Binding of Tetrabromobisphenol A and S to Human Serum Albumin Is Weakened by Coexisting Nanoplastics and Environmental Kosmotropes.

Human serum albumin (HSA) was used as a model protein to explore the effects of brominated flame retardant (BFR) binding and the corona formation on polystyrene nanoplastics (PNs). Under physiological conditions, HSA helped to disperse PNs but promoted the formation of aggregates in the presence of tetrabromobisphenol A (TBBPA, ΔDh = 135 nm) and S (TBBPS, ΔDh = 256 nm) at pH 7. At pH 4, these aggregates became larger with fewer electrostatic repulsion effects (ΔDh = 920 and 691 nm for TBBPA and TBBPS, respectively). However, such promotion effects as well as BFR binding are different due to structural differences of tetrabromobisphenol A and S. Environmental kosmotropes efficiently stabilized the structure of HSA and inhibited BFR binding, while the chaotropes favored bioconjugated aggregate formation. Such effects were also verified in natural seawater. The newly gained knowledge may help us anticipate the behavior and fate of plastic particles and small molecular pollutants in both physiological and natural aqueous systems.

Aged microplastics enhance their interaction with ciprofloxacin and joint toxicity on Escherichia coli.

Microplastics (MPs) in natural environments undergo complex aging processes, changing their interactions with coexisting antibiotics, and posing unpredictable ecological risks. However, the joint toxicity of aged MPs (aMPs) and antibiotics to bacteria, especially at the molecular level, is unclear. In this study, non-thermal plasma technology was used to simultaneously simulate various radical oxidation and physical reactions that occur naturally in the environment, breaking the limitation of simple aging process in laboratory aging technologies. After aging, we investigated the altered properties of aMPs, their interactions with ciprofloxacin (CIP), and the molecular responses of E. coli exposed to pristine MPs (13.5 mg/L), aMPs (13.5 mg/L), and CIP (2 µg/L) individually or simultaneously. aMPs bound far more CIP to their surfaces than pristine MPs, especially in freshwater ecosystems. Notably, the growth of E. coli exposed to aMPs alone was inhibited, whereas pristine MPs exposure didn't affect the growth of E. coli. Moreover, the most differentially expressed genes in E. coli were induced by the coexposure of aMPs and CIP. Although E. coli depended on chemotaxis to improve its flagellar rotation and escaped the stress of pollutants, the coexposure of aMPs and CIP still caused cell membrane damage, oxidative stress, obstruction of DNA replication, and osmotic imbalance in E. coli. This study filled the knowledge gap between the toxicity of aMPs and pristine MPs coexisting with antibiotics at the transcription level, helping in the accurate assessment of the potential risks of MPs to the environment.

Probing the potential toxicity by characterizing the binding mechanism of sodium dehydroacetate to human serum albumin.

BACKGROUND: Sodium dehydroacetate (DHA-S) is a common food additive, which can combine with serum proteins in the plasma, but the interaction mechanism between DHA-S and human serum albumin (HSA) is unclear. In this study, multiple spectroscopy techniques, isothermal titration calorimetry (ITC), molecular docking and esterase activity test were employed to investigate the interaction mechanism of DHA-S and HSA. RESULTS: A DHA-S-HSA complex was formed and the structure of HSA were altered by DHA-S. Since DHA-S changed the tight structure of the hydrophobic subdomain IIA where tryptophan (Trp) was placed, the hydrophobicity of the microenvironment of HSA was enhanced. With the addition of DHA-S, the skeleton structure of HSA became loose and the solvent shell on the HSA surface was destroyed. DHA-S altered the secondary structure of HSA, resulting in the decreased α-helix and increased ß-sheet contents. The interaction was exothermic and spontaneous driven by van der Waals and hydrogen bonding. DHA-S inhibited the esterase activity of HSA. Molecular docking demonstrated that the binding site of DHA-S on HSA located at the cavity of subdomains IIA and IIIA, but the amino acids related to esterase activity of HSA were not in the binding pocket, indicating that the mechanism by which DHA-S inhibited HSA esterase activity was the change in protein structure. CONCLUSION: This study illustrated that DHA-S interacted with HSA and the structure and function of HSA were affected by DHA-S. This research could help to understand the toxicity of DHA-S and provide basic data for safe use of food additives. © 2021 Society of Chemical Industry.

Binding mechanism of maltol with catalase investigated by spectroscopy, molecular docking, and enzyme activity assay.

Maltol is a flavor additive that is widely used in the daily diet of humans, and its biosafety attention is concomitantly increasing. Catalase (CAT) is an antioxidant enzyme to maintain homeostasis in the tissue's environment of human body and protect cells from oxidative damages. The adverse effects of maltol to CAT activity within mouse hepatocytes as well as the structural and functional changes of CAT on molecular level were investigated by multiple spectroscopy techniques, enzyme activity experiments, and molecular docking. Results suggested that when the maltol concentrations reached to 8 × 10-5 mol L-1 , the viability of hepatocytes decreased to 93%, and CAT activity was stimulated by maltol to 111% than the control group after exposure for 24 hours. Changes in CAT activity on molecular level were consistent with those on cellular level. The fluorescence quenching of CAT by maltol was static with the forming of maltol-CAT complex. Moreover, ultraviolet-visible (UV-visible) absorption, synchronous fluorescence, and circular dichroism (CD) spectra reflected that the presence of maltol caused conformational change of CAT and made the CAT molecule skeleton loose and increased α-helix of CAT. Maltol mainly bound with CAT through hydrogen bond, and binding site that is near the heme ring in the enzyme activity center did not interact with its main amino acid residues. This study explores the combination between maltol and CAT, providing references for evaluating health damages caused by maltol.

In vitro toxicity and molecular interacting mechanisms of chloroacetic acid to catalase.

Chloroacetic acid (CAA), one of typical disinfection by-products (DBPs), has attracted considerable concerns for its biological safety. Antioxidant enzyme catalase (CAT) plays a crucial part in the regulation of redox state balance. Herein, CAA was used to test its adverse effects on CAT and explore the underlying mechanism. The cell viability of mouse primary hepatocytes decreased under CAA exposure. A bell-shaped response to CAA exposure was observed in intracellular CAT activity, whose change was partly influenced by molecular CAT activity. CAA binds to CAT mainly via van der Waals forces and hydrogen bonds with a stoichiometry of 9.2. The binding caused structural changes in CAT with the unfolding of polypeptide chains and the decrease of α-helical content. CAA interacts with the amino acid residues surrounding the active sites and substrate channel of CAT. These interactions result in the decrease of molecular CAT activity, which could be restored by high ionic strength. This study has provided a combined molecular and cellular tactics for studying the adverse effects of DBPs on biomarkers and the underlying mechanisms.

Characterization on the toxic mechanism of two fluoroquinolones to trypsin by spectroscopic and computational methods.

Ciprofloxacin (CPFX) and enrofloxacin (ENFX), two of the most widely used fluoroquinolones (FQs), pose a great threat to humans and the ecosystem. In this study, the toxic mechanisms between the two FQs and trypsin were evaluated by means of multiple spectroscopic methods, as well as molecular docking. During the fluorescence investigations, both FQs quenched the intrinsic fluorescence of trypsin effectively, which was due to the formation of moderately strong complexes (mainly through van der Waals forces and hydrogen bonds). The binding of two FQs not only caused the conformational and micro-environmental changes of trypsin, but also changed its molecular activity; shown by the UV-Visible absorption spectroscopy, synchronous fluorescence spectroscopy, and functional tests. The established methods in this work can help to comprehensively understand the transport of FQs in the human body.

Interactions of three bisphenol analogues with hemoglobin investigated by spectroscopy and molecular docking.

Bisphenol F (BPF), bisphenol S (BPS), and bisphenol B (BPB) have been extensively used in food packaging, plasticizer, and paper products, causing more concern about their biosafety. The mechanism of these bisphenols' toxicity was investigated by determining diverse effects of them on common protein bovine hemoglobin (BHb). The effects at the molecular level were determined by ultraviolet-visible, circular dichroism, resonance light scattering, fluorescence spectroscopy, and molecular docking. The irreversible cross-linking results of bisphenols and BHb demonstrate that hydrogen (H) bonding and hydrophobic forces play major roles in the interaction. Both BPF and BPS decreased the amount of α-helix, leading to the loosening of protein skeleton while BPB made little change. In the loose structure, BPF exposed the internal amino acids to a hydrophobic environment and BPS (above 10µM) obviously quenched characteristic fluorescence. The variant effects of BPF, BPS, and BPB may arise from different structural formula. Accordingly, BPB could be used as a better substitute for bisphenol A, and it is necessary to control the concentration of BPS and BPF below 10µM in application. This study provided important basis for application of BPB and safe use of bisphenol analogues in industry.

A study on the interaction between cadmium and α-chymotrypsin and the underlying mechanisms.

Because cadmium might interact with proteins and, thus, exert toxicity in organisms, it is vital to understand the molecular mechanism of the interaction between cadmium and biologically relevant proteins as well as the structural and functional changes in these proteins. In this study, the interaction between α-chymotrypsin (α-ChT) and cadmium chloride (CdCl2 ) was investigated by performing enzyme activity determinations, multispectroscopic measurements, isothermal titration calorimetry, and molecular docking studies. It was demonstrated that CdCl 2 binds to α-ChT mainly via electrostatic forces with (21.0 ± 0.982) binding sites, leading to the increase of α-helix and the decrease of ß-sheet. The interaction between CdCl 2 and α-ChT loosened the protein skeleton and increased the molecular volume of α-ChT. CdCl 2 first binds to the interface of α-ChT and then interacts with the key residues His 57 or Asp 102 or both in the active sites, leading to the activity inhibition of α-ChT under the exposure of high CdCl 2 concentrations.

Characterizing the noncovalent binding behavior of tartrazine to lysozyme: A combined spectroscopic and computational analysis.

Tartrazine is a stable water-soluble azo dye widely used as a food additive, which could pose potential threats to humans and the environment. In this paper, we evaluated the response mechanism between tartrazine and lysozyme under simulated conditions by means of biophysical methods, including multiple spectroscopic techniques, isothermal titration calorimetry (ITC), and molecular docking studies. From the multispectroscopic analysis, we found that tartrazine could effectively quench the intrinsic fluorescence of lysozyme to form a complex and lead to the conformational and microenvironmental changes of the enzyme. The ITC measurements suggested that the electrostatic forces played a major role in the binding of tartrazine to lysozyme with two binding sites. Finally, the molecular docking indicated that tartrazine had specific interactions with the residues of Trp108. The study provides an important insight within the binding mechanism of tartrazine to lysozyme in vitro.

Perfluorodecanoic acid-induced oxidative stress and DNA damage investigated at the cellular and molecular levels.

Perfluorodecanoic acid (PFDA) has been widely used in production of many daily necessities because of its special nature. Althoughtoxic effects of PFDA to organisms have been reported, there is little research on the genotoxicity induced by oxidative stress of PFDA on the cellular and molecular levels simultaneously. Thus, we investigated the DNA oxidative damage caused by PFDA in mouse hepatocytes. On the cellular level, an increase in ROS content indicated that PFDA caused oxidative stress in mouse hepatocytes. In addition, after PFDA exposure, the comet assay confirmed DNA strand breaks and an increased 8-OHdG content demonstrated DNA oxidative damage. On the molecular level, the microenvironment of aromatic amino acids, skeleton and secondary structure of catalase (CAT) were varied after PFDA exposure and the enzyme activity was reduced because PFDA bound near the heme groups of CAT. Moreover, PFDA was shown to interact with DNA molecule by groove binding. This study suggests that PFDA can cause genotoxicity by inducing oxidative stress both on the cellular and molecular levels.

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.

Investigation on the interaction of catalase with sodium lauryl sulfonate and the underlying mechanisms.

As a classic type of anionic surfactants, sodium lauryl sulfonate (SLS) might change the structure and function of antioxidant enzyme catalase (CAT) through their direct interactions. However, the underlying molecular mechanism is still unknown. This study investigated the direct interaction of SLS with CAT molecule and the underlying mechanisms using multi-spectroscopic methods, isothermal titration calorimetry, and molecular docking studies. No obvious effects were observed on CAT structure and activity under low SLS concentration exposure. The particle size of CAT molecule decreased and CAT activity was slightly inhibited under high SLS concentration exposure. SLS prefers to bind to the interface of CAT mainly via van der Waals' forces and hydrogen bonds. Subsequently, SLS interacts with the amino acid residues around the heme groups of CAT via hydrophobic interactions and might inhibit CAT activity.

Molecular interaction of triclosan with superoxide dismutase (SOD) reveals a potentially toxic mechanism of the antimicrobial agent.

In this article, the interaction mechanism between the superoxide dismutase (SOD) and the triclosan (TCS), a kind of antimicrobial agent which is of widely application with potential effects both on environment and human health, was explored through a series of spectroscopic methods, animal experiment and the molecular docking simulation. The negative free energy change ∆G, enthalpy change (∆H = 162.21 kJmol-1) and entropy change (∆S = 615 Jmol-1K-1) demonstrated that TCS could combine with SOD spontaneously through hydrophobic interaction to form a complex. The binding constants of Ka293 and Ka313 were 1.706 × 103 and 1.2 × 105 Lmol-1, respectively. Furthermore, the interaction could also influence the skeleton structure and secondary contents of SOD. The molecular docking analysis revealed the TCS located between two subunits of SOD, and there was a hydrogen bond between TCS and the residue Asn51 of SOD, which influenced the structure of protein and resulted in a decrease of enzyme activity. This work could help understand the interaction mechanism between SOD and TCS. Moreover, it could also be used to consult for toxicity assessment of TCS at molecular level.

Comparison of the toxicity of the dyes Sudan II and Sudan IV to catalase.

The mechanisms of the toxicity of Sudan dyes to the key antioxidant enzyme catalase (CAT) were investigated by spectroscopic methods, calorimetry techniques, enzyme activity assay, and molecular docking. Results showed that Sudan dyes bound to CAT through hydrophobic force, which changed the microenvironment of tryptophan and tyrosine residues, leading to a conformational alteration and shrinkage of the protein. Enzyme activity assay and molecular docking revealed that the activity of CAT was slightly inhibited in the presence of Sudan dyes. In comparison, the binding of Sudan II with CAT was slightly stronger than Sudan IV. Also, Sudan II and Sudan IV showed a different impact on the microenvironment of aromatic amino acid residues. But the dyes had very similar effects on conformation and activity of the protein. This work provides an essential reference for the evaluation of Sudan dyes' effects on body's antioxidant defense system and safe use of Sudan dyes.

Molecular mechanism investigation of the neutralization of cadmium toxicity by transferrin.

Cadmium adversely affects the biological function of the liver. Transferrin might be involved in the detoxification system of cadmium. However, owing to the lack of investigation of the molecular mechanism of cadmium conjugating to transferrin, the role of transferrin in cadmium detoxification in the liver and how transferrin undergoes conformational and functional changes upon cadmium binding are not clear. In this article, we demonstrated the potential role of transferrin in the protection of the mouse primary hepatocytes against cadmium toxicity. After the incubation of hepatocytes with 10 and 100 µM CdCl2, pretreatment with transferrin significantly attenuated the reduction of cell viability in comparison with the samples treated with CdCl2 alone. Furthermore, a detailed molecular mechanism investigation of the interaction of CdCl2 with transferrin was reported using biophysical methods. Multi-spectroscopic measurements showed that CdCl2 formed complexes with transferrin and caused structural and conformational changes of transferrin. Isothermal titration calorimetry measurements revealed that transferrin has two classes of binding sites with different binding constants for CdCl2 binding. Hydrophobic forces and electrostatic forces are the major driving forces of the interaction. Preferred specific binding sites on transferrin were identified by dialysis experiments, molecular docking studies and molecular dynamics simulations. Upon low CdCl2 concentration exposure, no content of iron was released from transferrin because CdCl2 preferentially binds to the surface of transferrin molecules. Upon higher CdCl2 concentration exposure, the release of iron content from transferrin was observed due to the interaction of CdCl2 with the key residues around iron binding sites.

Novel biomarker pipeline to probe the oxidation sites and oxidation degrees of hemoglobin in bovine erythrocytes exposed to oxidative stress.

Research on biomarkers for protein oxidation might give insight into the mechanistic mode of oxidative stress. In the work present here, a novel pipeline was established to probe the oxidation mechanism of bovine hemoglobin (Hb) with its oxidation products serving as the biomarkers. Reactive oxygen species generated by irradiation were used to mimic oxidative stress conditions to oxidize Hb in bovine erythrocytes. After Hb extraction and digestion, oxidized peptides in the tryptic fragments were assigned by comparison with the extracted ion chromatography spectra of native peptide from the control sample. Subsequent tandem mass spectrometry analysis of these peptides proved that oxidation was limited to partially exposed amino acid residues (α-Phe36 , ß-Met1 , ß-Trp14 , for instance) in Hb. Quantitation analysis on these oxidized peptides showed that oxidation degrees of target sites had positive correlations with the extended oxidation dose and the oxidation processes were also controlled by residues types. Compared with the conventional protein carbonyl assay, the identified oxidized products were feasibility biomarkers for Hb oxidation, indicating that the proposed biomarker pipeline was suitable to provide specific and valid information for protein oxidation. Copyright © 2015 John Wiley & Sons, Ltd.

Characterization of the binding of 2-mercaptobenzimidazole to bovine serum albumin.

2-Mercaptobenzimidazole (MBI) is widely utilized as a corrosion inhibitor, copper-plating brightener and rubber accelerator. The residue of MBI in the environment is potentially harmful to human health. In this article, the interaction of MBI with bovine serum albumin (BSA) was explored using spectroscopic and molecular docking methods under physiological conditions. The positively charged MBI can spontaneously bind with the negatively charged BSA through electrostatic forces with one binding site. The site marker competition experiments and the molecular docking study revealed that MBI bound into site II (subdomain IIIA) of BSA, which further led to some secondary structure and microenvironmental changes of BSA. This work provides useful information on understanding the toxicological actions of MBI at the molecular level.

Non-negligible inhibition effect of microcystin-LR biodegradation products target to protein phosphatase 2A.

Though biodegradation is an important regulation pathway for microcystins (MCs) pollution, more consideration needs to be given to the potential risk associated with related biodegradation products (MC-BDPs). In this work, typical MCLR-BDPs were prepared and their toxicity was evaluated by protein phosphatases (PPs) inhibition assay. Results showed the initial ring opening of MCLR played a crucial role in detoxification. However, partial MCLR-BDPs still retained the critical structures and thus exhibited certain toxicity (2.8-43.5% of MCLR). With the aid of molecular simulation, the mechanism for the potential toxicity of BDPs targeting PP2A was elucidated. The initial ring opening made the loss of hydrogen bond Leu2←Arg89, and pi-H bond Adda5-His191, which was responsible for the significant reduction in the toxicity of MCLR-BDP. However, the key hydrogen bonds MeAsp3←Arg89, Glu6←Arg89, Adda5←Asn117, Adda5←His118, Arg4→Pro213, Arg4←Arg214, Ala1←Arg268, and Mdha7←Arg268, metal bond Glu6-Mn12+, and ionic bonds Glu6-Arg89, and Glu6-Mn22+ were preserved in varying degrees. Above preserved interactions maintained the interactions between PP2A and Mn2+ ions (reducing the exposure of Mn2+ ions). Above preserved interactions also hindered the combination of phosphate groups to Arg214 residual and thus exhibited potential toxicity.

Novel evidence on iodoacetic acid-induced immune protein functional and conformational changes: Focusing on cellular and molecular aspects.

Elevated levels of iodide occur in raw water in certain regions, where iodination disinfection byproducts are formed during chloramine-assisted disinfection of naturally iodide-containing water. Iodoacetic acid (IAA) is one of the typical harmful products. The mechanisms underlying IAA-induced immunotoxicity and its direct effects on biomolecules remained unclear in the past. Cellular, biochemical, and molecular methods were used to investigate the mechanism of IAA-induced immunotoxicity and its binding to lysozyme. In the presence of IAA, the cell viability of coelomocytes was significantly reduced to 70.8 %, as was the intracellular lysozyme activity. Upon binding to IAA, lysozyme underwent structural and conformational changes, causing elongation and unfolding of the protein due to loosening of the backbone and polypeptide chains. IAA effectively quenched the fluorescence of lysozyme and induced a reduction in particle sizes. Molecular docking revealed that the catalytic residue, Glu 35, which is crucial for lysozyme activity, resided within the docking range, suggesting the preferential binding of IAA to the active site of lysozyme. Moreover, electrostatic interaction emerged as the primary driving force behind the interaction between IAA and lysozyme. In conclusion, the structural and conformational changes induced by IAA in lysozyme resulted in impaired immune protein function in coelomocytes, leading to cellular dysfunction.

New mechanistic insights of nanoplastics synergistic cadmium induced overactivation of trypsin: Joint analysis from protein multi-level conformational changes and computational modeling.

Nanoplastics (NPs) are emerging global contaminants that can exacerbate the animal toxicity and cytotoxicity of cadmium (Cd). However, the mechanisms by which NPs influence the toxic effects of Cd on key functional proteins within the body remain unknown. In this study, trypsin, a protein that is prone to coexist with NPs in the digestive tract, was selected as the target protein. The effects and mechanisms of NPs on Cd2+-induced structural damage at multiple levels and alterations in the biological function of trypsin were investigated using multi-spectroscopy techniques, enzyme activity assays, and computational modeling. Results indicated that the Cd2+-induced decrease and red shift of the trypsin backbone peak were exacerbated by the presence of NPs, leading to more serve backbone loosening. Furthermore, compared to Cd2+, NPs@Cd2+ caused a more pronounced reduction in the α-helix content of trypsin. These structural changes led to the opening of the trypsin pocket and the overactivation of the enzyme (NPs@Cd2+: 227.22%; Cd2+: 53.35%). Ultimately, the formation of a "protein corona" around NPs@Cd2+ and the metal contact of Cd2+ to the trypsin surface were identified as the mechanisms by which NPs enhanced the protein toxicity of Cd2+. This study elucidates, for the first time, the effects and underlying mechanisms of NPs on the toxicity of key functional proteins of Cd2+. These findings offer novel mechanistic insights and critical evidence essential for evaluating the risks associated with NPs.