrations. Thermodynamic analysis suggests that hydrogen bonds and van der Waal's forces are the main forces between pepsin and acteoside. According to the theory of Förster's non‐radiation energy transfer, the binding distance between pepsin and acteoside was calculated to be 2.018 nm, which implies that energy transfer occurs between acteoside and pepsin. In addition, experimental results from UV/vis absorption spectra, 3D fluorescence spectra and synchronous fluorescence spectra imply that pepsin undergoes a conformation change when it interacts with acteoside. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative studies on the interactions of dihydroartemisinin and artemisinin with bovine serum albumin using spectroscopic methods

The interactions of dihydroartemisinin (DHA) and artemisinin (ART) with bovine serum albumin (BSA) have been investigated using fluorescence, UV/vis absorption and Fourier transform infrared (FTIR) spectra under simulated physiological conditions. The binding characteristics of DHA/ART and BSA were determined by fluorescence emission and resonance light scattering (RLS) spectra. The quenching mechanism between BSA and DHA/ART is static. The binding constants and binding sites of DHA/ART–BSA systems were calculated at different temperatures (293, 298, 304 and 310 K). According to Förster non‐radiative energy transfer theory, the binding distance of BSA to DHA/ART was calculated to be 1.54/1.65 nm. The effect of DHA/ART on the secondary structure of BSA was analyzed using UV/vis absorption, FTIR, synchronous fluorescence and 3D fluorescence spectra. In addition, the effects of common ions on the binding constants of BSA–DHA and BSA–ART systems were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Exploration of interaction of canthaxanthin with human serum albumin by spectroscopic and molecular simulation methods

The interaction between the food colorant canthaxanthin (CA) and human serum albumin (HSA) in aqueous solution was explored by using fluorescence spectroscopy, three‐dimensional fluorescence spectra, synchronous fluorescence spectra, UV–vis absorbance spectroscopy, circular dichroism (CD) spectra and molecular docking methods. The thermodynamic parameters calculated from fluorescence spectra data showed that CA could result in the HSA fluorescence quenching. From the K_SV change with the temperature dependence, it was concluded that HSA fluorescence quenching triggered by CA is the static quenching and the number of binding sites is one. Furthermore, the secondary structure of HSA was changed with the addition of CA based on the results of synchronous fluorescence, three‐dimensional fluorescence and CD spectra. Hydrogen bonds and van der Waals forces played key roles in the binding process of CA with HSA, which can be obtained from negative standard enthalpy (ΔH) and negative standard entropy (ΔS). Furthermore, the conclusions were certified by molecular docking studies and the binding mode was further analyzed with Discovery Studio. These conclusions can highlight the potential of the interaction mechanism of food additives and HSA.     

Spectroscopic analysis on structure–affinity relationship in the interactions of different oleanane‐type triterpenoids with bovine serum albumin

Oleanane‐type triterpenoids serve as an important group of plant secondary metabolites with a variety of biological activities and the C‐3 position substitution pattern is a significant structural feature for their biological activities. Three selected oleanane‐type triterpenoids (glycyrrhizin, glycyrrhetinic acid, and carbenoxolone) bearing different substituents (glucuronic acid dimer, hydroxyl, and succinyl groups) at the C‐3 position were studied for their affinities to bind bovine serum albumin (BSA) by steady‐state fluorescence, synchronous, three‐dimensional fluorescence and ultraviolet–visible (UV–vis) absorption spectra. The binding mechanism of the triterpenoids to BSA is due to the formation of the triterpenoids–BSA complex and the binding affinity is strongest for carbenoxolone and ranked in the order carbenoxolone > glycyrrhetinic acid > glycyrrhizin. The thermodynamic parameters calculated at different temperatures showed that triterpenoids binding to BSA primarily depended on hydrophobic interaction and hydrogen bonding. The distance between the bound triterpenoid and BSA was determined on the basis of the Förster's energy transfer theory. Displacement experiments using phenylbutazone and ibuprofen showed the binding site of triterpenoids on BSA at subdomain IIA (Sudlow's site I). The effect of triterpenoids on BSA conformation was analyzed by UV–vis absorption, and synchronous and three‐dimensional fluorescence spectra. These results revealed that the C‐3 position substitution pattern significantly affects the structure–affinity relationships of oleanane‐type triterpenoid binding to BSA and further affects the bioavailability of triterpenoids in the blood circulatory system. Copyright © 2014 John Wiley & Sons, Ltd.     

Spectroscopic investigation on the toxic interaction of melamine with herring sperm DNA

The toxic interaction of melamine with herring sperm DNA (hs‐DNA) was investigated by using fluorescence and UV–vis absorption spectra techniques. The experimental results showed that the toxic interaction between melamine and hs‐DNA occurred. Fluorescence quenching experiments indicated the existence of electrostatic binding between melamine and hs‐DNA. The binding constants K_A and the binding site numbers were calculated by means of the Stern–Volmer equation and were 9.8 × 10⁴ L mol^?1 and 1.3, respectively. Both the results of fluorescence spectra and UV–vis absorption spectra verified that there are electrostatic binding between melamine and hs‐DNA. The possibility in the presence of a classical intercalation binding mode could be ruled out by using DNA unwinding experiments. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:323–329, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20341     

Structural Changes of Human Serum Albumin Induced by Cadmium Acetate

The structural changes of human serum albumin (HSA) induced by the addition of cadmium acetate were systematically investigated using UV–vis absorption, circular dichroism (CD), synchronous, and three‐dimentional (3D) fluorescence methods. The fluorescence spectra suggested the formation of cadmium acetate–HSA complex. UV absorption result indicated that the interaction between cadmium acetate and HSA could lead to the alteration of the protein skeleton. The structural analysis according to CD method showed that the cadmium acetate binding altered HSA conformation with a major reduction of α‐helix, inducing a partial protein unfolding. Synchronous fluorescence spectra suggested that cadmium acetate was situated closer to tryptophan residue compared to tyrosine residues, making tryptophan residue locate in a more hydrophobic environment. 3D fluorescence demonstrated that cadmium acetate could induce the HSA aggregation and cause a slight unfolding of the polypeptide backbone of the protein.     

Mechanistic and conformational studies on the interaction of sulfamethazine with human immunoglobulin G by molecular modeling and multi‐spectroscopic approach in vitro

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ ? HIgG complex. Fluorescence spectroscopy, ultraviolet–visible (UV–vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?19.12 kJ · mol^?1 and 20.22 J · mol^?1 · K^?1, respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ ? HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three‐dimensional fluorescence spectroscopy, UV–vis absorption spectroscopy and CD spectroscopy. Copyright © 2014 John Wiley & Sons, Ltd.     

Spectroscopic investigation and in vitro cytotoxic activity toward HepG2 cells of a copper compound complexed with human serum albumin

The human serum albumin (HSA) interaction of a mixed‐ligand copper compound (1) with an imidazole and taurine Schiff base derived from salicylaldehyde and taurine was investigated using fluorescence spectroscopy, UV–vis spectroscopy, time‐resolved fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared (FT‐IR) spectroscopy and a molecular docking technique. The results of fluorescence and time‐resolved fluorescence spectroscopy indicated that 1 can effectively quench the HSA fluorescence by a static mechanism. Binding constants (K) and the number of binding sites (n ≈ 1) were calculated using modified Stern–Volmer equations. The thermodynamic parameters were calculated. UV–vis, CD and FT‐IR spectroscopy measurements confirm the alterations in the HSA secondary structure induced by 1. The site marker competitive experiment confirms that 1 is located in subdomain IB of HSA. The combination of molecular docking results and fluorescence experimental results reveal that hydrophobic interaction and hydrogen bonds are the predominant intermolecular forces stabilizing the 1–HSA complex. The 1–HSA complex increases approximately three times its cytotoxicity in cancer cells but has no effect on normal cells in vitro. Compared with unbound 1, the 1–HSA complex promotes HepG2 cells apoptosis and also has a stronger capacity for cell cycle arrest at the S phase of HepG2 cells.     

Effect of CdTe Quantum Dots Size on the Conformational Changes of Human Serum Albumin: Results of Spectroscopy and Isothermal Titration Calorimetry

Quantum dots (QDs) are recognized as some of the most promising candidates for future applications in biomedicine. However, concerns about their safety have delayed their widespread application. Human serum albumin (HSA) is the main protein component of the circulatory system. It is important to explore the interaction of QDs with HSA for the potential in vivo application of QDs. Herein, using spectroscopy and isothermal titration calorimetry (ITC), the effect of glutathione-capped CdTe quantum dots of different sizes on the HSA was investigated. After correction for the inner filter effect, the fluorescence emission spectra and synchronous fluorescence spectra showed that the microenvironment of aromatic acid residues in the protein was slightly changed when the glutathione (GSH)–cadmium telluride (CdTe) QDs was added, and GSH–CdTe QDs with larger particle size exhibited a much higher effect on HSA than the small particles. Although a ground-state complex between HSA and GSH–CdTe QDs was formed, the UV–vis absorption and circular dichroism spectroscopic results did not find appreciable conformational changes of HSA. ITC has been used for the first time to characterize the binding of QDs with HSA. The ITC results revealed that the binding was a thermodynamically spontaneous process mainly driven by hydrophobic interactions, and the binding constant tended to increase as the GSH–CdTe QDs size increased. These findings are helpful in understanding the bioactivities of QDs in vivo and can be used to assist in the design of biocompatible and stable QDs.     

Structural and optical properties of Cu‐doped ZnS nanoparticles formed in chitosan/sodium alginate multilayer films

Chitosan/alginate multilayers were fabricated using a spin‐coating method, and ZnS:Cu nanoparticles were generated within the network of two natural polysaccharides, chitosan and sodium alginate. The synthesized nanoparticles were characterized using an X‐ray diffractometer (XRD), transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS) and atomic force microscopy (AFM). The results showed that cubic zinc blende‐structured ZnS:Cu nanoparticles with an average crystal size of ~ 3 nm were uniformly distributed. UV–vis spectra indicate a large quantum size effect and the absorption edge for the ZnS:Cu nanoparticles slightly shifted to longer wavelengths with increasing Cu ion concentrations. The photoluminescence of the Cu‐doped ZnS nanoparticles reached a maximum at a 1% doping level. The ZnS:Cu nanoparticles form and are distributed uniformly in the composite multilayer films with a surface average height of 25 nm. Copyright © 2013 John Wiley & Sons, Ltd.     

Spectroscopic investigation into the interaction of a diazacyclam‐based macrocyclic copper(ii) complex with bovine serum albumin

Cyclam‐based ligands and their complexes are known to show antitumor activity. This study was undertaken to examine the interaction of a diazacyclam‐based macrocyclic copper(II) complex with bovine serum albumin (BSA) under physiological conditions. The interactions of different metal‐based drugs with blood proteins, especially those with serum albumin, may affect the concentration and deactivation of metal drugs, and thereby influence their availability and toxicity during chemotherapy. In this vein, several spectral methods including UV–vis absorption, fluorescence and circular dichroism (CD) spectroscopy techniques were used. Spectroscopic analysis of the fluorescence quenching confirmed that the Cu(II) complex quenched BSA fluorescence intensity by a dynamic mechanism. In order to further determine the quenching mechanism, an analysis of Stern–Volmer plots at various concentrations of BSA was carried out. It was found that the K_SV value increased with the BSA concentration. It was suggested that the fluorescence quenching process was a dynamic quenching rather than a static quenching mechanism. Based on Förster's theory, the average binding distance between the Cu(II) complex and BSA (r) was found to be 4.98 nm; as the binding distance was less than 8 nm, energy transfer from BSA to the Cu(II) complex had a high possibility of occurrence. Thermodynamic parameters (positive ΔH and ΔS values) and measurement of competitive fluorescence with 1‐anilinonaphthalene‐8‐sulphonic acid (1,8‐ANS) indicated that hydrophobic interaction plays a major role in the Cu(II) complex interaction with BSA. A Job's plot of the results confirmed that there was one binding site in BSA for the Cu(II) complex (1:1 stoichiometry). The site marker competitive experiment confirmed that the Cu(II) complex was located in site I (subdomain IIA) of BSA. Finally, CD data indicated that interaction of the Cu(II) complex with BSA caused a small increase in the α‐helical content. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescent bovine serum albumin interacting with the antitussive quencher dextromethorphan: a spectroscopic insight

The interaction of dextromethorphan hydrobromide (DXM) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV–vis absorption, synchronous fluorescence spectra (SFS), 3D fluorescence spectra, Fourier transform infrared (FTIR) spectroscopy and circular dichroism under simulated physiological conditions. DXM effectively quenched the intrinsic fluorescence of BSA. Values of the binding constant, K_A, are 7.159 × 10³, 9.398 × 10³ and 16.101 × 10³ L/mol; the number of binding sites, n, and the corresponding thermodynamic parameters ΔG°, ΔH° and ΔS° between DXM and BSA were calculated at different temperatures. The interaction between DXM and BSA occurs through dynamic quenching and the effect of DXM on the conformation of BSA was analyzed using SFS. The average binding distance, r, between the donor (BSA) and acceptor (DXM) was determined based on Förster's theory. The results of fluorescence spectra, UV–vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of DXM. Copyright © 2015 John Wiley & Sons, Ltd.     

Study on the synthesis of sulfonamide derivatives and their interaction with bovine serum albumin

Three sulfonamide derivatives (SAD) were first synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfadimidine, sulfamethoxazole and sulfachloropyridazine sodium) and were characterized by elemental analysis, ¹H NMR and MS. The interaction between bovine serum albumin (BSA) and SAD was studied using UV/vis absorption spectroscopy, fluorescence spectroscopy, time‐resolved fluorescence spectroscopy and circular dichroism spectra under imitated physiological conditions. The experimental results indicated that SAD effectively quenched the intrinsic fluorescence of BSA via a static quenching process. The thermodynamic parameters showed that hydrogen bonding and van der Waal's forces were the predominant intermolecular forces between BSA and two SADs [4‐((4‐(N‐(4,6‐dimethylpyrimidin‐2‐yl)sulfamoyl)phenyl)carbamoyl)phenyl acetate and 4‐((4‐(N‐(5‐methylisoxazol‐3‐yl)sulfamoyl)phenyl)carbamoyl)phenyl acetate], but hydrophobic forces played a major role in the binding process of BSA and 4‐((4‐(N‐(6‐chloropyridazin‐3‐yl)sulfamoyl)phenyl) carbamoyl)phenyl acetate. In addition, the effect of SAD on the conformation of BSA was investigated using synchronous fluorescence spectroscopy and circular dichroism spectra. Molecular modeling results showed that SAD was situated in subdomain IIA of BSA. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation into the interaction of methylparaben and erythromycin with human serum albumin using multispectroscopic methods

In this paper, the interaction of methylparaben and erythromycin with human serum albumin (HSA) was studied for the first time using spectroscopic methods including Fourier transform infrared (FTIR) spectroscopy and UV absorption spectroscopy in combination with fluorescence quenching under physiological conditions. The binding parameters were evaluated using a fluorescence quenching method. Based on Förster's theory of non‐radiation energy transfer, the binding average distance, r between the donor (HSA) and the acceptor (methylparaben and erythromycin) was evaluated. UV/vis absorption, FTIR, synchronous and 3D spectral results showed that the conformation of HSA was changed in the presence of methylparaben and erythromycin. The thermodynamic parameters were calculated according to the van't Hoff equation and are discussed. The effect of some biological metal ions and site probes on the binding of methylparaben and erythromycin to HSA were further examined. Copyright © 2015 John Wiley & Sons, Ltd.