Bovine Serum Albumin Binding Research Articles

4-Dimethylamino-beta-nitrostyrene, a fluorescent solvatochromic probe to estimate the apparent dielectric constant in serum albumin: Experimental and molecular dynamics studies

The spectroscopic properties of 4-dimethylamino-beta-nitrostyrene (DANS) were theoretically and experimentally correlated with the polarity of organic solvents and applied to estimate the overall apparent dielectric constant in the binding sites of bovine serum albumin (BSA). The linear correlation between McRae's solvent polarity function and the wavelength of maximum emission (R2 = 0.8899) showed the suitability of DANS as a fluorescent solvatochromic probe. The interaction between BSA and DANS was evaluated (Ka = 3.1 × 104 mol−1 L), and based on the DANS/BSA emission, the apparent dielectric constant of BSA was estimated (ε = 5.5 ± 0.8). The experimental and theoretical absorption and emission maxima were correlated using density-functional theory. Molecular dynamics was used to calculate the mean electrostatic energy between DANS and the organic solvent molecules. These results were associated with the experimental fluorescence and applied to the BSA/DANS system, resulting in electrostatic energy (−37 kJ mol−1). The application of this model generated a dielectric constant of 5.8 ± 1.3 to BSA, which was consistent with the values obtained by McRae's solvent polarity function. The mean electrostatic energy of DANS in BSA binding sites was calculated directly from molecular dynamics simulations and resulted in −40 ± 2, −47 ± 3, and −48 ± 3 kJ mol−1 for sites I, II, and III. These results showed that the environment offered by the site I presented the value of electrostatic energy closest to the obtained with the solvatochromic model. In conclusion, DANS can be used to estimate the apparent polarity of albumins.

Binding behavior of ibuprofen-based ionic liquids with bovine serum albumin: Thermodynamic and molecular modeling studies

• Nine ibuprofen-based ionic liquids containing cations amino acid alkyl esters [AAOR] were synthesized and characterized. • The [AAOR][Ibu] are not toxic against mouse fibroblasts and keratinocytes. • Ibu-ILs bind spontaneously to bovine serum albumin with moderate affinities (Ka 1–3.7 × 10 5 L/mol) at 25 °C. • Depending on their structures, [AAOR] showed differences at the preferred binding site of bovine serum albumin. Ionic liquids based on active-pharmaceutical ingredients are an attractive alternative to classical drugs because they are able to overcome problems with drug solubility and polymorphism, hence bioavailability and as well as to propose different routes of administration and/or improve the pharmacokinetics of the drug. In this manuscript, we report the synthesis of nine amino acid alkyl esters of ibuprofen [AAOR][Ibu]. The estimated half-maximum inhibitory constants toward murine embryotic fibroblasts and human dermal keratinocytes are in the millimolar range, which makes them practically not toxic. The conversion of ibuprofen into ionic liquids does not affect the binding affinity of ibuprofen to bovine serum albumin (BSA). Molecular modeling studies have shown that bulky cations such as L-PheOEt and L-PheOPr are able to bind effectively in three different BSA binding sites. On the other hand, smaller cations preferably bind to Sudhow’s site I, the same that binds ibuprofen.

Heterodinuclear Ru-Pt Complexes Bridged with 2,3-Bis(pyridyl)pyrazinyl Ligands: Studies on Kinetics, Deoxyribonucleic Acid/Bovine Serum Albumin Binding and Cleavage, In Vitro Cytotoxicity, and In Vivo Toxicity on Zebrafish Embryo Activities.

Di- and poly-homo/heteronuclear complexes have great potential as anticancer drugs. Here, we report their reactivity, deoxyribonucleic acid (DNA)/bovine serum albumin (BSA) binding and cleavage interactions, in vitro cytotoxicity, and in vivo zebrafish embryo toxicity of [(phen)2Ru(μ-L)PtCl2]2+ (phen = 1,10-phenanthroline and L = 2,3-bis(2-pyridyl)pyrazine, bpp, C1; 2,3-bis(2-pyridyl)quinoxaline, bpq, C2ial; 2,3-bis(2-pyridyl)benzo[g]quinoxaline, bbq, C3) anticancer prodrugs. The substitution reactivity increases from C1 to C3 owing to an increase in the π-conjugation on the bridging chelate which facilitates π-back bonding. As a result, the electrophilicity index on the C3 complex increases than that on the complex C2 followed by C1 which leads to higher rates of substitution and thus the reactivity order follows C1 < C2 < C3. The coordination of Ru at one end of each of the complexes enhances water solubility. Moreover, the charge addition of the two metal ions increases their reactivity toward substitution in addition to ensuring electrostatic interactions at target sites such as the DNA/BSA. Spectroscopic (UV–vis absorption and fluorescence quenching) titration and viscosity measurement results of the interactions of C1/2/3 with CT-DNA established the formation of stable, nonconvent C1/2/3-DNA adducts with DNA most likely via the intercalative binding mode. Furthermore, studies with BSA showed a good binding affinity of these complexes owing to hydrophobic interactions with the coordinated ligands. The interactions of these complexes with DNA/BSA are in line with the reactivity trend, and all these experimental findings were further supported by molecular docking analysis. In vitro MTT cytotoxic activities on human breast cancer cell line MCF-7 revealed that all the complexes have high cytotoxicity activity (IC50 > 9 μM); furthermore, the selectivity index and SI values were higher (>3). Complex C3 showed the highest cytotoxicity with IC50 = 3.1 μM and SI value (5.55) against MCF7 cell lines and these values were comparable to those of the cisplatin (IC50 and SI values are 5.0 μM and 4.02, respectively). In vivo toxicological assessments on zebrafish embryos revealed that all the Ru–Pt complexes (CI/2/3) have poor embryo acute toxic effects over 96 h postfertilization, hpf with LC50 > 65.2 μM. The complex C3 has shown the lowest embryo toxicity (LC50 = 148.8 μM), which is comparable to that of commercial cisplatin (LC50 = 181.1 μM). Based on the cytotoxicity results, complexes C2 and C3 could be considered for further development as chemotherapeutic agents against MCF breast cancer cells.

Unravelling the role of [Ru(bpy)2(OH2)2]2+ complexes in photo-activated chemotherapy

Photoactivated chemotherapy (PACT) has emerged as a promising strategy to selectively target cancer cells by using light irradiation to generate cytotoxic complexes in situ through a mechanism involving ligand-loss. Due to their rich optical properties and excited state chemistry, Ru polypyridyl complexes have attracted significant attention for PACT. However, studying PACT is complicated by the fact that many of these Ru complexes can also undergo excited-state electron transfer to generate 1O2 species. In order to deconvolute the biological roles of possible photo-decomposition products without the added complication of excited-state electron transfer chemistry, we have developed a methodology to systematically investigate each product individually, and assess the structure-function relationship. Here, we synthesized a series of eight distinct Ru polypyridyl complexes: Ru-Xa ([Ru(NN)3]2+), Ru-Xb ([Ru(NN)2py2]2+), and Ru-Xc ([Ru(NN)(OH2)2]2+) where NN = 2,2′-bipyridine, 4,4′-dimethyl-2,2′-bipyridine, or dimethyl 2,2′-bipyridine-4,4′-dicarboxylate and py = pyridine. The cytotoxicity of these complexes was investigated in two cell lines amenable to PACT: H23 (breast cancer) and T47D (lung cancer). We confirmed that light irradiation of Ru-Xa and Ru-Xb complexes generate Ru-Xc complexes through UV–visible spectroscopy, and observed that the Ru-Xc complexes are the most toxic against the cancer cell lines. In addition, we have shown that ligand release and biological activity including bovine serum albumin (BSA) binding, lipophilicity, and DNA interaction are altered when different groups are appended to the bipyridine ligands. We believe that the methodology presented here will enhance the development of more potent and selective PACT agents moving forward.

Can One Novel Lanthanide Complex and Its Nano-Encapsulated Compounds Afford Advances in Biological Inorganic Chemistry? A Biological Applications Study for Dysprosium (III) Complex and Its Nano-Encapsulated Compounds

ABSTRACT In this paper, the biological applications of synthetic dysprosium(III) complex, with 4,5-diazafluoren-9-one (dafone) ligand, including DNA/BSA interaction, antibacterial and anticancer activity were studied in vitro. The bovine serum albumin (BSA) and fish DNA (FS-DNA) binding of the dysprosium complex were studied by multi-spectrophotometric as well as computational calculation. Its DNA and BSA binding ability were estimated by fluorescence, absorption, circular dichroism spectroscopy, and viscosity measurements (only for DNA). The Dy-complex binds to DNA and BSA presenting high binding constants. For both DNA/BSA binding, the negative signs of thermodynamic parameter confirmed that hydrogen bonds and van der Waals forces play a main role in the interaction process. The competitive experiments with ethidium bromide (EtBr) and rhodamine B exhibited that the Dy-complex interacts with DNA via groove binding. The BSA competitive experiments showed that Dy-complex interacts with site 3 of BSA, which was completely arranged by docking studies. This complex showed high antimicrobial and cytotoxicity. Besides, nanocarriers of Dy-complex were produced, and the anticancer activities of these compounds were measured. (This paper provides a manifestation of a new tradition by which Comments on Inorganic Chemistry starts publishing original research content that, nonetheless, preserves the Journal’s identity as a niche for a critical discussion of contemporary literature in inorganic chemistry) (For previous manifestations, see Comments Inorg. Chem. 2018, 38, 1–35; 2019, 39, 1–26; 2019, 39, 188–215; 2020, 40, 1–24; 2020, 40, 277–303; 2021, 1–46, doi: 10.1080/02603594.2021.1962310.)

The Relationship between Hydrophobicity and Drug-Protein Binding in Human Serum Albumin: A Quartz Crystal Microbalance Study

In this paper, the quartz crystal microbalance with dissipation monitoring (QCM-D) was used to investigate hydrophobicity and binding strength (KD) for 10 different drugs interacting with human serum albumin (HSA). Quantitative structure activity relationship (QSAR) analysis was used to determine the relationship between drug hydrophobicity (ClogP) and HSA binding strength log(1/KD). The results are compared to prior knowledge on bovine serum albumin (BSA) binding. We demonstrate a positive correlation between drug hydrophobicity and the strength of ligand-protein binding to HSA and show a statistically significant similarity with the trend reported in BSA. The findings presented in this work provide insight into the role that bound water plays in ligand-protein interactions. Further, the comparison between HSA and BSA provides quantitative justification for the use of these proteins interchangeably in the analysis of drug-based binding kinetics.

Bidentate acylthiourea ligand anchored Pd-PPh3 complexes with biomolecular binding, cytotoxic, antioxidant and antihemolytic properties

Acylthiourea-based Pd(II) complexes (1–5) with a PPh3 moiety bearing the general formula [PdCl(PPh3)(L-R)] [L-R = monoanionic bidentate acylthiourea ligand, where R = C6H5 (L1), C6H4CH3(o) (L2), C6H4OCH2CH3(p) (L3), C10H7 (L4) or C6H4Cl (L5)] have been synthesized and characterized by spectroscopic and analytical tools. The single crystal X-ray structures (1–3) revealed that the acylthiourea ligands coordinated to Pd(II) ion in an uncommon bidentate fashion through S and N atoms, forming a four-member ring. The Pd(II) ion exhibited a square planar geometry fulfilled by the ligand (N, S), one Cl‐ and one triphenylphosphine (PPh3). Calf thymus (CT) DNA and bovine serum albumin (BSA) binding of the complexes have been analyzed by spectroscopic and molecular docking studies. The complexes were tested for their in vitro cytotoxicity on three cancer (cervical, breast and lung) and one normal (human embryo) cell lines. Complex 4 bearing the naphthalene substitution exhibited the highest activity against three cancer cells with the half-maximal inhibitory concentration (IC50) values of 8.6 (cervical), 8.8 (breast) and 9.4 μM (lung). The acridine orange/ethidium bromide (AO/EB) and 4′,6-diamidino-2-phenylindole (DAPI) staining assays indicated that 4 induced cancer cell death through apoptosis. Among the complexes, 4 exhibited the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of 86.19%. All the complexes were subjected to the hemolysis assay which revealed their biocompatibility with red blood cells (RBCs) with a lysis rate of less than 5 %.

Carboxymethyl chitosan assembled piezoelectric biosensor for rapid and label-free quantification of immunoglobulin Y

Immunoglobulin Y (IgY) proves advantageous to IgG in prophylaxis and diagnosis. Quantification of IgY is therefore becoming a topic of interest. Here, we demonstrate a piezoelectric biosensor with carboxymethyl chitosan (CMCS) as the immobilization matrix. Gelation and hydrophilic nature of CMCS are favored to form a crosslinked matrix for antibody immobilization, and a comparison was made between carboxymethyl cellulose (CMC) and CMCS to investigate the benefits of such substitution. Calibration from 500 ng/mL to 200 μg/mL was established in buffer with the detection limit (LOD) down to 270 ng/mL, confirming its feasibility. As-prepared biosensor effectively prevents non-specific binding of bovine serum albumin (BSA) and lysozyme. Each real-time assay took 15 min including sensor regeneration, which can be further reduced to 4 min for signal readout only, ready for both repeated measurements after regeneration and disposable devices. Thus, as-prepared biosensor offers a rapid, label-free and cost-effective approach for IgY quantification.

Exciton-tryptophan coupling pulse behaviour along with corona formation, binding analysis, and interaction study of ZnO nanorod-serum albumin protein bioconjugate.

The bioconjugate of bovine serum albumin (BSA) and zinc oxide nanorods (ZnO NRs) is investigated to explore the behaviour of the tryptophan (Trp)-exciton coupling and corona formation. The pulse like nature of the coupled system between Trp of BSA and exciton of ZnO NRs has been observed after analysis of the optical parameters such as refractive index, susceptibility, and optical dielectric constant. The time constant for tryptophan, exciton surface binding (t1 ) and reorganization (t2 ) are found to be (t1 ) 8 min, 7 min and (t2 ) 150 min, 114.5 min, respectively. The close proximity binding of BSA with ZnO NRs via tryptophan as well as exciton is responsible for bioconjugate formation. The aggregated structure of BSA is observed from small-angle X-ray scattering study in interaction with ZnO NRs. The change in secondary structure and tertiary deformation of the serum protein have been studied from Fourier transform infrared and emission quenching analyses. The number of binding sites (n) signified to the enhancement of the cooperative binding. The binding has been found to be endothermic and favoured using unfavourable positive enthalpy with a favourable entropy change from the result of the isothermal titration calorimetry.

A muconate bridged bipyridyl appended binuclear Cu(II) complex reveals dissimilar affinities to DNA and BSA protein

A new muconate based Cu(II) metal complex, [Cu2(muco)(5,5′-Me2bpy)4]·2ClO4¯ (1; H2muco = trans, trans-1,3-butadiene-1,4-dicarboxylic acid or trans, trans-muconic acid and 5,5′-Me2bpy = 5,5′-dimethyl-2,2′-bipyridine), has been synthesized and structurally characterized. X-ray structure analysis reveals that 1 forms a binuclear complex with a five coordinated square pyramidal geometry of the central Cu(II) ion attached to a muco and two bipyridine ligands. Docking studies have been carried out which reveal the successful binding of the metal complex to the major groove of DNA and the planar aromatic bipyridyl ligand partially intercalates into the base pairs. The synthesized complex fits into the pocket of bovine serum albumin (BSA), while the maximum fraction submerges into the BSA binding pocket. An extra coordinate bond has been found, forming through the oxygen atom of 158 asparagines amino acid, which subsequently forms a new six coordinated octahedral complex. Experimental studies have been performed to validate all the data obtained from the docking studies. This study reveals highly diverse affinities and binding modes of the synthesized complex towards DNA and protein. Therefore, the synthesized complex may pave the way to be used as a specific protein binder (low affinity to DNA or RNA) bioactive compound.

CT-DNA/BSA Binding Studies of Thiosemicarbazone-Derivated Zn(II) Complex

Zn(II) complex of 2-hydroxy-5-methoxyacetophenone thiosemicarbazone { Zn(HMAT)2} was synthesized and characterized by 1H NMR, UV–Vis and FT-IR spectroscopies. Further, X-ray powder diffraction (XRD) analysis of Zn(HMAT)2 was carried out to point out the complexation. The binding affinities of Zn(HMAT)2 with calf thymus DNA (CT-DNA) have been studied by using fluorescence and absorption titration technics. In addition, bovine serum albumin (BSA) binding studies were recorded by fluorescence and UV–Vis spectroscopy. Zn(HMAT)2 is a strong binders of CT-DNA with binding constant (Kb) 3.65×107 M−1. The binding parameters KSV (for EB), Kq (for BSA) and Kb (for BSA) were determined as 8.2×107 M−1, 1.8×1014 M−1 s−1 and 2×107 M−1 respectively.

Theoretical and experimental investigation of 1,4-dihydropyridine-based hexahydroquinoline-3-carboxylates: Photophysics and bovine serum albumin binding studies

In this article, the binding affinity of 1,4-dihydropyridine-based hexahydroquinoline-3-carboxylates with bovine serum albumin (BSA) was studied by electronic spectroscopies, quantum calculations, and molecular docking. These compounds were obtained by the one-pot microwave-assisted method via a modified Hantzsch reaction. The photophysical characterization showed in organic solutions absorption maxima in the UV region, ascribed to spin and symmetry allowed electronic transitions 1π–π*. In addition, they presented a main fluorescence emission in the violet-blue regions (406–445 nm), with a relatively large Stokes shift (54–81 nm), depending on the structure of the compounds. BSA fluorescence quenching experiments based on the intrinsic fluorescence of the Trp residues were successfully applied to these compounds, indicating strong interaction with BSA by a static mechanism. The docking simulations corroborated the strong interaction between the studied compounds and BSA, where the surroundings of Trp213 seemed to be the preferred interaction site for all compounds and were responsible for the fluorescence quenching effects.

Multispectroscopic and computational evaluation of the binding of flavonoids with bovine serum albumin in the presence of Cu2+

Bovine serum albumin (BSA) has the potential application of establishing a delivery system for flavonoids. However, the effect of copper on the binding of flavonoids with BSA is unclear. Therefore, the binding of six flavonoids with BSA containing Cu2+ was investigated using UV–vis, fluorescence, and molecular docking. The UV–vis spectral changes demonstrated the formation of flavonoid-Cu2+ complexes. The fluorescence quenching results suggested that the chelation of Cu2+ increased the binding affinity of galangin and baicalin to the BSA but decreased the binding capacity of chrysin, baicalein, luteolin, and vitexin. Synchronous fluorescence data revealed that Cu2+ could influence the secondary structure conformation of BSA binding with flavonoids, which was further confirmed by ANS-binding fluorescence, circular dichroism, and molecular docking. These findings demonstrate that the complexation of Cu2+ significantly affects the binding of flavonoids with BSA, which provides the theoretical basis for the development of natural product-metal complex functional foods.

Experimental measurement of kinetic parameters using quantum plasmonic sensing

Kinetic models are essential for describing how molecules interact in a variety of biochemical processes. The estimation of a model’s kinetic parameters by experiment enables researchers to understand how pathogens, such as viruses, interact with other entities like antibodies and trial drugs. In this work, we report a simple proof-of-principle experiment that uses quantum sensing techniques to give a more precise estimation of kinetic parameters than is possible with a classical approach. The interaction we study is that of bovine serum albumin (BSA) binding to gold via an electrostatic mechanism. BSA is an important protein in biochemical research as it can be conjugated with other proteins and peptides to create sensors with a wide range of specificity. We use single photons generated via parametric down-conversion to probe the BSA–gold interaction in a plasmonic resonance sensor. We find that sub-shot-noise-level fluctuations in the sensor signal allow us to achieve an improvement in the precision of up to 31.8% for the values of the kinetic parameters. This enhancement can, in principle, be further increased in the setup. Our work highlights the potential use of quantum states of light for sensing in biochemical research.

Bovine Serum Albumin as a Potential Carrier for the Protection of Bioactive Quercetin and Inhibition of Cu(II) Toxicity.

Considering the protective ability of proteins and the potential toxicity of free Cu(II), it was proposed herein that the co-presence of protein could play an important role in suppressing the toxicity of free Cu(II) to the stability of bioactive quercetin if a flavonoid-protein-Cu(II) complex could be formed. In this study, the interaction between quercetin (a major flavonoid in the human diet) and bovine serum albumin (BSA) was investigated in the absence and presence of free Cu(II). The results demonstrated that both quercetin and free Cu(II) had a strong ability to quench the intrinsic fluorescence of BSA through a static procedure (i.e., formation of a BSA-monoligand complex). Site marker competitive experiments illustrated that the binding of both quercetin and Cu(II) to BSA mainly took place in subdomain IIA. The quenching process of free Cu(II) with BSA was easily affected by quercetin, and the increased binding capacity possibly resulted from the generation of a ternary quercetin-BSA-Cu(II) complex. The stability and free radical scavenging activity of bioactive quercetin during incubation was promoted in the BSA-diligand complex relative to a quercetin-Cu(II) complex. A quercetin-Cu(II) system could generate reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), which were significantly inhibited upon BSA binding. Consistently, the cytotoxicity of the quercetin-Cu(II) system to endothelial cells was decreased in the BSA-diligand complex, where the co-presence of BSA played an important role. These results suggest the possibility and advantage of developing albumin-based carriers for the protection of bioactive components and suppression of Cu(II) toxicity in their biomedical and nutritional applications.

Novel Zinc and Vanadium (V) Hydroquinonate Complexes: Synthesis and Biological Solution Evaluation

Four novel mononuclear complexes of Zn2+ and V5+ ions were synthesized by the chelation of tridentate ligands of hydroquinone derivatives in 1:1 M ratios. The synthesis of the two novel ligands and their metal complexes were identified and characterized by NMR, IR, UV–Vis, cyclic voltammetry, and conductivity measurements. Structural information was also collected using computational chemistry and density functional theory studies. The biological activity of these metal complexes was investigated by the ctDNA interaction process via UV–Vis and viscosity measurements. The synthesized compounds C1 – C4 were primarily screened for their in vitro anti-microbial activities against gram-positive, gram-negative bacteria, and their anticancer potential against MDA-MB-231 cells carried out by MTT assay. Furthermore, on the complex molecules, we performed DPPH antioxidant studies and BSA (bovine serum albumin) binding studies on the UV–Vis. Additionally, studies were performed in silico by molecular docking evaluation of the molecules against breast cancer-related proteins. The data show that complexes C1- C4, have anticancer activity even in cells that showed resistivity against cis-platin (C3 > C4 > C2, C1) and the complexes (C2 > C1), have antibacterial activity against gram-positive bacteria. Another remarkable piece of evidence is that the anticancer activity of the molecules does not occur only because of their prooxidant activity, since C4 compound resulted in antioxidant activity.

BSA binding affinity, free radical scavenging capacity and antibacterial activity of new Silver(I) complexes with Schiff base ligands

In this work, four silver(I) complexes with Schiff bases derived from ninhydrin and selected amino acids (methionine, histidine, cysteine, and phenylalanine) were synthesized. The aim of this study is to determine the potential biological activity of these complexes. FTIR and UV/VIS spectroscopy were used for structural characterization of the products. Antioxidant activity was examined in vitro using DPPH method. Antimicrobial screening was performed by diffusion technique on reference bacterial strains from the ATCC collection. Interaction with bovine serum albumin (BSA) was examined using UV/VIS spectroscopy. Based on the obtained spectral data, it is assumed that all Schiff bases coordinate the Ag(I) ion as a tridentate ONO donor ligand. The antioxidant activity of the synthesized compounds is extremely high, with a range of IC50 values of 0.023-0.06 mg/mL. Antimicrobial screening determined the inhibitory ability of almost all complexes for concentrations of 1 and 2 mg/mL, with zones of inhibition in the range of 9-19 mm. Examination of the interaction of BSA with Ag(I) complexes revealed the same change in the absorption maximum (hyperchromic shift) in the region of about 205 nm, which indicates that the interaction of BSA and the complex results in conformational changes of BSA.  Â

Synthesis, Molecular Docking, BSA, and In Vitro Reactivation Study of Imidazopyridine Oximes Against Paraoxon Inhibited Acetylcholinesterase.

To synthesize and evaluate the fused heterocyclic imidazo[1,2-a]pyridine based oxime as a reactivator against paraoxon inhibited acetylcholinesterase. Organophosphorus compounds (OPs) include parathion, malathion, chlorpyrifos, monocrotophos, and diazinon, which are commonly used in agriculture for enhancing agricultural productivity via killing crop-damaging pests. However, people may get exposed to OPs pesticides unintentionally/intentionally via ingestion, inhalation, or dermal. The current treatment regimen includes reactivator such as mono or bis-pyridinium oximes along with anticholinergic and anticonvulsant drugs that are recommended for the treatment of OP poisoning. Unfortunately, the drawback of the existing reactivator is the permanent charge present on the pyridinium, making them inefficient to cross the blood-brain barrier (BBB) and reactivate OP-inhibited central nervous system (CNS) acetylcholinesterase. Therefore, there is a need of a reactivator that could cross the BBB and reactivate the OP inhibited acetylcholinesterase. The objectives of the study were synthesis, molecular docking, BSA binding, and in-vitro estimation of oximes of various substituted imidazo [1,2-a]pyridine against paraoxon inhibited acetylcholinesterase. The reactivators were synthesized in three steps and characterized using various spectroscopic techniques. The molecular docking study was performed on 2WHP and 3ZLV PDB using the Glide-XP software. The acid dissociation constant (pKa) of oximes was calculated experimentally, and the drug-likeness properties of the oximes were calculated in silico using Molinspiration and Swiss ADME software. The binding of oximes with bovine serum albumin (BSA) was also investigated using a Fluorescence spectrophotometer. The reactivation potential of the oximes was determined by in vitro enzymatic assay. The In-silico study inferred that the synthesized molecules fulfilled the parameters required for a successful CNS drug candidate. Furthermore, in-vitro enzymatic assay indicated reasonable reactivation potential of the oximes against paraoxon-inhibited AChE. The binding of oximes with bovine serum albumin (BSA) revealed that there was a static quenching of intrinsic fluorescence of BSA by the oxime. The binding constant value and number of binding sites were found to be 0.24 x 104 mol-1 and 1, respectively. The results of the study concluded that this scaffold could be used for further designing of more efficient uncharged reactivators.

Oxidation state of graphene oxide nanosheets drives their interaction with proteins: A case of bovine serum albumin

In the present study, we explored the interaction of bovine serum albumin (BSA) with oxidized graphene oxide (GO) nanosheets. Nanosheets, synthesized with 4, 6, 8, 10 and 12 wt equivalents of KMnO4 as oxidant, were coded as GO-4, GO-6, GO-8, GO-10 and GO-12, respectively. After incubating sheets with a fixed concentration of BSA at room temperature, interactions were monitored with time. The analysis is based on UV–vis spectroscopy, fluorescence quenching, dynamic light scattering (DLS), small angle neutron scattering (SANS), Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) techniques. Binding of BSA over sheets was recorded in the following order; GO-04 >> GO-06 > GO-08 > GO-10 ≈ GO-12. Our observations suggest that these interactions are largely regulated by the availability of pure graphitic domains and density of oxygen functionalities on sheet surface. This led us to the conclusion that GO-protein interactions can be minimized by modulating the extent of sheet oxidation. Moreover, we show that adsorption of proteins as colloidal aggregates contributes to improved biosafety of sheets. The protein molecule did not exhibit depletive changes in its conformation. However, from the viewpoint of drug delivery applications, density of oxygen groups must be optimized for maximizing the loading efficiency of oxidized sheets.

Preparation and characterization of agarose-encapsulated ceramic hydroxyapatite particles for flow-through chromatography

ABSTRACT Composite adsorbents were prepared by encapsulating ceramic hydroxyapatite particles (CHT Type I) within inert agarose beads using an emulsion method as stationary phases for the removal of small protein and nucleic acid impurities from large proteins and bioparticles in a flow-through mode. The composite particles were prepared with 6% and 10% agarose and contain 35% and 25% (v/v) CHT, respectively, with effective pore radii of 15.1 and 8.8 nm. The functional properties were studied using yeast RNA, bovine serum albumin (BSA), thyroglobulin (Tg), IgM, and 30 and 50 nm silica nanoparticles (NP) as models. The RNA and BSA adsorption capacities are smaller than for unencapsulated CHT on a bead volume basis but comparable when normalized by the volume of CHT particles in the composite beads attaining values between 30 and 40 mg/mL. Confocal laser scanning microscopy shows that, while RNA and BSA have complete access to the encapsulated CHT particles, Tg, IgM, and the NPs are largely excluded. RNA and BSA breakthrough experiments in laboratory-scale columns at a 5-min residence time show RNA and BSA binding capacities comparable to those measured in a batch mode with either single component feeds or with feed mixtures containing 30 nm NPs.