Comparative study of the interactions between bisphenol-A and its endocrine disrupting analogues with bovine serum albumin using multi-spectroscopic and molecular docking studies

Interaction studies of bisphenol analogues; biphenol-A (BPA), bisphenol-B (BPB), and bisphenol-F (BPF) with bovine serum albumin (BSA) were performed using multi-spectroscopic and molecular docking studies at the protein level. The mechanism of binding of bisphenols with BSA was dynamic in nature. SDS refolding experiments demonstrated no stabilization of BSA structure denatured by BPB, however, BSA denatured by BPA and BPF was found to get stabilized. Also, CD spectra and molecular docking studies revealed that BPB bound more strongly and induced more conformational changes in BSA in comparison to BPA. Hence, this study throws light on the replacement of BPA by its analogues and whether the replacement is associated with a possible risk, raising a doubt that perhaps BPB is not a good substitute of BPA.     

Effects of N‐Acetyl‐L‐Cysteine‐Capped CdTe Quantum Dots on Bovine Serum Albumin and Bovine Hemoglobin: Isothermal Titration Calorimetry and Spectroscopic Investigations

The interactions of N‐acetyl‐L‐cysteine‐capped CdTe quantum dots (QDs) with bovine serum albumin (BSA) and bovine hemoglobin (BHb) were investigated by isothermal titration calorimetry (ITC), fluorescence, synchronous fluorescence, fluorescence lifetime, ultraviolet–visible absorption, and circular dichroism techniques. Fluorescence data of BSA–QDs and BHb–QDs revealed that the quenching was static in every system. While CdTe QDs changed the microenvironment of tryptophan in BHb, the microenvironment of BSA kept unchanged. Adding CdTe QDs affected the skeleton and secondary structure of the protein (BSA and BHb). The ITC results indicated that the interaction between the protein (BSA and BHb) and QDs‐612 was spontaneous and the predominant force was hydrophobic interaction. In addition, the binding constants were determined to be 1.19 × 10⁵ L mol^?1 (BSA–QDs) and 2.19 × 10⁵ L mol^?1 (BHb–QDs) at 298 K. From these results, we conclude that CdTe QDs have a larger impact on the structure of BHb than BSA.     

Application of a novel mass spectrometric (MS) method to examine exposure to Bisphenol-A and common substitutes in a maternal fetal cohort

The use of Bisphenol A (BPA) has widely been replaced in consumer products by analogs BPB, BPE, BPF, BPS, and BPAF. Recent studies have linked these substitutes to similar adverse health outcomes as BPA, including disruption of endocrine pathways in animal and human studies. We designed a novel MS method, developed specifically for this study, to capture the most relevant BPA alternatives, BPB, BPE, BPF, BPS, BPAF and 4-NP in human blood and urine to quantify potential in utero exposures. To our knowledge, this is the first study to explore in utero exposure to these BPA analogs and the first U.S. study to test for BPA in maternal/fetal pairs. The method was run on 30 paired maternal urine and fetal cord blood samples from mothers undergoing elective Caesarean sections. 90% of mothers and 77% of babies tested positive for at least one BP analog. 83% of mothers tested positive for BPAF, 60% for BPS, 57% for BPB, 17% for BPF and 7% for BPA. 57% of babies tested positive for BPAF and 50% for BPF. BPA and BPB were detected in one cord blood sample each. BPS was not detected in cord blood. BPE was not detected in any fetal cord blood or maternal urine samples. These findings demonstrate the pervasiveness of some BP analogs in pregnant women and their babies at birth.     

Spectroscopic characterization,calorimetric study and molecular docking to evaluate the bioconjugation of maltol with hemoglobin

Maltol, a food additive, is extensively used in our daily life. To date, its biological safety is still debated. In this article, binding interaction of maltol with bovine hemoglobin (BHb), an important functional protein, was studied by molecular docking research and spectroscopic and calorimetric measurements. We found that maltol could cause structural changes of BHb. By interacting with Glu 101 (1.27 Å) and Lys 104 (2.49 Å) residues, maltol changed the cavity structure and induced a microenvironment change around tryptophan (Trp) residue. Thermodynamic parameters obtained from isothermal titration calorimetry (ITC) measurement showed that hydrophobic forces were the main forces existing in this system. The association constant of K (8.0 ± 3.4 × 10⁴ M^?1) shows the mild ligand–protein binding for maltol with BHb. The α‐helix amount in BHb increased (59.6–62.6%) with different concentrations of maltol and the intrinsic fluorescence intensity was quenched by maltol, indicating the conformation changes and denaturation of BHb. This work presents the interactions of maltol with BHb at the molecular level and obtains evidence that maltol induces adverse effects to proteins in vitro.     

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.     

Experimental and computational characterization on the binding of two fluoroquinolones to bovine hemoglobin

Ciprofloxacin (CPFX) and enrofloxacin (ENFX) are 2 representatives of widely used fluoroquinolones (FQs) with many human and veterinary applications. The residues of FQs in the environment are potentially harmful. Recently, great concern has been paid to their persistence and fate in the environment because of the potential adverse effects on humans and ecosystem functions. In the present study, we examined the interactions of bovine hemoglobin (BHb) with these 2 FQs by means of multiple spectroscopic and molecular docking methods under physiological conditions. The experimental results revealed that both FQs could bind with BHb to form complexes mainly through electrostatic interactions. And CPFX posed more of an affinity threat to BHb than ENFX. On the basis of molecular docking, both FQs could bind into the central cavity of BHb and interact with the residue Trp 37, resulting in the remarkable fluorescence quenching of protein. Additionally, as shown by the synchronous fluorescence, UV‐visible absorption and circular dichroism data, both CPFX and ENFX could lead to the conformational and microenvironmental changes of BHb, which may affect its physiological functions. The work is beneficial for understanding the biological toxicity of FQs in vivo.     

The study on interactions between levofloxacin and model proteins by using multi-spectroscopic and molecular docking methods

The interactions of levofloxacin (LEV) with lysozyme (LYZ), trypsin and bovine hemoglobin (BHb) were investigated, respectively, by using multi-spectral techniques and molecular docking in vitro. Fluorescence studies showed that LEV quenched LYZ/trypsin fluorescence in a combined quenching ways and BHb fluorescence in a static quenching with binding constants of .14, .51 and .20 × 10⁵ L mol^?1 at 298 K, respectively. The thermodynamic parameters demonstrated that hydrophobic forces, hydrogen bonds, and van der Waals forces played the major role in the binding process. The binding distances between LEV and the inner tryptophan residues of LYZ, trypsin, and BHb were calculated to be 4.04, 3.38, and 4.52 nm, respectively. Furthermore, the results of circular dichroism spectra (CD), UV–vis, and three-dimensional fluorescence spectra indicated that the secondary structures of LYZ, trypsin, and BHb were partially changed by LEV with the α-helix percentage of LYZ-LEV system increased while that of BHb-LEV system was decreased, the β-sheet percentage of trypsin-LEV system increased from 41.3 to 42.9%. UV–vis spectral results showed that the binding interactions could cause conformational and some micro-environmental changes of LYZ, trypsin, and BHb. The results of molecular docking revealed that in LYZ and trypsin systems, LEV bound to the active sites residues GLU 35 and ASP 52 of LYZ and trypsin at the active site SER 195, and in BHb system, LEV was located in the central cavity, which was consistent with the results of synchronous fluorescence experiment. Besides, LEV made the activity of LYZ decrease while the activity of trypsin increased.     

The interaction of blood proteins with brucine

The features of brucine (BC) binding to two blood proteins, bovine hemoglobin (BHb), and bovine serum albumin (BSA), were investigated via fluorescence, circular dichroism and UV/Vis absorption spectroscopy. The results revealed that BC caused the fluorescence quenching of blood proteins by the formation of BC–protein complex. The corresponding thermodynamic parameters were measured at different temperatures. The process of binding BC molecule on protein was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The molecular docking has been employed to explore the binding site of the BC in BHb and BSA on the Autodock 4.2. The distances r between BC and protein were calculated to be 4.93 and 5.08 nm for BHb, and BSA, respectively. The effect of BC on the conformation of blood proteins was analyzed using CD, synchronous fluorescence and three-dimensional fluorescence spectra.     

Ultraviolet irradiation-dependent fluorescence enhancement of hemoglobin catalyzed by reactive oxygen species

Ultraviolet (UV) light has a potent effect on biological organisms. Hemoglobin, an oxygen-transport protein, plays an irreplaceable role in sustaining life of all vertebrates. In this study we scrutinize the effects of ultraviolet irradiation (UVI) as well as visible irradiation on the fluorescence characteristics of bovine hemoglobin (BHb) in vitro. Data show that UVI results in fluorescence enhancement of BHb in a dose-dependant manner. Furthermore, UVI-induced fluorescence enhancement is significantly increased when BHb is pretreated with hydrogen peroxide (H(2)O(2)), a type of reactive oxygen species (ROS). Meanwhile, The water-soluble antioxidant vitamin C suppresses this UVI-induced fluorescence enhancement. In contrast, green light irradiation does not lead to fluorescence enhancement of BHb no matter whether H(2)O(2) is acting on the BHb solution or not. Taken together, these results indicate that catalysis of ROS and UVI-dependent irradiation play two key roles in the process of UVI-induced fluorescence enhancement of BHb.     

Effects of an antimalarial quinazoline derivative on human erythrocytes and on cell membrane molecular models

Plasmodium, the parasite which causes malaria in humans multiplies in the liver and then infects circulating erythrocytes. Thus, the role of the erythrocyte cell membrane in antimalarial drug activity and resistance has key importance. The effects of the antiplasmodial N(6)-(4-methoxybenzyl)quinazoline-2,4,6-triamine (M4), and its inclusion complex (M4/HPβCD) with 2-hydroxypropyl-β-cyclodextrin (HPβCD) on human erythrocytes and on cell membrane molecular models are herein reported. This work evidences that M4/HPβCD interacts with red cells as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a 10μM concentration; b) in isolated unsealed human erythrocyte membranes (IUM) a concentration as low as 1μM induced sharp DPH fluorescence anisotropy decrease whereas increasing concentrations produced a monotonically decrease of DPH fluorescence lifetime at 37°C; c) X-ray diffraction studies showed that 200μM induced a complete structural perturbation of dimyristoylphosphatidylcholine (DMPC) bilayers whereas no significant effects were detected in dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of lipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively; d) fluorescence spectroscopy data showed that increasing concentrations of the complex interacted with the deep hydrophobic core of DMPC large unilamellar vesicles (LUV) at 18°C. All these experiments are consistent with the insertion of M4/HPβCD in the outer monolayer of the human erythrocyte membrane; thus, it can be considered a promising and novel antimalarial agent.     

Quantification and confocal imaging of protein specific molecularly imprinted polymers

We have employed FITC--albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labeled template is reported, with subsequent characterization of the smart material to show that the HydroMIP possesses a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC--albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of the imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine hemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols and document the use of fluorescence as a useful tool in template labeling/detection and novel imaging strategies.     

Studies of the interaction between paraquat and bovine hemoglobin

The interaction between paraquat (PQ) and bovine hemoglobin (BHb) was investigated using fluorescence and UV/vis absorption spectroscopy. The reactivity of the heme centers with superoxide anions formed by PQ was judged on the basis of the decrease of the Soret band. The experimental results showed that the fluorescence quenching of BHb by PQ was a result of the formation of PQ-BHb complex; static quenching was confirmed to result in the fluorescence quenching. The binding site number n, apparent binding constant K(A) and corresponding thermodynamic parameters were measured at different temperatures. The process of binding PQ molecule on BHb was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic and electrostatic interactions played a major role in stabilizing the complex. The effect of PQ on the conformation of BHb was analyzed using synchronous fluorescence spectroscopy.     

Molecular interactions of thymol with bovine serum albumin: Spectroscopic and molecular docking studies

Thymol is the main monoterpene phenol present in the essential oils which is used in the food industry as flavoring and preservative agent. In this study, the interaction of thymol with the concentration range of 1 to 6 μM and bovine serum albumin (BSA) at fixed concentration of 1 μM was investigated by fluorescence, UV‐vis, and molecular docking methods under physiological‐like condition. Fluorescence experiments were performed at 5 different temperatures, and the results showed that the fluorescence quenching of BSA by thymol was because of a static quenching mechanism. The obtained binding parameters, K, were in the order of 10⁴ M^?1, and the binding number, n, was approximately equal to unity indicating that there is 1 binding site for thymol on BSA. Calculated thermodynamic parameters for enthalpy (ΔH), entropy (ΔS), and Gibb's free energy (ΔG) showed that the reaction was spontaneous and hydrophobic interactions were the main forces in the binding of thymol to BSA. The results of UV‐vis spectroscopy and Arrhenius' theory showed the complex formation in the interaction of thymol and BSA. Negligible conformational changes in BSA by thymol were observed in fluorescence experiments, and the same results were also obtained from UV‐vis studies. Results of molecular docking indicated that the subdomain IA of BSA was the binding site for thymol.     

Novel thymine-functionalized polystyrenes for applications in biotechnology. 2. Adsorption of model proteins

This paper investigates the adsorption of bovine serum albumin (BSA) and bovine hemoglobin (BHb) model proteins onto novel thymine-functionalized polystyrene (PS-VBT) microspheres, in comparison with polystyrene (PS) microspheres. Maximum adsorption was obtained for both proteins near their corresponding isoelectric points (pI at pH = 4.7 for BSA and 7.1 for BHb). FTIR and adsorption isotherm analysis demonstrated that, although both proteins were physisorbed onto PS through nonspecific hydrophobic interactions, adsorption onto the functionalized copolymers occurred by both physisorption and chemisorption via hydrogen bonding. FTIR analysis also indicated conformational changes in the secondary structure of BSA and BHb adsorbed onto PS, whereas little or no conformation change was seen in the case of adsorption onto PS-VBT. Atomic force microscopy (AFM), consistent with the isotherm results, also demonstrated monolayer adsorption for both proteins. AFM images of BSA adsorbed onto copolymers with 20 mol % surface VBT loading showed exclusively end-on orientation. Adsorption onto copolymers with lower functionality showed mixed end-on and side-on orientation modes of BSA, and only the side-on orientation was observed on PS. The AFM results agreed well with theoretically calculated and experimentally obtained adsorption capacities. AFM together with calculated and observed adsorption capacity data for BHb indicated that this protein might be highly compressed on the copolymer surface. Adsorption from a binary mixture of BSA and BHb onto PS-VBT showed good separation at pH=7.0; approximately 90% of the adsorbed protein was BHb. The novel copolymers have potential applications in biotechnology.     

A probe to study the toxic interaction of tartrazine with bovine hemoglobin at the molecular level

Tartrazine is an artificial azo dye commonly used in food products, but tartrazine in the environment is potentially harmful. The toxic interaction between tartrazine and bovine hemoglobin (BHb) was investigated using fluorescence, synchronous fluorescence, UV–vis absorption, circular dichroism (CD) and molecular modeling techniques under simulated physiological conditions. The fluorescence data showed that tartrazine can bind with BHb to form a complex. The binding process was a spontaneous molecular interaction, in which van der Waals' forces and hydrogen bonds played major roles. Molecular docking results showed that the hydrogen bonds exist between the oxygen atoms at position 31 of tartrazine and the nitrogen atom NZ7 on Lys99, and also between the oxygen atoms at position 15 of tartrazine and the nitrogen atom NZ7 on Lys104, Lys105. The results of UV–vis and CD spectra revealed that tartrazine led to conformational changes in BHb, including loosening of the skeleton structure and decreasing α helix in the secondary structure. The synchronous fluorescence experiment revealed that tartrazine binds into the hemoglobin central cavity, and this was verified using a molecular modeling study. Copyright © 2013 John Wiley & Sons, Ltd.     

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.     

Binding of phospholipids to beta-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, beta-Lactoglobulin (beta-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC10PE) to beta-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, KB, was (1.2+/-0.2)x10(6) M(-1) at 25 degrees C, pH 7.4 and I=0.15 M. Dependence of KB on pH and on the monomer-dimer equilibrium of beta-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the beta-barrel in the protein. The monomer-dimer equilibrium of beta-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, KD, was (7.0+/-1.5)x10(5) M(-1) at 25 degrees C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC10PE between beta-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. beta-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Complex formation between Chromatium vinosum ferric cytochrome c' and bromophenol blue

An unusual complex has been observed between the common electrophoresis tracer bromophenol blue (BPB) and the cytochrome c' from Chromatium vinosum during polyacrylamide gel electrophoresis. Complex formation results in a shift and increase in the intensity of the visible absorption band of BPB. Differential spectrophotometric titration of BPB with cytochrome c' indicates that one BPB binds to each of the two subunits of cytochrome c' with a binding constant of 4.2(0.5) x 10(5). The absence of a significant effect of ionic strength on the binding constant and the effect of Triton X-100 on the spectrum of BPB suggest that hydrophobic interactions are important to binding. An analysis of the structure of C. vinosum cytochrome c' shows the presence of a surface hydrophobic patch which may participate in the binding interaction. Many of the hydrophobic amino acids in the patch are well conserved by type among all known sequences of cytochrome c' and are found in loop elements of the 3D structure, suggesting a functional basis for conservation. It is proposed that the binding of BPB may mimic a relevant interaction involving the cytochrome c' biological function.     

Applicability of airlift draft-tube fluidized bioreactors for binary protein mixture bioseparation

An airlift draft-tube fluidized bioreactor has been designed and tested for applications in protein bioseparation. Operating parameters and geometrical dimensions of the bioreactor were optimized to ensure fluid circulation in a defined cyclic pattern between the riser and the downcomer. The overall directionality of liquid flow generates homogeneous field of low shear and achieves good mixing efficiency. Bioseparation of proteins was achieved from solutions containing both BSA and BHb at different initial concentrations and at pH 7. Similar adsorption capacities of both proteins were observed in single protein adsorption experiments at pH 7. Compressibility of BHb allowed for high adsorption capacity, in addition to the hydrophobic interaction forces. Apparently the homogeneous and lower shear generated by the airlift bioreactor reduces the compressibility of adsorbed BHb. This allowed for higher BSA adsorption from solutions containing BSA and BHb mixtures. Conventional batch adsorption experiments showed more adsorption of BHb, which reduces bioseparation efficiency.     

Gamma-linolenic acid improves the constancy of contractions in uteri from sprayed rats and is metabolized to prostaglandin E1 but not to bisenoic prostanoids

The effects of gamma-linolenic acid (GLA) on the time-dependent constancy of spontaneous contractions (isometric developed tension = IDT and frequency of contractions = FC) in uterine strips isolated from spayed rats, were explored. Moreover, the influence of the unsaturated fatty acid on the basal generation and release of tissue prostaglandins (PGs) as well as the conversion of labelled GLA into prostanoids by the uterine tissue and the effects of p-bromo-phenacyl-bromide (BPB), were also studied. GLA (10(-7)M), attenuated significantly the spontaneous decrement of contractile constancy exhibited by control preparations during a period of 180 min of activity in isolation, whereas BPB (10(5) M) resulted in an augmented and faster decrement of inotropic constancy. Spontaneous changes in the constancy of uterine motility as time progressed involved similarly both IDT and FC. After 180 min of activity in isolation a basal generation and release of PGs E and F of the series 1 and 2, were detected. The challenge with 10(-7) M GLA (delivered immediately after isolation) enhanced significantly the output of PGE1 but did not influence the generation and release of PGE2 or PGF2 alpha. BPB (10(-5) M) had no significant effect on the basal output of PGE1, PGE2 or PGF2 but completely prevented the enhancing action of GLA on the synthesis and release of PGE1. Labelled GLA was mainly converted to PGE1 by rat uterine segments and negligible counts in the 2-series of prostanoids, were observed. In presence of BPB (10(-5) M) the conversion of 1-14C-GLA, to PGE1 was almost completely abolished. The foregoing evidences suggest that exogenous GLA is metabolized by the spayed rat uterus via an elongase, forming di-homogamma-linolenic acid (DHLA), which in turn is substrate for cyclo-oxygenase peroxidase reactions yielding finally PGE1. No evidence of a delta 5-desaturase activity, converting DHLA into arachidonate and further derivatives, was detected. Coincidently, exogenous GLA was able to support a better contractile constancy as a function of time than that evidenced in untreated uterine strips isolated from castrated rats.