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•Interaction mechanism of NMB with SAs examined through experimental and Insilico approach.•Emission studies established the static quenching mechanism between SAs and NMB.•Molecular ...docking analysis revealed the stabilization of NMB inside the binding pocket Subdomain IIA (Site I) of SAs.•Insilico findings were correlates well with the emission results.
Herein, the interaction mechanism of new methylene blue (NMB) with human serum albumin (HSA) and bovine serum albumin (BSA) was carefully investigated both experimentally and conceptually, employing experimental and insilico analysis. The steady-state emission spectral studies showed that the emission intensity of HSA and BSA was quenched significantly by NMB. The findings of the Stern–Volmer and double logarithmic plot revealed that the observed emission quenching process was through a static quenching mechanism and the measured binding constant values (Kb) for HSA–NMB and BSA–NMB are 2.766 and 1.187 × 105 dm3 mol−1 respectively. The time-resolved fluorescence lifetime measurement and UV–vis absorption investigation further verify the complex formation between NMB and HSA/BSA. The assessment of thermodynamic parameters disclosed the binding process was spontaneous driven by hydrogen bonds (H-bond) and van der Waals interactions, which contributed a significant role in the complexation. Moreover, the secondary structural conformation and microenvironment of HSA/BSA were modified in the presence of NMB, as evidenced by circular dichroism and synchronous fluorescence data. Molecular docking study predicted a plausible binding mode of NMB inside the binding pocket of HSA and BSA. These results demonstrated that the stabilized NMB is found at the Subdomain IIA (site I) of both the proteins and the results were correlated well with the competitive binding assay. Additionally, the principal components analysis revealed less variation of docked poses for HSA, while, more dispersed docked poses were observed for the BSA model. This also highlights the effects of docking towards a modeled protein (BSA). Molecular dynamic (MD) simulation based binding free energy (ΔGmmgbsa) estimation obtained at 298, 303, 308 and 313 K, were in good agreement with our experimental (ΔGbind) values.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
42.
Fetal bovine serum-a cell culture dilemma van der Valk, Jan
Science (American Association for the Advancement of Science),
2022-Jan-14, 2022-01-14, 20220114, Volume:
375, Issue:
6577
Journal Article
Peer reviewed
Ethical and possible reproducibility issues arise when using fetal bovine serum in cell culture media.
► Photobehavior of MC 540 in presence of BSA and HSA. ► Deviation of monomer–dimer equilibrium of MC540 towards monomer in presence of SAs. ► MC 540 binds both sites I and II of SAs and interactions ...are entropy driven. ► Binding affinity of MC 540 is more with hydrophobic environment of HSA than BSA. ► Secondary structure of BSA only changes in presence of MC 540.
Photophysical studies on binding interactions of a negatively charged anti-tumor photosensitizer, Merocyanine 540 (MC 540), with serum proteins, bovine serum albumin (BSA) and human serum albumin (HSA), have been performed using absorption and steady-state as well as time-resolved fluorescence techniques. Formation of ground state complex has been confirmed from the detailed studies of absorption spectra of MC 540 in presence of SAs producing isosbestic points. Binding between the proteins and MC 540, which perturbs the existing equilibrium between the fluorescent monomer and its non-fluorescent dimer, induces a remarkable enhancement in fluorescence anisotropy and intensity of MC 540 along with a red shift of its maximum. The binding stoichiometry of MC 540 and SAs are more than 1.0 which depicts that two types of complexes, i.e., 1:1 and 2:1 are formed with addition of varied concentration of protein. Both the steady-state and time-resolved fluorescence results show that in 2:1 complex one of the MC 540 molecules is exposed towards aqueous environment with a greater extent when bound with HSA compared to BSA due to the structural flexibility of that protein. Thermodynamic analyses using van’t Hoff plot indicate that the binding between MC 540 and individual SA is an entropy-driven phenomenon. The probable hydrophobic binding site has been located by denaturation of proteins, micropolarity measurement and Förster resonance energy transfer and that is further supported by molecular docking studies. Changes in circular dichroism spectra of BSA in presence of MC 540 depict secondary structural changes of the protein. The induced-CD shows that BSA due to its rigid structure generates chirality in MC 540 much more efficiently compared to HSA.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway ...or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL x h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Free drug concentration in the blood sera is crucial for its appropriate activity. Serum albumin, the universal blood carrier protein, is responsible for transporting drugs and releasing them into ...the bloodstream. Therefore, a drug's binding to SA is especially important for its bioavailability and it is a key problem in the drug design process. In this paper, we present crystal structures of three animal serum albumin complexes: ovine, caprine, and leporine, with diclofenac, a popular non-steroidal anti-inflammatory drug that is used in therapy of chronic and acute pain. Details of diclofenac binding mode by the presented serum albumins are compared with analogous complexes of human and equine serum albumins. The analysis of the occupied binding pockets in crystal structures of the investigated serum albumins from different mammals shows that they have two common and a number of unique diclofenac binding sites. The most intriguing is the fact that the albumins from the described species are able to bind different numbers of molecules of this popular anti-inflammatory drug, but none of the binding sites overlap with ones in the human serum albumin.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Recently, enormous attention has been focused on the development of protein-molecularly imprinted polymers (MIPs). In this sense, bovine serum albumin (BSA) is well regarded as a favorite template in ...various MIPs-based biochemical/analytical assays mainly due to its low price, easy availability, and high structural homology to human serum albumin (HSA). Equally, the implications of BSA in the pathology of different human-related disorders necessitate the development of methods for its precise detection in biological samples. Accordingly, the current review seeks to provide an update on the design, synthesis, and characterization of the developed MIPs which have used BSA as template protein. Also, the recognition and quantification of BSA in different real samples using the prepared MIPs are discussed. Additionally, main strategies, such as surface imprinting, epitope-MIPs, microcontact imprinting and other methods to overcome the problems associated with the molecular printing of BSA are discussed here. The final discussion provides a comparative exploration of different approaches developed, emphasizing their relative advantages and disadvantages and underlining developments and possible future directions.
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•Different types of recently developed BSA-MIPs are overviewed here.•Design, synthesis, and characterization of the BSA-MIPs are described.•Main strategies adopted for overcoming the BSA-MIPs obstacles are highlighted.•Other approaches considered in BSA-MIPs advancement are also discussed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The pharmacokinetic properties of small biotherapeutics can be enhanced via conjugation to cross‐reactive albumin‐binding ligands in a process that improves their safety and accelerates testing ...through multiple pre‐clinical animal models. In this context, the small and stable heavy‐chain‐only nanobody NbAlb1, capable of binding both human and murine albumin, has recently been successfully applied to improve the stability and prolong the in vivo plasma residence time of multiple small therapeutic candidates. Despite its clinical efficacy, the mechanism of cross‐reactivity of NbAlb1 between human and murine serum albumins has not yet been investigated. To unveil the molecular basis of such an interaction, we solved the crystal structure of human serum albumin (hSA) in complex with NbAlb1. The structure was obtained by harnessing the unique features of a megabody chimeric protein, comprising NbAlb1 grafted onto a modified version of the circularly permutated and bacterial‐derived protein HopQ. This structure showed that NbAlb1 contacts a yet unexplored binding site located in the peripheral region of domain II that is conserved in both human and mouse serum albumin proteins. Furthermore, we show that the binding of NbAlb1 to both serum albumin proteins is retained even at acidic pH levels, thus explaining its extended in vivo half‐life. The elucidation of the molecular basis of NbAlb1 cross‐reactivity to human and murine albumins might guide the design of novel nanobodies with broader reactivity toward a larger panel of serum albumins, thus facilitating the pre‐clinical and clinical phases in humans.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. ...5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR's pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR-HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern-Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR-HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA's binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR-HSA complexes, reaffirming that AICAR binds to both site I and site II.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach ...concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
Typical porous silica (SBA-15) has been modified with pore expander agent (1,3,5-trimethylbenzene) and fluoride-species to diminish the length of the channels to obtain materials with different ...textural properties, varying the Si/Zr molar ratio between 20 and 5. These porous materials were characterized by X-ray Diffraction (XRD), N
adsorption/desorption isotherms at -196 °C and X-ray Photoelectron Spectroscopy (XPS), obtaining adsorbent with a surface area between 420-337 m
g
and an average pore diameter with a maximum between 20-25 nm. These materials were studied in the adsorption of human blood serum proteins (human serum albumin-HSA and immunoglobulin G-IgG). Generally, the incorporation of small proportions was favorable for proteins adsorption. The adsorption data revealed that the maximum adsorption capacity was reached close to the pI. The batch purification experiments in binary human serum solutions showed that Si sample has considerable adsorption for IgG while HSA adsorption is relatively low, so it is possible its separation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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