Interaction of tebuconazole with bovine serum albumin: determination of the binding mechanism and binding site by spectroscopic methods

Abstract

This study investigates the interaction between tebuconazole and bovine serum albumin (BSA) in a physiological buffer (pH = 7.4) using the fluorescence quenching method to obtain the apparent binding constants (K) and number of binding sites (n) in the interaction between tebuconazole and BSA. The results revealed that tebuconazole can quench the intrinsic fluorescence of BSA through a static quenching procedure. It also shows that the thermodynamic parameters of enthalpy change (ΔH) and entropy change (ΔS) are negative, indicating that the interaction of tebuconazole with BSA is mainly driven by van der Waals forces and hydrogen bonds. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance of r between the donor (BSA) and acceptor (tebuconazole) was calculated to be 0.68?nm based on Forster’s non-radiative energy transfer theory. Analysis of synchronous fluorescence, three-dimensional fluorescence and circular dichroism (CD) spectra demonstrates that tebuconazole can induce conformational changes of BSA.     

Potential toxicity of amphenicol antibiotic: binding of chloramphenicol to human serum albumin

Antibiotics are widely used in daily life but their abuse has posed a potential threat to human health. To evaluate the toxicity of chloramphenicol (CAP) at the protein level, the interaction between CAP and human serum albumin (HSA) was investigated by fluorescence, Ultraviolet–visible (UV–Vis) absorption, Fourier transform infrared (FT-IR) spectroscopy and molecular docking methods. Fluorescence data revealed that the fluorescence quenching of HSA by CAP was the result of the formation of CAP–HSA complex, and the binding constant was determined to be 3.196?×?10⁴ L mol^?1 at 310 K. The thermodynamic determination indicated that the interaction was driven by enthalpy change and entropy change together, where the multiple hydrogen bonds (CAP and the residues Arg 222 and His 242 of HSA) and van der Waals forces were the dominant binding force. The site marker competition revealed that CAP bound into sub-domain IIA of HSA. The binding of CAP induced the drastic reduction in α-helix conformation and the significant enhancement in β-sheet conformation of HSA. Molecular docking study further confirmed the binding mode obtained by experimental study. This work provides a new quantitative evaluation method for antibiotics to cause the protein damage.     

Methyl-triclosan binding to human serum albumin: Multi-spectroscopic study and visualized molecular simulation

Methyl-triclosan (MTCS), a transformation product and metabolite of triclosan, has been widely spread in environment through the daily use of triclosan which is a commonly used anti-bacterial and anti-fungal substance in consumer products. Once entering human body, MTCS could affect the conformation of human serum albumin (HSA) by forming MTCS–HSA complex and alter function of protein and endocrine in human body. To evaluate the potential toxicity of MTCS, the binding mechanism of HSA with MTCS was investigated by UV–vis absorption, circular dichroism and Fourier transform infrared spectroscopy. Binding constants, thermodynamic parameters, the binding forces and the specific binding site were studied in detail. Binding constant at room tempreture (T = 298 K) is 6.32 × 10³ L mol⁻¹; ΔH⁰, ΔS⁰ and ΔG⁰ were 22.48 kJ mol⁻¹, 148.16 J mol⁻¹ K⁻¹ and −21.68 kJ mol⁻¹, respectively. The results showed that the interactions between MTCS and HSA are mainly hydrophobic forces. The effects of MTCS on HSA conformation were also discussed. The binding distance (r = 1.2 nm) for MTCS–HSA system was calculated by the efficiency of fluorescence resonance energy transfer. The visualized binding details were also exhibited by molecular modeling method and the results could agree well with that from the experimental study.     

Investigation on the interaction between triclosan and bovine serum albumin by spectroscopic methods

Abstract

Multi-spectroscopic and molecular docking methods were used to study the interaction between triclosan (TCS) and bovine serum albumin (BSA). The results indicated that the fluorescence quenching of BSA by TCS was due to the formation of TCS–BSA complex through static quenching. This result was also demonstrated by time-resolved fluorescence experiment. The binding constants and number of binding sites between TCS and BSA were 1.30?×?10⁵ M^?1 and 1.17 at 298?K, respectively. The thermodynamic parameters were studied in detail which suggested that hydrophobic forces and hydrogen bond played major roles in the TCS–BSA interaction. Moreover, the site marker competitive experiments and docking studies revealed that TCS could bind BSA into site I in subdomain IIA. All the results of UV–vis spectrophotometry, circular dichroism spectroscopy and synchronous fluorescence spectroscopy showed that interaction between TCS and BSA induced conformation changes of BSA.     

New insights into the toxic interactions of polyvinyl chloride microplastics with bovine serum albumin

Environmental Science and Pollution Research - The binding interaction between emerging pollutant polyvinyl chloride microplastics (PVC MPs) and bovine serum albumin (BSA) was studied by...     

Binding of aluminum to human serum transferrin,human serum albumin and rat serum proteins

Abstract

Human serum transferrin (HSTF), human serum albumin (HSA) and rat serum were compared for their interaction with AlCl₃ , in a Tris‐HCl buffer solutions (pH 7.4). The AlCl₃ was tested in series of concentrations in the range of 50 μM up to 500 μM . HSTF, HSA and their 1:1 mixture and rat serum were incubated at 37°C with series of AlCl₃ concentrations. The protein profile of the incubated solutions were compared to control using SDS‐PAGE and FPLC tests. The results indicated that HSTF was more specifically responsive to AlCl₃showing a characteristic increase in its UV absorption, peak and area dimensions. Simultaneously, HSA was less affected, but it showed a significant shift with an increase in molecular weight accompanied with a change in its profile. The respective bands of transferrin and albumin in rat serum behaved similarly. The SDS‐PAGE and FPLC data coincided and confirmed the preferential affinity of HSTF to bind with Al³⁺ . These results support the suggestion of using HSTF for monitoring levels of Al³⁺ in human blood samples of exposed population. The importance of further developing such a biomarker is the increased demand for early detection of the hazardous levels of Al³⁺ in relation to its long term neurotoxic adverse effects.     

Investigation of the binding and simultaneous quantifications of propanil and bromoxynil herbicide concentrations in human serum albumin

This study reported the use of UV–visible and fluorescence spectroscopy and partial-least-square (PLS) multivariate regression for accurate and simultaneous quantifications of two widely used herbicides, propanil, 3′,4′-dichloropropionanilide (PPL) and bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile (BXL) in human serum albumin (HSA) at physiological conditions. The binding affinity and thermodynamic properties of PPL-HSA and BXL-HSA complexes were also investigated. Partial-least-square (PLS) regression was used to collate the variability in the absorption or emission spectra of PPL-HSA and BXL-HSA complexes with PPL and/or BXL concentrations in HSA samples. The binding constants of 7.66× 10⁸ M^?1 for PPL-HSA and 4.88× 10⁶ M^?1 for BXL-HSA complexes were calculated at physiological conditions (temperature, 310 K; pH 7.4). Thermodynamic parameter values: enthalpy (ΔH) (13.99 kJ mol^?1), entropy (ΔS) (0.078 kJ mol^?1 K^?1), and Gibbs free energy (ΔG) (?10.19 kJ mol^?1) were determined for PPL-HSA complexation at physiological conditions. However, differences in thermodynamic property values of: ΔH (?214.3 kJ mol^?1), ΔS (?0.563 kJ mol^?1 K^?1), and ΔG (?39.70 kJ mol^?1) were observed for BXL–HSA complexes. The binding constants and negative ΔG values indicated strong binding affinity and thermodynamically favorability of PPL–HSA and BXL–HSA complex formation. Results of the PLS regression calibration showed good linearity (R² ≥ 0.998289), high sensitivity, and impressive low limit-of-detections (LODs) of 1.38× 10^?8 M for PPL and 1.68× 10^?8 M for BXL that are comparable and/or lower than many previously reported LODs for herbicide and pesticide analyses. Most importantly, PLS regression is capable of simultaneous quantifications of PPL and BXL concentrations in HSA samples with good accuracy and low errors of 3.66%. UV–visible spectrophotometers and spectrofluorometers are fairly inexpensive, easy to use, and are readily available in almost every laboratory, making this protocol excellent and affordable for routine analysis of weed/pest control chemical residues in humans. The results of this study are significant and remarkable that will provide critical insight into the binding mechanism of herbicide toxicity in humans and non-target organisms, which are of special interest in the area of biomedical study, environmental risk assessment, and ecotoxicology.     

Binding of triclosan to human serum albumin: insight into the molecular toxicity of emerging contaminant

Purpose

The interaction between triclosan (TCS) and human serum albumin (HSA) was investigated in order to obtain the binding mechanism, binding constant, the type of binding force, the binding distance between the donor and acceptor, and the effect of TCS on the conformation change of HSA.

Methods

A HSA solution was added to the quartz cell and then titrated by successive addition of TCS. The fluorescence quenching spectra and synchronous spectra were recorded with the excitation and emission slits of the passage of band set at 10 and 20 nm. Three-dimensional fluorescence spectra of HSA were recorded before and after the addition of TCS. The capillary electrophoresis was conducted with the pressure injection mode at 0.5 psi for 5 s, separation under 25 kV, and detection at 214 nm.

Results

Fluorescence data indicated the fluorescence quenching of HSA by TCS was static quenching, and the quenching constants (K _a ) were 1.14?×?10⁵, 8.75?×?10⁴, 6.67?×?10⁴, and 5.00?×?10⁴ at 293, 298, 303, and 309 K, respectively. The thermodynamic parameters, enthalpy change (??H) and entropy change (??S) for the interaction were calculated to be ?37.9 kJ mol^?1 and 32.6 J?mol^?1 K^?1. The binding distance between TCS and tryptophan residues of HSA was obtained to be 1.81 nm according to F??rster nonradioactive energy transfer theory. The UV-Vis absorption spectroscopy, the synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, and circular dichroism spectroscopy revealed the alterations of HSA secondary structure in the presence of TCS. Finally, the interaction between TCS and HSA was further confirmed by capillary electrophoresis.

Conclusions

TCS was bound to HSA to form the TCS-HSA complex, with the binding distance of 1.81 nm. Hydrophobic interaction and hydrogen bond were dominated in the binding. TCS could change the secondary conformation of HSA. This work provides an insight into noncovalent interaction between emerging pollutants and protein, helping to elucidate the toxic mechanism of such pollutants.     

Physiological mechanism of plant roots exposed to cadmium

Physiological experiments on plant roots exposed to cadmium were conducted on carrot and radish using a liquid culture and a pot experiment with a series of cadmium applications. Activities of four enzymes (catalase, peroxidase, polyphenol oxidase, superoxide dismutase), and concentrations of free proline and malonaldehyde in the roots of both plants were investigated. Results showed that the germination rate and growth of roots of both plants were inhibited at the concentration of 20 mg Cd/l, and the inhibition was increased with the increasing concentrations of cadmium, both in the liquid culture and in the pot experiment; activities of the four enzymes declined similarly in both species. The concentration of proline in roots reached the maximum when the application of cadmium was at the level of 20 mg/l in the liquid culture (or 20 mg/kg in soil), and then it declined slowly with the increasing concentration of cadmium. However, the reverse trend was observed for the concentration of malonaldehyde. All of bio-indicators measured here was quite sensitive to the addition of cadmium.     

Thermodynamics of metal cation binding by a solid soil-derived humic acid: binding of Fe(III), Pb(II), and Cu(II)

Metal binding and release by solid humic acids (HAs) in soils and sediments can affect metal mobility and bioavailability. Isotherms for tight binding of Fe(III), Pb(II) and Cu(II) by a solid humic acid at pH2.0 fit the Langmuir binding model. Low pH was chosen to protonate the HA carboxylate groups and avoid metal cation hydrolysis. Binding of Fe(III), Pb(II) and Cu(II) occurs in one detectable step labeled A. Site capacities nu(A) are temperature-independent from 10.0 to 40.0 degrees C and point to binding by charge-neutralization to form solid complexes M(OOC-R)(n)(s), where n appears to be 2 for Pb(II) and 3 for Fe(III). Thermodynamic data pairs (DeltaH(A), DeltaS(A)) for metal binding are linearly correlated with previous data for Ca(II), Co(II) and Mg(II) binding by solid HAs.     

The response of Lemna trisulca L. to cadmium

Lemna trisulca was grown, using aseptic culture techniques in a filter-sterilized medium, a portion of which was replaced regularly during experiments. L. trisulca responded to the addition of 0.64 microM Cd with a reduction in multiplication rate (MR) 2 +/- 1 days after exposure. The internal Cd content reached 1000 +/- 140 microg Cd/g (dry wt) within 2 days exposure to 0.64 microM Cd. The final yield was reduced by an average of c.8% for each day of exposure to 0.64 microM Cd in a 14 day experiment. This implies that an equilibration period should be used for short-term bioassay tests before the effect of a toxicant is determined. Pretreating L. trisulca with 0.08 or 0.32 microM Cd for 6 weeks had no significant effect on MR or Cd uptake when plants were subsequently exposed to a range of Cd concentrations or grown in a control medium. This suggests that L. trisulca does not become acclimated to elevated Cd concentrations. The MR of L. trisulca fluctuated over a period of almost 600 days and the doubling time ranged from 1.6 to 2.4 days. This produced more than a fivefold difference in final yield in experiments of 14 days duration. The reduction in MR in response to 0.32 microM Cd during this same 600 days period averaged 24% with a coefficient of variation of 38%, and varied with the MR of control cultures. Fluctuations in the intrinsic growth rate and the effect of a toxicant on L. trisulca could potentially confound the assessment of toxicity and must be carefully considered when designing test protocols for aquatic plants.     

Phosphorus and cadmium interactions in Kandelia obovata (S. L.) in relation to cadmium tolerance

This study focused on the cadmium (Cd) tolerance of mangroves with application of phosphate (P) in order to explore whether exogenous P can alleviate Cd stress on these intertidal species. Kandelia obovata (S. L.) seedlings were cultivated in rhizoboxes under different levels of Cd and P concentrations. The speciation distributions of Cd in the rhizosphere and non-rhizosphere sediments were examined by sequential extraction procedures; organic acid in plant tissues and soil solution was measured by high-performance liquid chromatography; Cd and P accumulation in the plants was also determined. Results showed that considerable differences existed in Cd speciation distributions between rhizosphere and non-rhizosphere sediments. Root activity influenced the dynamics of Cd, P application increased the organic acid content in root tissues, P also increased Cd accumulation in roots whilst lowering Cd translocation from root to the above-ground tissues, and a significant positive correlation was found between Cd and P in roots (r?=?0.905). It is postulated that Cd detoxification of K. obovata (S. L.) is associated with higher Cd immobilization in the presence of higher P and organic acid contents in root tissue.     

Potential contribution of arbuscular mycorrhiza to cadmium immobilisation in soil

The contribution of arbuscular mycorrhiza (AM) to immobilisation of Cd in substrate was studied in two experiments. In the first experiment, substrates prepared by cultivating tobacco, either non-mycorrhizal or inoculated with the AM fungus Glomus intraradices were enriched with a range of Cd concentrations, and Cd toxicity in the substrates was assessed using root growth tests with lettuce as a test plant. The tests revealed lower Cd toxicity in the mycorrhizal than in the non-mycorrhizal substrate, and the difference increased with increasing total Cd concentration in the substrates. In the second experiment, extraradical mycelium (ERM) of G. intraradices exposed in vivo to Cd was collected and analysed on Cd concentration. The ERM accumulated 10–20 times more Cd per unit of biomass than tobacco roots. While Cd concentrations were lower in the biomass of mycorrhizal plants than of non-mycorrhizal plants, Cd immobilisation by ERM did not affect the total Cd content in mycorrhizal tobacco.

It is concluded that mycorrhiza may decrease Cd toxicity to plants by enhancing Cd immobilisation in soil. The results therefore suggest a potential role of AM symbiosis in the phytostabilisation of contaminated soils, where high Cd availability inhibits plant growth.     

Effect of chronic exposure to cadmium on hepatic drug metabolism

In an attempt to examine the chronic effect of low levels of cadmium on hepatic drug-metabolizing enzyme system, an experiment was carried out in which growing male rats were given 0, 5, 10, and 20 ppm of cadmium in drinking water for a period of 8 weeks. An ip administration of a hypnotic dose of pentobarbital to the cadmium-treated and the control rats 24 hr following the termination of the experiment exhibited that there was no significant difference in the drug metabolism in control and any of the treated groups. Next, liver microsomes were isolated from animals in all groups to study their ability to metabolize drugs in vitro. The results indicated that the activity of benzphetamine N-demethylase and aniline hydroxylase, and the concentration of microsomal cytochrome P-450 were almost identical in the control and treated groups. On the other hand, a single ip dose of cadmium (2 mg/kg) caused significant decrease in the vivo and in vitro microsomal drug metabolism. These results suggest that although a single ip dose of cadmium (2 mg/kg) causes significant inhibition of drug metabolism, chronic exposure to cadmium up to 20 ppm in drinking water over a period of 8 weeks is unlikely to affect hepatic drug metabolism.     

Combined effects of cadmium and composted manure to aquatic organisms

To evaluate the interactive toxicity of cadmium (Cd) and composted manure to aquatic organisms 96 h static bioassays were conducted in the laboratory with fry of common carp (Cyprinus carpio), copepod (Diaptomusforbesi) and oligochaete worm (Branchiura sowerbyi). Five concentrations of composted manure (0, 0.25, 0.5, 1.0 and 6.7 g/l) were prepared from the aquatic weed, Pistia stratiotes and each of them was combined with several concentrations of Cd to determine 96 h LC-50 values of Cd for the test organisms. Addition of composted manure, irrespective of concentration, significantly reduced the LC-50 value of Cd to the copepod and common carp fry while it increased the LC-50 value of Cd to the worm. Increased susceptibility of the worm to combined treatment of composted manure and small concentrations of Cd could be revealed only from the dose mortality curve. Results of acute toxicity bioassays were different from the results of bioassays conducted with small concentrations of Cd. Worms, exposed to 2.5 mg/l Cd, accumulated more Cd than did the carp fry and copepod. Accumulation of Cd by worms was increased by the addition of 6.7 g/l composted manure while it decreased in the carp fry and copepod. Food consumption rate of common carp fingerling was significantly reduced relative to the control by exposure to 2.5 mg/l Cd. No change in feeding rate was observed when Cd was combined with composted manure (6.7 g/l).     

Effect of chronic exposure to cadmium on hepatic drug metabolism

Abstract

In an attempt to examine the chronic effect of low levels of cadmium on hepatic drug‐metabolizing enzyme system, an experiment was carried out in which growing male rats were given 0, 5, 10, and 2 0 ppm of cadmium in drinking water for a period of 8 weeks. An ip administration of a hypnotic dose of pentobarbital to the cadmium‐treated and the control rats 2 4 hr following the termination of the experiment exhibited that there was no significant difference in the drug metabolism in control and any of the treated groups. Next, liver microsomes were isolated from animals in all groups to study their ability to metabolize drugs in vitro. The results indicated that the activity of benzphetamine N‐demethylase and aniline hydroxylase, and the concentration of microsomal cytochrome P‐450 were almost identical in the control and treated groups. On the other hand, a single ip dose of cadmium (2 mg/kg) caused significant decrease in the in vivo and in vitro microsomal drug metabolism. These results suggest that although a single ip dose of cadmium (2 mg/kg) causes significant inhibition of drug metabolism, chronic exposure to cadmium up to 2 0 ppm in drinking water over a period of 8 weeks is unlikely to affect hepatic drug metabolism.     

Feasibility of phytoextraction to remediate cadmium and zinc contaminated soils

A Cd and Zn contaminated soil was mixed and equilibrated with an uncontaminated, but otherwise similar soil to establish a gradient in soil contamination levels. Growth of Thlaspi caerulescens (Ganges ecotype) significantly decreased the metal concentrations in soil solution. Plant uptake of Cd and Zn exceeded the decrease of the soluble metal concentrations by several orders of magnitude. Hence, desorption of metals must have occurred to maintain the soil solution concentrations. A coupled regression model was developed to describe the transfer of metals from soil to solution and plant shoots. This model was applied to estimate the phytoextraction duration required to decrease the soil Cd concentration from 10 to 0.5 mg kg⁻¹. A biomass production of 1 and 5 t dm ha⁻¹ yr⁻¹ yields a duration of 42 and 11 yr, respectively. Successful phytoextraction operations based on T. caerulescens require an increased biomass production.     

Feasibility of phosphate fertilizer to immobilize cadmium in a field

To reduce effectively cadmium (Cd) phytoextractability by phosphate fertilizer in Cd contaminated soil, fused and superphosphate (FSP) was applied at the rate of 0, 33.5 (recommendation level), 167.5, and 335 kg P ha⁻¹ for radish (Raphanus sativa L.). Unlike from what we expected, soil Cd extractability and Cd concentration in radish increased with increasing FSP application in the field. To determine the effect of FSP on Cd immobilization, FSP was mixed with the selected soil at the rate of 0, 200, 400, 800, and 1600 mg P kg⁻¹ and then incubated for 8 weeks. As observed in the field study, NH₄OAc extractable Cd concentration increased slightly with FSP addition up to 400 mg P kg⁻¹ and thereafter dramatically decreased upon increasing its application rate. Soil pH and negative charge were decreased at low level of FSP application up to 400 mg P kg⁻¹, but thereafter continually increased with increasing application level. This could be indirect evidence that net soil negative charge was increased by the specific adsorption of phosphate at the high rate of FSP application over 400 mg P kg⁻¹. The labile Cd fraction (water soluble and exchangeable + acidic fraction) increased with increasing FSP application by 400 mg P kg⁻¹ and thereafter gradually decreased with corresponding increase in unlabile fraction (oxidizable and residual fraction). Based on these results, FSP might be applied with a very high rate over 800 mg P kg⁻¹ to decrease Cd extractability in the selected field. However, this level is equivalent to 1440 kg P ha⁻¹, which is about 43 times higher than the recommendation levels for radish production and resulted in a significant increase in water soluble P concentration creating a new environmental problem. Therefore, the feasibility of FSP to reduce Cd extractability in the field is very low.     

Antioxidant metabolism of coffee cell suspension cultures in response to cadmium

The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to cadmium (Cd) were investigated. Cd accumulated very rapidly in the cells and this accumulation was directly correlated with an increase in applied CdCl(2) concentration in the external medium. At 0.05mM CdCl(2), growth was stimulated, but at 0.5mM CdCl(2), the growth rate was reduced. An alteration in activated oxygen metabolism was detected by visual analysis as well as by an increase in lipid peroxidation at the higher CdCl(2) concentration. Catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) activity increased, particularly at the higher concentration of CdCl(2). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was increased at the lower CdCl(2) concentration used, but could not be detected in cells growing in the higher CdCl(2) concentration after 24h of growth, whilst guaiacol peroxidase (GOPX; EC 1.11.1.7) did not show a clear response to Cd treatment. An analysis by non-denaturing PAGE followed by staining for enzyme activity, revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differently affected by CdCl(2) treatment and one GR isoenzyme was shown to specifically respond to CdCl(2). The results suggest that the higher concentrations of CdCl(2) may lead to oxidative stress. The main response appears to be via the induction of SOD and CAT activities for the removal of reactive oxygen species (ROS), and by the induction of GR to ensure the availability of reduced glutathione for the synthesis of Cd-binding peptides, which may also be related to the inhibition of APX activity probably due to glutathione and ascorbate depletion.