Conformation Of Bovine Serum Albumin Research Articles

Study of transport, tissue distribution, depletion, and hepatotoxicity of Cyadox, a quinoxaline-1,4-dioxide derivative.

Cyadox (CYA) is a derivative of quinoxaline 1,4-dioxide and a safe and effective synthetic antibacterial agent. This study aimed to explore the drug transport in blood, distribution, depletion and hepatotoxicity of drugs in animals. The transport of CYA in blood was studied using fluorescence, circular dichroism (CD) and molecular docking methods. Tissue distribution and depletion of CYA in rats were evaluated following oral administration of [3H]-CYA at different doses. Hepatotoxicity of drugs evaluated by transcriptomics. During transport in the bloodstream, the drug binds to bovine serum albumin (BSA) by hydrogen bonding and has only one binding site. Hydrogen bonds were formed between O (2) of CYA and ARG208, O (3) of CYA and LEU480, VAL481. The secondary protein conformation of BSA changed after binding with an increase in α-helix and a decrease in β-strand. After a single oral administration of [3H]-CYA, it was excreted rapidly within 7days, with 34.81% from the urine and 60.25% from the feces. Higher and sustained levels of radioactivity were detected in the liver during the post-dose period, suggesting that the drug may concentrate in the liver. The transcriptomic data indicates that CYA exhibits low hepatotoxicity. However, there are indications that it may have an impact on steroid biosynthesis. This study could serve as a basis for conducting further studies on the use of CYA in food animals and improving the pharmacologic, pharmacokinetic, and toxicologic effects of CYA on food animals.

Effect of the Protein Chain Conformation on the Collapse into Nanoparticles.

Protein single-chain nanoparticles can outperform synthetic nanoparticles in biomedical applications due to enhanced biocompatibility. Compared to synthetic (co)polymers, the chemical complexity of proteins challenges chain conformation control. Here, we investigate the impact of the precursor chain conformation of bovine serum albumin (BSA) on the nanoparticle structure after intramolecular cross-linking. We explore the urea concentration (denaturant), pH, salt, cross-linker length, and concentration. Small-angle neutron scattering and dynamic light scattering experiments reveal a shrinking chain conformation upon cross-linking. However, the ability to collapse depends on solvent conditions: more expanded chains collapse more, whereas proteins that are already compact barely change in size upon cross-linking. Static light scattering measurements demonstrate that binding is primarily intramolecular. The use of a shorter cross-linker does not lead to collapse of extended chains. Overall, BSA exhibits a similar behavior to that of polymer nanoparticles, which then allows to harness the precursor conformation for morphological control.

Interactions between deep eutectic solvents and bovine serum albumin studied by spectroscopic and electrochemical methods

Deep eutectic solvent (DES) has become a potential protein extractant as its good biocompatibility, environmental friendliness and accessibility. In this study, two types of DESs, ferric chloride hexahydrate: oxalic acid (FeCl3·6H2O: H2C2O4) and choline chloride: zinc chloride (ChCl: ZnCl2), were prepared. The interactions between these two types of DESs and bovine serum albumin (BSA) were investigated by utilizing spectroscopic and electrochemical methods. It was found that both FeCl3·6H2O: H2C2O4 and ChCl: ZnCl2 are able to form complexes with BSA by a relatively high affinity. During the process, no significant change in BSA conformation was observed. The binding ratios of BSA in the FeCl3·6H2O: H2C2O4 and ChCl: ZnCl2 systems are up to 98.59 % and 98.14 %, respectively, indicating that the two DESs are both promising extractants for BSA. The interactions between the DESs and BSA are mainly hydrogen bond and van der Waals force, which are strong enough to extract BSA but not too strong for regeneration, making the DESs more applicable in the industry. Moreover, it was found that the binding ratio of BSA in the FeCl3·6H2O: H2C2O4-BSA system remains above 91.7 % in the temperature range of 298–313 K, illustrating the potential of FeCl3·6H2O: H2C2O4 to be used in BSA extraction under a wider range of temperature. In contrast, ChCl: ZnCl2 will be better used under lower temperatures.

Interactions of three berberine mid-chain fatty acid salts with bovine serum albumin (BSA): Spectroscopic analysis and molecular docking

In this paper, the interaction of three berberine mid-chain fatty acid salts ([BBR][FAs]), viz. berberine caproate ([BBR][CAP]), berberine heptylate ([BBR][HEP]) and berberine octoate ([BBR][OCT]), with bovine serum albumin (BSA) was studied by means of UV–visible absorption spectroscopy, fluorescence spectroscopy, fourier transform infrared spectroscopy (FT-IR) and molecular docking techniques. Fluorescence experiments revealed that three berberine salts quench the fluorescence of BSA by static quenching mechanism resulted from a stable [BBR][FAs]-BSA complex formation. The stoichiometric numbers of [BBR][FAs]-BSA complexes were found to be 1:1. Synchronous and three-dimensional fluorescence spectra as well as FT-IR demonstrated that the binding of [BBR][FAs] altered the microenvironment and conformation of BSA. The binding average distance from [BBR][FAs] to BSA (3.2–3.5 nm) was determined according to Förster energy transfer theory. Site probe investigation showed that [BBR][FAs] bound to BSA active site I (sub-domain IIA). The binding promotes the esterase-like activity of BSA. The molecular docking results confirmed the fluorescence competition findings and provided the type of binding forces. Furthermore, the relationship between the anionic chain length of [BBR][FAs] and the interaction was explored, and the positive correlation was found.

Albumin-mediated molecular competition of supramolecular photosensitizers for NIR-II imaging-guided phototherapy

Heptamethine cyanines (Cy7) typically exhibit strong near-infrared (NIR) fluorescence signals when in monodisperse states, but their distinctive π-conjugated structure leads to limited solubility in aqueous solutions, ultimately resulting in inherent fluorescence self-quenching. Enhancing the fluorescence quantum yield of these agents within aqueous environments, in order to achieve high-resolution biological imaging, presents a significant challenge. Herein, a benzothiazole-based Cy7-derivative (Cy7-BTH) is developed, exhibiting unique H-aggregation properties in aqueous solutions. Bovine serum albumin (BSA) binds to Cy7-BTH (BSA@Cy7-BTH) to modulate its molecular stacking and energy transfer, boosting its fluorescence accompanied by bright NIR-II tail emission. The competitive binding alters the planarity of Cy7-BTH, restricting its intramolecular rotation, and simutaneously changes the conformation of BSA, leading to BSA@Cy7-BTH aggregation to form hierarchical-structured nanoparticles. Notably, the BSA@Cy7-BTH nanoparticles significantly enhanced photothermal response of Cy7-BTH while maintaining its controlled reactive oxygen species (ROS) generation, offering NIR-II imaging-guided surgical removal of primary tumors, and simultaneously inhibiting cancer cell metastasis by synergistic PDT/PTT dissection of metastatic tumor cells in sentinel lymph nodes (SLNs). This study provides an innovative but facile strategy for their potential clinical applications by precisely regulating the intermolecular competitive interactions of NIR theranostic agents.

Investigation on the binding behaviors of two dihydrochalcones to bovine serum albumin and their anti-glycation activities

The interactions of two dihydrochalcones (naringin dihydrochalcone (NGDC) and neohesperidin dihydrochalcone (NHDC)) with bovine serum albumin (BSA) and their anti-glycation effects were studied utilizing multiple spectral methods and molecular simulation technique. The findings proved that NGDC/NHDC could use a static quenching mechanism to reduce the intrinsic fluorescence of BSA, and the interaction between NGDC/NHDC and BSA was a spontaneous behavior with the binding constant ranging from 104 to 105M−1. Furthermore, NGDC and NHDC mainly interacted with the site I (subdomain IIA) of BSA via hydrophobic interactions and hydrogen bonds, endorsed by molecular docking study. A conjoint analysis of circular dichroism, three-dimensional fluorescence, dynamic light scattering, and UV-vis absorption spectra confirmed that NGDC/NHDC could induce some alterations in the spatial conformation and microenvironment of BSA. Additionally, NGDC and NHDC inhibited the non-enzymatic glycation of BSA by decreasing the contents of advanced glycation end products (AGEs), trapping methylglyoxal/glyoxal and maintaining the stability of the natural conformation of the protein. Overall, these findings were highly beneficial in disclosing the interaction mechanisms between dihydrochalcones and BSA and providing valuable information for the application of dihydrochalcones as functional food ingredients.

Fluorescence study of the interaction between albumin and layered double hydroxides

Layered double hydroxides nanoparticles (LDH-NP) are increasingly studied for biomedical applications. Nevertheless, their interaction with biomolecules such as proteins needs further exploration for an effective application. In this work, the adsorption of bovine serum albumin (BSA) on LDH-NP and the conformation changes of the protein upon adsorption were characterized using fluorescence spectroscopy. First, the quenching of tryptophan residues of BSA by chloride-intercalated LDH-NP was explored and the BSA adsorption capacity of LDH-NP were determined. Then, the structural conformation of the protein was analyzed by fluorescence spectroscopy (including synchronous, polarization and quenching studies) at different surface coverages. Finally, the proclivity of adsorbed BSA molecules to assemble as amyloid fibril was evaluated. Due to the positive charging and low curvature of LDH-NP, BSA molecules were strongly adsorbed, which produced a quenching of the protein fluorescence and a large adsorption capacity. The effect on BSA conformation was dependent on surface coverage (SC): at low values ,t he tryptophan residues were in more hydrophobic environments and more accessible to quenchers than al high ones. At low SC, there is space between the BSA molecules to spread on the surface, which led to a conformation change. Contrarily, the native conformation around tryptophan residues of BSA was preserved at high SC due to the tight packing of the adsorbed protein molecules. As a result, BSA molecules are stabilized against the formation of amyloid fibrils at high SC, while at low SC they present a similar fibrillation than free BSA.

Probing berberine syringate-bovine serum albumin interactions by multi-spectroscopic, conductimetric, volumetric and molecular docking methods

In this paper, a new berberine-based salt berberine syringate ([BBR][SA]) was synthesized and [BBR][SA]-bovine serum albumin (BSA) interactions were investigated by various techniques like UV–visible absorption, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR), volumetry and conductimetry. Using the experimental values of density and conductivity, the specific volume of BSA and the critical aggregation concentration of [BBR][SA] were derived. From the variation trends of specific volume for BSA with [BBR][SA] concentrations, it was observed the predominate ionic interaction/dehydration effect at small [BBR][SA] concentration and the non-polar interaction/hydration phenomenon at higher [BBR][SA] concentration for the binding of [BBR][SA] with BSA, as well as hydration shell structure changes at different temperatures in the medium with different pH values. Aggregation of [BBR][SA] is slowed by increasing concentration of BSA and temperature. The thermodynamic parameters of aggregation process have been exploited to gain more information on the interaction of [BBR][SA] with BSA. UV–vis absorption, multiple fluorescence spectroscopy (steady-state spectroscopy, synchronous fluorescence, three-dimensional fluorescence), and FT-IR have been utilized to understand the interaction mechanisms and binding strength between the two molecules. [BBR][SA] quenches the fluorescence of BSA through static quenching mechanism. The 1:1 complex between [BBR][SA] and BSA with a strong affinity was formed. The changes in microenvironment and conformation of BSA caused by [BBR][SA] were revealed by synchronous fluorescence spectra, 3D fluorescence maps and FT-IR. The binding average distance from [BBR][SA] to BSA (2.83 nm) was obtained based on the theory of Förster energy transfer. Competitive binding experiment confirms [BBR][SA] binding to Sudlow site I (sub-domain IIA) of BSA. In addition, molecular docking was generated to provide valuable information about the available binding sites of [BBR][SA] on BSA and the types of binding forces.

Active Dual-Protein Coating Assisted by Stepwise Protein-Protein Interactions Assembly Reduces Thrombosis and Infection.

Universal protein coatings have recently gained wide interest in medical applications due to their biocompatibility and ease of fabrication. However, the challenge persists in protein activity preservation, significantly complicating the functional design of these coatings. Herein, an active dual-protein surface engineering strategy assisted by a facile stepwise protein-protein interactions assembly (SPPIA) method for catheters to reduce clot formation and infection is proposed. This strategy is realized first by the partial oxidation of bovine serum albumin (BSA) and lysozyme (LZM) for creating stable nucleation platforms via hydrophobic interaction, followed by the assembly of nonoxidized BSA (pI, the isoelectric point, ≈4.7) and LZM (pI ≈11) through electrostatic interaction owing to their opposite charge under neutral conditions. The SPPIA method effectively preserves the conformation and functionality of both BSA and LZM, thus endowing the resultant coating with potent antithrombotic and bactericidal properties. Furthermore, the stable nucleation platform ensures the adhesion and durability of the coating, resisting thrombosis and bacterial proliferation even after 15 days of PBS immersion. Overall, the SPPIA approach not only provides a new strategy for the fabrication of active protein coatings but also shows promise for the surface engineering technology of catheters.

Influence of protein nativity on the stability of bovine serum albumin coated microbubbles

Protein-coated microbubbles have become one of the emerging platforms in biomedical research as theranostic agents. In recent years, microbubbles have been extensively used as ultrasound contrast agents and carriers of molecular cargoes, pertaining to which several studies have focused on tuning the properties of these bubbles to achieve a higher degree of biocompatibility and extended stability. Synthesis of microbubbles has so far been traditionally carried out with pre-heated proteins like bovine serum albumin (BSA) as shell coatings, owing to the ease in making BSA crosslinked structures through disulfide bridge formation. We, however, have performed experiments to demonstrate that air core microbubbles formed with native BSA are more stable compared with those formed using denatured BSA. The experimental observations have been supported with analytical modeling and computational studies, which offer insights into the effect of BSA conformation in stabilizing the microbubbles shells and prolonging their lifetimes.

Effects of polystyrene nanoplastics on the binding of ciprofloxacin to bovine serum albumin

Nanoplastics (NPs) and antibiotics have attracted more attention due to the potential toxicity to humans. Studies on their interactions as well as their subsequent combinations can provide useful information for understanding their health risks. In this study, bovine serum albumin (BSA) was used as the model protein to study the effects of polystyrene nanoplastics (PSNPs) on the binding of ciprofloxacin (CPFX) to BSA. The results showed that both the esterase-like activity of BSA in the presence and absence of PSNPs presented an increasing trend when increasing CPFX, and the enhancement when adding PSNPs could be attributed to the alteration of BSA conformation caused by PSNPs. The spectral analysis results indicated that the intrinsic fluorescence quenching of BSA in presence of CPFX is less affected by PSNPs, and PSNPs cannot directly affect the interactions between BSA and CPFX. Additionally, the binding experiments indicated that the presence of PSNPs is likely to promote the binding of CPFX to BSA molecules. This work will provide useful information on the influences of NPs to the health risks of antibiotics, and new insights into the toxic effects in the scene of co-existence.

Ratiometric luminescent sensor based on BSA-coated gold/silver nanoclusters for the selective determination and spatiotemporal imaging of gallic acid in plants.

Anewfluorescence sensing strategyhas been developed. Four bimetallic nanoclusters, gold/silver, gold/copper, gold/molybdenum and gold/cobalt, were prepared using bovine serum albumin (BSA) as a reducing and stabilizing agent. The fluorescence properties of four nanoclusters were explored by solid-state UV and XPS. The gold/silver nanoclusters (BSA-Au/Ag NCs) with the best ratiometric fluorescence properties for gallic acid (GA) in plants were selected to realize the sensitive detection of GA. GA affected the conformation of BSA, thereby disrupting the luminescent environment of the nanoclusters, resulting in a pronounced fluorescence quenching at 566nm. The ratiometric fluorescence signal (I566/I453) was used for trace detection of GAin plants. It has a wide response range of 1.25-40.0μM and a low detection limit of 45.27nM. GA was detected at 19.49μM in the plant extract, and the spiked recoveries ranged from 96.09 to 104.6%. In addition, due to the non-toxic and biocompatible properties of BSA, BSA-Au/Ag NCs have also been validated for fluorescence imaging of plant tissues. It realized the comparison of GA content in different parts of plants and the difference of GA content in plants after abiotic stress. Therefore, the developed strategy offers potential application for the analytical study of active substances in plants.

Probing the biomolecular interactions of oxidized Ti3C2Tx nanosheets: How surface chemistry influences serum protein binding

Ti3C2Tx has promising applications in the biomedical field due to its biocompatibility, hydrophilicity, and antibacterial activity. However, its effects on health are still being explored. This study analyzes the interaction of bovine serum albumin (BSA) with Ti3C2Tx at different levels of oxidation. Zeta potential measurements showed that the colloidal stability of Ti3C2Tx varied with the degree of oxidation, with unoxidized Ti3C2Tx (U-Ti3C2Tx) being the most stable, followed by fully oxidized Ti3C2Tx (F-Ti3C2Tx) and partially oxidized Ti3C2Tx (P-Ti3C2Tx). UV–Vis absorption and fluorescence spectroscopy confirmed that the formation of Ti3C2Tx-BSA complexes resulted in fluorescence quenching. In addition, synchronous fluorescence spectroscopy, Fourier transform infrared spectroscopy and circular dichroism showed that the interaction between F-Ti3C2Tx and BSA altered the secondary conformation of BSA, and that U-Ti3C2Tx had a weaker effect on the conformation of BSA. Molecular docking results emphasized that hydrogen bonding is the main driving force between Ti3C2Tx and BSA.

Ascorbic and salicylic acids modulate the binding interactions of an emergency contraceptive pill levonorgestrel to a model transport protein: Insights from spectroscopy and molecular docking analysis

Serum proteins generally help to transport and distribute drug molecules within the body. In this study, the binding characteristics of bovine serum albumin (BSA) with levonorgestrel (LVG), an emergency contraceptive pill, and the influences of ascorbic acid (ASC) and salicylic acid (SAL) on the binding behaviour and protein structure were elucidated using multi-spectroscopic techniques and molecular docking. The results showed that levonorgestrel decreased BSA intrinsic fluorescence via static quenching mechanism. Binding constant (Ka) values for BSA-LVG complexes were 103 to 104 M-1, indicating their high stabilities. Site probing/docking analysis indicated LVG bound between BSA subdomains IIA and IIIA. UV–visible absorption, Fourier Transform-Infrared and 3D fluorescence spectroscopies affirmed LVG-induced changes in BSA structure, especially in α-helix and β-sheet contents. ASC and SAL influenced BSA conformation for LVG binding and reduced the Ka values by 3.37 and 5.43-folds, respectively. LVG altered the microenvironments of tyrosine residues, interacted with Arg-217, Lys-221, Val-292, Glu-443 etc. within the binding domains. The process was spontaneous (ΔG<0), entropy driven (TΔS>ΔH) and involved van der Waals forces and hydrogen bonding. The findings of the study offered details on the binding interaction between BSA and LVG, and also indicated that prior intake of ASC or SAL could suppress the binding affinity of BSA for levonorgestrel.

Probing the interaction between encapsulated ethoxyquin and its β-cyclodextrin inclusion complex with bovine serum albumin

Ethoxyquin (EQ) is a synthetic antioxidant that is derived from quinolines and found in many meat products. EQ is strictly regulated in feed due to its potential health implications. An investigation of the interaction mechanism between EQ and transporter protein before and after β-cyclodextrin (β-CD) encapsulation was conducted with the use of multi-spectroscopy, cyclic voltammetry, and molecular docking. EQ formed complexes with bovine serum albumin (BSA), and affected secondary structure and microenvironment polarity of BSA. However, at 298 K, EQ's fluorescence quenching constants decreased from (9.81 ± 0.05) × 103 L mol−1 to (4.94 ± 0.09) × 103 L mol−1, binding constants decreased from (10.28 ± 0.02) × 103 L mol−1 to (2.08 ± 0.07) × 103 L mol−1, after encapsulation in β-CD as well as the binding distance increased. β-CD contains part of EQ in its hydrophobic cavity, inhibiting its binding to BSA. β-CD inclusion complex prevented adverse effects of EQ on BSA conformation. However, β-CD encapsulation had no effect on EQ's antioxidant activity.

Effect of Soft Preheating of Bovine Serum Albumin on the Complexation with Oligochitosan: Structure and Conformation of BSA in the Complex.

Phase analysis, spectroscopic and light scattering methods are applied to investigate the peculiarities of the interaction of oligochitosan (OCHI) with native and preheated BSA as well as the conformational and structural changes of BSA in BSA/OCHI complex. As shown, untreated BSA binds with OCHI mainly forming soluble electrostatic nanocomplexes, with the binding causing an increase in BSA helicity without a change in the local tertiary structure and thermal stability of BSA. In contrast, soft preheating at 56°C enhances the complexation of BSA with OCHI and slightly destabilizes the secondary and local tertiary structures of BSA within the complex particles. Preheating at 64°C (below the irreversible stage of BSA thermodenaturation) leads to further enhancement in the complexation and formation of insoluble complexes stabilized by both Coulomb forces and hydrophobic interactions. The finding can be promising for the preparation of biodegradable BSA/chitosan-based drug delivery systems. This article is protected by copyright. All rights reserved.

Probing the interaction of tert-butylhydroquinone and its β-cyclodextrin inclusion complex with bovine serum albumin

Tert-butylhydroquinone (TBHQ) is a synthetic antioxidant found widely in foods such as cooking oil, fried baked goods and meat products, however, if consumed in excessive quantities, it may cause nutritional problems and chronic diseases. The binding of TBHQ to transport protein before and after β-cyclodextrin (β-CD) encapsulation was investigated by multiple spectroscopies and molecular docking technology. At 298 K, the binding constant, enthalpy, and entropy of bovine serum albumin (BSA) with TBHQ were (1.18 ± 1.50) × 104 M−1, (−110.11 ± 15.29) kJ mol−1 and (−293.36 ± 50.87) J mol−1 K−1 at 298 K, respectively. By forming a stable complex with BSA through van der Waals forces and hydrogen bonds, TBHQ altered BSA's secondary structure and microenvironmental polarity. However, the binding affinity of TBHQ with BSA decreased after encapsulation by β-CD, but the binding distance increased. Because of the encapsulation of β-CD, the interference of TBHQ with the BSA conformation was also inhibited. Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscope study confirmed the formation of an inclusion complex between CD and TBHQ. According to UV spectroscopy, TBHQ and β-CD have a binding constant of (1.21 ± 0.03) × 103 M−1. The formation of TBHQ–β-CD inclusion complex inhibited the binding of TBHQ and BSA, possibly because the inclusion of β-CD prevented TBHQ from entering the hydrophobic cavity of BSA, which prevented TBHQ from binding to BSA, alleviating the negative effects of TBHQ on BSA conformation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging results showed that the antioxidant activity of TBHQ was not affected by β-CD encapsulation.

2-Phenylquinoline-polyamine conjugate (QPC): Interaction with bovine serum albumin (BSA)

A novel 2-phenylquinoline-polyamine conjugate (QPC) was synthesized and characterized, its interaction with bovine serum albumin (BSA) was evaluated using UV–Vis, fluorescence and circular dichroism (CD) spectroscopy. The results showed that QPC caused a whole train of spectral variation, including enhancement of UV–vis absorption and reduction of fluorescence (FL), indicating QPC-BSA complex formed. FL results showed that the type of FL quenching waslarge static quenching, which was also accompanied with a process of dynamic quenching. Binding constants, thermodynamic parameters and docking results showed that the interaction between QPC and BSA was basically a Van der Waals, hydrogen bond and hydrophobic interaction. Synchronous and 3D-FL analysis revealed that QPC resulted in unapparent conformational alteration of BSA. The docking study suggested QPC was situated at the binding sites II of BSA, and 2-phenylquinoline moiety contributed to the hydrophobic interaction. The results of molecular dynamics revealed QPC altered the conformation of BSA, which showed that the inconsistency between experimental data and theoretical calculation results may be due to the instability of the compound.

Study of the effect of accelerated electrons on the structural characteristics of the bovine serum albumin using liquid chromatography-mass spectrometry and high-resolution tandem mass spectrometry

A method for quantification of the dose effect of ionizing radiation on the structural characteristics of bovine serum albumin (BSA) in aqueous solution through identification of unique peptides of protein domain structures using high-resolution liquid chromatography-mass spectrometry is proposed. BSA with the initial concentration of 500 mg/liter in a physiological solution was exposed to irradiation at a dose rate of 18. 5 Gy/sec using an accelerated electron beam with the maximum energy of 1 MeV at an average beam current of 1 uA. The absorbed dose in the sample volume was estimated using a Fricke (ferrous sulphate) dosimeter. After irradiation of BSA solution at 0.3, 0.6,1.8, and 20 kGy we analyzed the structural integrity of the protein native form and then quantified the content. For this, masses more than 30 kDa were removed using centrifugation. Then BSA was subjected to enzymatic hydrolysis with the addition of trypsin solution, and the resulting peptides with a mass of more than 10 kDa were repeatedly removed. The resultant samples were then examined using liquid chromatography mass spectrometry (LC-MS) and high-resolution tandem mass spectrometry (HRMS-MS/MS). The content of intact protein molecules was assessed by determining the concentrations of unique peptides corresponding to each of the three domains into which the amino acid sequence of BSA was divided. Using the developed methodology, a change in the natural conformation of bovine serum albumin (denaturation) in water samples induced by ionizing radiation at a dose ranging from 0.3 to 20 kGy was revealed on average in 71% of protein molecules exposed to doses up to 1 kGy in 79% of molecules exposed to doses of 4 kGy and in 99 % to 100% of molecules exposed to doses of 8 and 20 kGy.

New insights into the pH dependence of anthocyanin-protein interactions by a case study of cyanidin-3-O-glucoside and bovine serum albumin

Anthocyanins are natural pigments that can undergo structural transformations depending on pH, and their interactions with proteins have received considerable attention in recent decades. This work intended to unravel the pH-dependence of anthocyanin-protein binding mechanisms by examining interactions between cyandin-3-O-glucoside (C3G) and bovine serum albumin (BSA) at different pH. Fluorescence quenching (FQ) and microscale thermophoresis (MST) demonstrated that their binding affinity was pH 7 > pH 5 > pH 3, with dissociation constant (Kd) at pH 7 of 43.1 μM for FQ and 33.0 μM for MST. The predominant C3G forms were determined by measuring UV–Vis absorption spectra with pH-jump experiments. Additionally, circular dichroism, Trp fluorescence, zeta potential and particle size measurements demonstrated the “molten globule” state of BSA at pH 3, and C3G had a negligible effect on BSA conformation. The binding mechanisms were investigated using molecular dynamics simulation, which revealed the importance of electrostatic interaction. Flavylium cation rarely bound to BSA due to electrostatic repulsion at pH 3, whereas the uncharged forms of C3G at all pH values bound to BSA surface due to hydrophobic interactions and hydrogen bonds, but possibly in a weak and nonspecific manner. Anionic quinoidal bases, the most common C3G forms at pH 7, mostly bound to the positively charged pockets of BSA and formed several hydrogen bonds with surrounding amino acids, resulting in higher binding affinity. These findings provide insights into the interactions of proteins with different anthocyanin forms, which may guide the future research and applications in this field.