Human serum albumin (HSA) serves as a crucial indicator for therapeutic monitoring and biomedical diagnosis. In this study, a near infrared (NIR) fluorescent probe, termed BTPA, characterized a donor-π-acceptor (D-π-A) structure based on bridged triphenylamine (TPA) was developed. BTPA exhibited outstanding sensitivity and selectivity towards HSA among various analysts, with a remarkable 50-fold fluorescence enhancement with a significant Stokes shift (∼190 nm) and a wide linear detection range of 0-20 μM of HSA. Especially, BTPA displayed selectivity for discrimination of HSA from BSA. Job\'s Plot analysis suggested a 1:1 stoichiometry for the formation of the BTPA-HSA complex. Displacement assays and molecular docking demonstrated that BTPA binds to subdomain IB of HSA which could effectively avoid interference from most drugs. Besides, BTPA have good biocompatibility and could detect of exogenous HSA with a relatively low fluorescence background. For practical applications, BTPA was tested for detecting HSA levels in human urine without any pretreatment, showing detection capability in the range of 0-10 μM with a fast response (<30 s), a limit of detection (LOD) of 0.12 μM and good recoveries (81.7-92.9 %), highlighting the high performance of bridged triphenylamine-based probe BTPA.

The competition among drugs for binding to plasma proteins is regarded as a pharmacokinetic drug interaction. Competition between antitumor agents and other drugs for plasma protein binding can alter the free concentration of the drug, potentially impacting its efficacy and increasing the risk of toxic side effects. Through a range of spectroscopic techniques, this study examined the interaction between limonin and human serum albumin (HSA) in the context of berberine (Ber) and curcumin (Cur) under physiological conditions to clarify the binding mechanisms of binary and ternary systems at the molecular level. As demonstrated by fluorescence quenching experiments, Static quenching was identified as the mechanism of interaction between HSA and limonin. The results of site competition experiments indicated that the binding site between limonin and HSA was site I, a result further supported by molecular docking simulations. Through the use of thermodynamic data calculations, it was determined that limonin forms a stable complex with HSA by establishing hydrogen bonds and van der Waals forces. Circular dichroism (CD) spectroscopy, three-dimensional (3D) fluorescence spectroscopy, and synchronous fluorescence spectroscopy (SFS) employed to validate the notion that limonin perturbed the microenvironment of amino acids and induced conformational changes in HSA. What\'s more, the presence of Ber or Cur was found to have further modified the alterations observed in the interaction between the original HSA-limonin binary system. In vitro cellular experiments showed that interaction with HSA reduced the antitumor activity of limonin. In contrast, adding Ber or Cur increased the inhibition rate of tumor cells. The coexistence of both Ber and Cur significantly diminished limonin\'s binding affinity to HSA. The current investigation enhances comprehension regarding the binding characteristics and interaction mechanisms involving limonin, Ber, Cur, and HSA. It explores the potential of HSA as a versatile drug carrier and furnishes theoretical underpinnings for co-administrative strategies.

YM201636 is the potent PIKfyve inhibitor that is being actively investigated for liver cancer efficacy. In this study, computer simulations and experiments were conducted to investigate the interaction mechanism between YM201636 and the transport protein HSA. Results indicated that YM201636 is stably bound between the subdomains IIA and IIIA of HSA, supported by site marker displacement experiments. YM201636 quenched the endogenous fluorescence of HSA by static quenching since a decrease in quenching constants was observed from 7.74 to 2.39 × 104 M-1. UV-vis and time-resolved fluorescence spectroscopy confirmed the YM201636-HSA complex formation and this binding followed a static mechanism. Thermodynamic parameters ΔG, ΔH, and ΔS obtained negative values suggesting the binding was a spontaneous process driven by Van der Waals interactions and hydrogen binding. Binding constants ranged between 5.71 and 0.33 × 104 M-1, which demonstrated a moderately strong affinity of YM201636 to HSA. CD, synchronous, and 3D fluorescence spectroscopy revealed that YM201636 showed a slight change in secondary structure. The increase of Kapp and a decrease of PSH with YM201636 addition showed that YM201636 changed the surface hydrophobicity of HSA. The research provides reasonable models helping us further understand the transportation and distribution of YM201636 when it absorbs into the blood circulatory system.

Pyridoxal-S-methyl-isothiosemicarbazone (PLITSC) is a member of an important group of ligands characterized by different complexation modes to various transition metals. In this contribution, a new complex containing two differently protonated PLITSC ligands ([Fe(PLITSC-H)(PLITSC)]SO4)∙2.5H2O was obtained. The crystal structure was solved by the X-ray analysis and used further for the optimization at B3LYP/6-311++G(d,p)(H,C,N,O,S)/def2-TZVP(Fe) level of theory. Changes in the interaction strength and bond distance due to protonation were observed upon examination by the Quantum Theory of Atoms in Molecules. The protein binding affinity of [Fe(PLITSC-H)(PLITSC)]SO4 towards transport proteins (Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA)) was investigated by the spectrofluorimetric titration and molecular docking. The interactions with the active pocket containing fluorescent amino acids were examined in detail, which explained the fluorescence quenching. The interactions between complex and DNA were followed by the ethidium-bromide displacement titration and molecular docking. The binding along the minor groove was the dominant process involving complex in the proximity of DNA.

Radiotherapy plays a vital role in cancer therapy. However, the hypoxic microenvironment of tumors greatly limits the effectiveness, thus it is crucial to develop a simple, efficient, and safe radiosensitizer to reverse hypoxia and ameliorate the efficacy of radiotherapy. Inspired by the structure of canonical nanodrug Abraxane, herein, a native HSA-modified CaO2 nanoparticle system (CaO2-HSA) prepared by biomineralization-induced self-assembly is developed. CaO2-HSA will accumulate in tumor tissue and decompose to produce oxygen, altering the hypoxic condition inside the tumor. Simultaneously, ROS and calcium ions will lead to calcium overload and further trigger immunogenic cell death. Notably, its sensitizing enhancement ratio (SER = 3.47) is much higher than that of sodium glycididazole used in the clinic. Furthermore, in animal models of in situ oral cancer, CaO2-HSA can effectively inhibit tumor growth. With its high efficacy, facile preparation, and heavy-metal free biosafety, the CaO2-HSA-based radiosensitizer holds enormous potential for oral cancer therapy.

Through molecular recognition, drugs can interact and complex with macromolecules circulating in the body. The serum albumin transport protein, found in several mammals, has several interaction sites where these molecules can be located. The drug sulfasalazine (SSZ) is known in the literature to complex at drug site 1 (DS1) in human serum (HSA) and bovine serum (BSA) proteins. This complexation can be studied using various spectroscopic techniques. With the techniques used in this work, absorption in the ultraviolet and visible regions (UV-Vis) and electronic circular dichroism (ECD), a significant difference was observed in the results involving HSA and BSA. The application of theoretical methodologies, such as TD-DFT and molecular docking, suggests that the conformation that SSZ assumes in DS1 of the two proteins is different, which exposes it to different amino acid residues and different hydrophobicities. This difference in conformation may be related to the location of DS1 where the drug interacts or to the possibility of SSZ moving in the BSA site, due to its larger size, and moving less freely in HSA.

Per- and poly-fluorinated compounds constitute a wide group of fluorocarbon chemicals with widespread industrial applications, ranging from non-stick coating in cookware to water surfactants, from fire-fighting foams to water-repellent coatings on textiles. Presently, over 12,000 PFAS are known worldwide. In recent years, extensive research has focused on investigating the biological effects of these molecules on various organisms, including humans. Here, we conducted in silico simulations to examine the potential binding of a representative selection of PFAS to various human proteins known to be involved in chemical transportation and accumulation processes. Specifically, we targeted human serum albumin (HSA), transthyretin (TTR), thyroxine binding protein (TBG), fatty acid binding proteins (FABPs), organic anion transporters (OATs), aiming to assess the potential for bioaccumulation. Molecular docking simulations were employed for this purpose, supplemented by molecular dynamics (MD) simulations to account for protein flexibility, when necessary. Our findings indicate that so-called \"legacy PFAS\" such as PFOA or PFOS exhibit a higher propensity for interaction with the analysed human protein targets compared to newly formulated PFAS, characterised by higher branching and hydrophilicity, and possibly a higher accumulation in the human body.

The Shark-derived immunoglobulin new antigen receptors (IgNARs) have gained increasing attention for their high solubility, exceptional thermal stability, and intricate sequence variation. In this study, we immunized whitespotted bamboo shark (Chiloscyllium plagiosum) to create phage display library of variable domains of IgNAR (VNARs) for screening against Human Serum Albumin (HSA), a versatile vehicle in circulation due to its long in vivo half-life. We identified two HSA-binding VNAR clones, 2G5 and 2G6, and enhanced their expression in E. coli with the FKPA chaperone. 2G6 exhibited a strong binding affinity of 13 nM with HSA and an EC50 of 1 nM. In vivo study with a murine model further provided initial validation of 2G6\'s ability to prolong circulation time by binding to HSA. Additionally, we employed computational molecular docking to predict the binding affinities of both 2G6 and its humanized derivative, H2G6, to HSA. Our analysis unveiled that the complementarity-determining regions (CDR1 and CDR3) are pivotal in the antigen recognition process. Therefore, our study has advanced the understanding of the potential applications of VNARs in biomedical research aimed at extending drug half-life, holding promise for future therapeutic and diagnostic progressions.

BACKGROUND: Glycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.
METHODS: We enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC-MS/MS.
RESULTS: Human albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.
CONCLUSIONS: Albumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Synthetic phenolic antioxidants (SPAs) and relevant transformation products (TPs) are potentially neurotoxic pollutants to which humans are widely exposed. However, their penetration behavior across the brain barrier and associated exposure to the central nervous system (CNS) remain unknown. This study is the first to investigate a wide range of 30 SPAs and TPs, including emerging SPAs, in matched serum and cerebrospinal fluid (CSF) samples from children in Guangzhou, China. Sixty-two children of either sex aged <14 years with nonbloody CSF and complete clinical information were included. The findings demonstrated the ubiquitous occurrence of many SPAs and TPs, particularly BHT, 2,4-di-tert-butylphenol (DBP), AO 1010, AO 1076, BHT-Q, and BHT-quinol, not only in serum but also in the CSF. Median total concentrations of SPAs and TPs were up to 22.0 and 2.63 ng/mL in serum and 14.5 and 2.11 ng/mL in CSF, respectively. On calculating the penetration efficiencies across the blood-CSF barrier (BCSFB) (RCSF/serum, CCSF/Cserum) for selected SPAs and TPs, their RCSF/serum values (median 0.52-1.41) were highly related to their physicochemical properties, indicating that passive diffusion may be the potential mechanism of BCSFB penetration. In addition, the RCSF/serum values were positively correlated with the barrier permeability index RAlb (AlbuminCSF/Albuminserum), indicating that barrier integrity is an important determinant of BCSFB penetration. Overall, these results will improve our perception of human internal exposure to SPAs and lay a solid foundation for assessing the risk of CNS exposure to various SPAs.