Binding Sites Of Human Serum Albumin Research Articles

The displacement study of 99m Tc-DTPA-Human serum albumin binding in presence of furosemide and metformin by using equilibrium dialysis and molecular docking.

The 99m Tc-DTPA (Technetium99m diethylenetriaminepentaacetic acid), is a radiopharmaceutical used in renal scintigraphy. The human serum albumin (HSA) binding site(s) for the 99m Tc-DTPA have never been characterized. This study will cover in vitro the binding rates of 99m Tc-DTPA on HSA and the 99m Tc-DTPA competition interactions with two drugs having known human serum albumin binding sites. Furosemide (FUR) and metformin (MET) were added to 99m Tc-DTPA solution (weight ratios 1/1 vol:vol) followed by the quantification of 99m Tc-DTPA binding rates to HSA (40 g/L) using equilibrium dialysis and the qualification of this binding using Molecular Modeling methods. The 99m Tc-DTPA binding rates to human serum albumin increased with the highest concentration. Both drugs FUR and MET displaced 99m Tc-DTPA binding. 99m Tc-DTPA could bind to human serum albumin in many locations in site I and I-II, but strongly bound to site I through hydrogen bonds.

Abstract 4660: Molecular mechanisms by which a bioactivated human carcinogen is transported to target tissues

Abstract Aristolochic acid (AA) is a potent human carcinogen and nephrotoxin found in preparations of Aristolochia plants used in Chinese Traditional Medicine. Following biotransformation to form N-sulfonyloxyaristolactam (AL-I-NOSO3), this intermediate undergoes heterolytic cleavage of the sulfate group to generate a reactive cyclic nitrenium ion, the ultimate DNA binding species. Recently, we showed that primary human hepatocytes significantly increase renal toxicity of AA in the integrated human liver-kidney “organs on-chips” model. Therefore, we propose that AA is activated in the liver by forming AL-I-NOSO3, which is transported to the kidney protected from decomposition by binding to serum albumin. We employed mass spectrometric, fluorimetric and X-crystallography based approaches to dissect mechanisms of interactions between human serum albumin (HSA), AA, N-hydroxyaristolactam and AL-I-NOSO3. First, we demonstrate that HSA stabilizes otherwise labile N-sulfonyloxyaristolactam. Quenching of the native fluorescence of HSA due to the presence of a sole molecule of tryptophane-214, allowed us to conclude that all three compounds have similar affinities to IIA drug binding pocket of HSA. Subsequently, we obtained a high-resolution X-ray structure of AA bound to HSA in domain IB (1.9°A, pdb: 6HSC). Since prior to crystallization HSA was enriched with sodium myristate and site IB in circulation is occupied by fatty acids, our results imply that the IB pocket is the primary high affinity binding site for AA and its active forms. To assess whether AL-I-NOSO3 covalently binds protein, we incubated human plasma and purified HSA with this active AA. Immunoblotting of reacted HSA using antibodies that recognize aristolactam(AL)-adducted DNA suggests irreversible covalent adduction of AL to HSA. A combined approach using mass spectrometry instruments and enzymatic digestion revealed that AL is adducted to HSA at the following sites: Trp-214, Tyr-138 and Tyr-141. The former amino acid is located in the IIA drug binding site of HSA, while the latter two can be found in our HSA/AA structure in the site IB in the vicinity to AA molecule, corroborating our X-crystallography and fluoremetric data. Based on these studies we propose that AL-I-NOSO3 has a dual mode of interactions with HSA. If AL-I-NOSO3 decomposes prior to HSA binding, aristolactam will become irreversibly trapped with HSA. This binding to HSA would serve as mechanism of detoxication of AA species. However, if AL-I-NOSO3 binds to HSA prior to decomposition, it should be protected by HSA and transported to target tissues in its intact form. Citation Format: Sergei Pomyalov, Radha Bonala, Robert Rieger, Irina Zaitseva, Charles Iden, John Haley, Robert Turesky, Francis Johnson, Thomas Rosenquist, Arthur P. Grollman, Gil Shoham, Viktoriya S. Sidorenko. Molecular mechanisms by which a bioactivated human carcinogen is transported to target tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4660.

Insights into intramolecular charge transfer fluorescent probes for recognizing human serum albumin

Development of powerful intramolecular charge transfer (ICT) fluorescent probes that can recognize, detect, and visualize human serum albumin (HSA) is important in the fields of biology and biomedicine. Three ICT probes, namely, butyl gallate (BG), isobutyl gallate (IBG), and isoamyl gallate (IAG), which have simple structures, were selected and used to detect HSA. The probes selectively recognized HSA in aqueous solutions by forming stable complexes with the protein. All of the probes exhibited different changes in fluorescence, and HSA underwent significant changes in fluorescence intensity and decay time. We hypothesized that the probes formed electromagnetic coupling with the whole HSA. Not all biomolecules can offer favorable environment for electromagnetic coupling due to their complex structures. The probes shared the same main binding site (located in Sudlow site II) in HSA. The probes and warfarin fluorescence intensity evidently increased in different hydrophobic cavities of HSA. This finding indicated the lack of limit to the location of the probes. A more suitable electromagnetic environment in protein made the ICT probes all had better response ability in HSA than in BSA. Y-type alkyl chains in IBG and IAG were beneficial for their responsiveness. The binding and response abilities of a specific hydrophobic cavity of a protein should be comprehensively considered when designing a novel fluorescent probe.

Spectroscopic and Molecular Docking Approaches for Investigation of Interaction of Phellopterin with Human Serum Albumin

Abstract The mechanism of interaction between human serum albumin (HSA) and natural product phellopterin (PL) from Angelica dahurica was investigated by spectroscopic techniques with molecular docking under simulated physiological conditions. The experimental results showed that the fluorescence of HSA was regularly quenched by PL, and the quenching constants (KSV) decreased with increasing temperature, which indicated that the quenching mechanism was a static quenching procedure. The binding constants (KA) were larger than 10−5 M−1 and the number of binding sites (n) was approximate to 1 at different temperatures, which indicated that the binding affinity was hige and there was just one main binding site in HSA for PL. According to thermodynamic parameters from Van't Hoff equation, the binding process of PL with HSA was spontaneous and exothermic process due to ΔG 0. The binding distance (r) was calculated to be about 3.35 nm, which implied that the energy transfer from HSA to PL occurred with high possibility according to the theory of Forster's non-radiation energy transfer. The microenvironment and conformation of HSA changed with the addition of PL based on the results of synchronous and three-dimensional fluorescence methods. The molecular docking analysis revealed the binding locus of PL to HSA in subdomain IIIA (Sudlow's site II).

In Vitro Cytotoxicity and Interaction of Noscapine with Human Serum Albumin: Effect on Structure and Esterase Activity of HSA.

Noscapine is effective to inhibit cellular proliferation and induced apoptosis in nonsmall cell, lung, breast, lymphoma, and prostate cancer. It also shows good efficiency to skin cancer cells. In the current work, we studied the mechanism of interaction between the anticancer drug noscapine (NOS) and carrier protein human serum albumin (HSA) by using a variety of spectroscopic techniques (fluorescence spectroscopy, time-resolved fluorescence, UV-visible, fluorescence resonance energy transfer (FRET), Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy), electrochemistry (cyclic voltammetry), and computational methods (molecular docking and molecular dynamic simulation). The steady-state fluorescence results showed that fluorescence intensity of HSA decreased in the presence of NOS via a static quenching mechanism, which involves ground state complex formation between NOS and HSA. UV-visible and FRET results also supported the fluorescence result. The corresponding thermodynamic result shows that binding of NOS with HSA is exothermic in nature, involving electrostatic interactions as major binding forces. The binding results were further confirmed through a cyclic voltammetry approach. The FRET result signifies the energy transfer from Trp214 of HSA to the NOS. Molecular site marker, molecular docking, and MD simulation results indicated that the principal binding site of HSA for NOS is site I. Synchronous fluorescence spectra, FTIR, 3D fluorescence, CD spectra, and MD simulation results reveal that NOS induced the structural change in HSA. In addition, the MTT assay study on a human skin cancer cell line (A-431) was also performed for NOS, which shows that NOS induced 80% cell death of the population at a 320 μM concentration. Moreover, the esterase-like activity of HSA with NOS was also done to determine the variation in protein functionality after binding with NOS.

Interaction of Cm(III) with human serum albumin studied by time-resolved laser fluorescence spectroscopy and NMR

The complexation of Cm(III) with human serum albumin (HSA) was investigated using time-resolved laser fluorescence spectroscopy (TRLFS). The Cm(III) HSA species is dominating the speciation between pH 7.0 and 9.3. The first coordination sphere is composed by three to four H2O molecules and five to six coordinating ligands from the protein. For the complex formation at pH 8.0 a conditional stability constant of logK = 6.16 ± 0.50 was determined. Furthermore, information on the Cm(III) HSA binding site were obtained. With increasing Cu(II) concentration the Cm(III) HSA complexation is suppressed whereas the addition of Zn(II) has no effect. This points to the complexation of Cm(III) at the N-terminal binding site (NTS) which is the primary Cu(II) binding site. NMR experiments with Cu(II), Eu(III) and Am(III) HSA show a decrease of the peak assigned to the His C2 proton of His 3, which is part of the NTS, with increasing metal ion concentration. This confirms the complexation of Eu(III) and Am(III) at the Cu(II) binding site NTS. The results presented in this study contribute to a better understanding of relevant biochemical reactions of incorporated actinides.

Effects of conjugation metabolism on radical scavenging and transport properties of quercetin – In silico study

Quercetin (Q) is a natural polyphenol with high radical scavenging capacity, but low in vivo bioavailability. It is extensively transformed by host phase II metabolism and microbiota. Herein, effects of major in vitro and in vivo conjugation transformations of Q on its radical scavenging capacity and human serum albumin (HSA) binding were studied by using appropriate computational approaches, DFT (U)B3LYP/6-31 + G(d,p) and molecular docking, respectively. With regard to radical scavenging capacity of Q, conjugation transformations generally reduce its antioxidant capacity including regeneration efficiency through disproportionation of an intermediate radical species since these structural modifications occur mainly at its radical scavenging –OH groups. They were also found to alter dominant radical scavenging mechanism in a specific way dependent upon conjugation type and site. Concerning distribution by HSA, binding to this main plasma transporter protein may not be dominant transport mechanism for Q and its metabolites in vivo. Like Q aglycon, most of its metabolites are bound non-specifically at multiple binding sites of HSA, with relatively weak affinities. Only sulfo-conjugates including plasma abundant isomer Q-3′-O-SO3–, were predicted to bind specifically in warfarin-like manner, but also with relatively low binding affinity.

Binding affinity studies of 1,2,3-triazole copper(II) complexes to human serum albumin

Two copper(II) complexes with tetradentate 1,4-disubstituted-1,2,3-triazole ligands, [CuL(MeCN)](ClO4)2 (1) and [CuL′](ClO4)2 (2), have been prepared and characterized by different techniques, including X-ray structure determination, spectroscopic, and electrochemical measurements, as reported elsewhere. Herein, we report the interactions of these complexes, and corresponding free ligands, with human serum albumin (HSA) verifying their relative thermodynamic stability and differences in binding to this protein. Interactions with HSA were verified by CD measurements monitored at 564 nm, up to stoichiometric ratio 2:1 [Complex]:[protein], according to competitive equilibria involving the insertion of copper at the selective N-terminal metal binding site in HSA, and additionally at a secondary nonselective site. Further interactions of these complexes with L-tryptophan residues, and probable supplementary site(s) for the binding, were followed by fluorescence measurements. Analogous experiments with the free L and L′ indicated much weaker interactions. Protein oxidation damage was observed for both complexes, monitored by carbonyl groups formation in the presence of H2O2, probably with the participation of reactive oxygen species. Density functional theory calculations exhibit metal-ligand binding interaction energies similar to [Cu(HSA-Nterminal)]+, and reinforced the experimental results, showing clearly that such triazole ligands are competitive toward copper(II) in biological medium.

Design and tuning of a cell-penetrating albumin derivative as a versatile nanovehicle for intracellular drug delivery

Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell-penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl-poly-arginine peptides (CPPs) and an evaluation of their cell-penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D-dodecaarginines, palmitoyl-cyclic-(D-Arg)12 was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell-penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA-paclitaxel complex, b) an HSA-doxorubicin covalent conjugate and c) an HSA-thioredoxin fusion protein. The results showed that cell-penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl-cyclic-(D-Arg)12/HSA is a versatile cell-penetrating drug delivery system with great potential for use as a nano-carrier for a wide diversity of pharmaceutical applications.

Characterization of the interaction between human serum albumin and diazinon via spectroscopic and molecular docking methods.

Human serum albumin (HSA) is a soluble blood protein which binds to small molecules (such as drugs and toxins) and transfers them within the blood circulation. In this research, the interaction of diazinon, as a toxic organophosphate, with HSA was investigated. Various biophysical methods such as fluorescence, ultraviolet-visible (UV-vis), Fourier transform infrared spectroscopy, and molecular docking were utilized to characterize the binding properties of diazinon to HSA under physiological-like condition. The UV-vis spectroscopy showed that the absorption increased and the fluorescence intensity of HSA decreased regularly with regard to the gradual increases of the concentrations of diazinon. Due to the binding constant of ( ka = 3.367 × 10+4 M-1), the α-helix structure for the first day and 35 days of incubation were obtained 66.09-55.4% and 59.99-46.48%, respectively, and their amounts in other secondary structures (β-sheet, β-anti, and random (r) coils) were increased. The molecular docking revealed a good binding site in HSA (Trp-214) for diazinon which was related to the considerable alterations in HSA secondary and tertiary structures. There is a close relationship between the secondary structure of protein and its biological activity and after 35 days of incubation, the high toxic concentrations of diazinon can make HSA to be partially unfolded and lose its structure.

Multiple Spectroscopic and Theoretical Approaches to Study the Interaction between HSA and the Antiparasitic Drugs: Benznidazole, Metronidazole, Nifurtimox and Megazol

The interaction between four antiparasitic drugs (benznidazole (BZL), metronidazole (MTZ), nifurtimox (NFX) and megazol (MZ)) with human serum albumin (HSA), the main vehicle of biodistribution of xenobiotics, hydrophobic, small and endogenous molecules in the bloodstream, was evaluated by multiple spectroscopic techniques and theoretical calculations. In all cases quenching of the fluorescence of HSA by these drugs involve a static mechanism, due to ground state association. There is just one main binding site in HSA for these four ligands (Sudlow’s site I); binding is spontaneous, moderate, does not have any effect on the polarity around the Tyr and Trp residues and does not perturb significantly the secondary structure of the protein. Molecular docking studies suggest hydrogen bonding and hydrophobic interactions as the main binding forces, i.e., BZL associates with the Trp-214 residue via hydrophobic interactions and with Gln-220, Arg-221 and Glu-449 residues via hydrogen bonding; whereas MTZ associates with Leu-197 and Leu-480 residues via hydrophobic interactions and with Trp-214, Glu-449 and Ser-453 via hydrogen bonding. Furthermore, electrostatic interactions were also suggested for HSA:MZ and HSA:NFX.

Computational simulations determining disulfonic stilbene derivative bioavailability within human serum albumin.

Disulfonic stilbene (DS) derivatives are a member of the large family of compounds widely employed in medicine and biology as modulators for membrane transporters or inhibitors of a protein involved in DNA repair. They constitute interesting compounds that have not yet been investigated within the bioavailability framework. No crystallographic structures exist involving such compounds embedded in the most common drug carrier, human serum albumin (HSA). The present work studies, for the first time, the physico-chemical features driving the inclusion of three DS derivatives (amino, nitro and acetamido, named DADS, DNDS and DATDS, respectively) within the four common HSA binding sites using combined molecular docking and molecular dynamics simulations. A careful analysis of each ligand within each of the studied binding sites is carried out, highlighting specific interactions and key residues playing a role in stabilizing the ligand within each pocket. The comparison between DADS, DNDS and DATDS reveals that depending on the binding site, the conclusions are rather different. For instance, the IB binding site shows a specificity to DADS compounds while IIIA is the most favorable site for DNDS and DATDS.

Multispectral and molecular docking studies on the interaction of human serum albumin with iohexol

Interaction between iohexol and human serum albumin (HSA) was studied by using some spectroscopic and molecular docking methods at different temperatures (298, 303, 310 and 318K) under physiological pH. Fluorescence quenching was the result of the collision between fluorophore and the iohexol and dynamic quenching mechanism was dominant. The binding constant (K~103) and the number of binding sites (n~1) were also obtained from fluorescence titration data. The thermodynamic parameters namely enthalpy change (ΔH), Gibbs free energy change (ΔG), and entropy change (ΔS) were calculated and the results suggested that hydrophobic interaction was the significant intermolecular force between HSA and iohexol. FT-IR analyses showed that interaction iohexol with HSA caused a conformational change of the protein. The CD spectra revealed major alterations in the secondary structure of HSA with increase of α-helices content and reduction of β-turn. Binding distance between iohexol and HSA was calculated by the Förster Resonance Energy Transfer (FRET) method. The site marker competitive experiments suggested that the binding site of iohexol and HSA was probably located on sites I and II. Results determined by molecular docking method were in agreement with thermodynamic and spectroscopic data and confirmed the binding mechanism of iohexol to HSA.

Pre-S deletions of hepatitis B virus predict recurrence of hepatocellular carcinoma after curative resection.

The relationship between hepatitis B virus (HBV) and the prognosis of hepatocellular carcinoma (HCC) after surgery remains uncertain. A retrospective cohort study was performed to evaluate the impact of pre-S deletions, T1762/A1764, and A1896 mutations on prognosis of HCC after curative resection. A total of 113 patients with positive serum HBV DNA (>200 IU/mL) who had underwent curative resection of pathologically proven HCC were recruited to determine the risk factors affecting the prognosis.The median follow-up time was 36.5 months and recurrence was detected in 67 patients (59.3%). The cumulative recurrence rates and overall survival rates at 1-, 3-, and 5-year after curative resection were 18.0%, 49.7%, 70.3%, and 93.7%, 61.0%, 42.5%, respectively. Patients with pre-S deletions showed significantly higher recurrence rates compared with those with wild type infection (HR: 1.822, P = .018), but not related with a significantly poor survival (HR: 1.388, P = .235). Subgroup analysis indicated that the patients with type III deletion had significant higher tumor recurrence rates than other deletion types (HR: 2.211, 95% confidence intervals [CI]: 1.008–4.846, P = .048). Multivariate analysis revealed that pre-S deletion, tumor size >3 cm in diameter, and the presence of microvascular invasion were independent risk factors for tumor recurrence. HBV pre-S deletions were found to be clustered primarily in the 5′ end of pre-S2 region and were more often found between amino acids 120 and 142 of the pre-S2 domain. The domains most frequently potentially involved were the transactivator domain in pre-S2 and polymerized human serum albumin binding site.Our cohort showed that pre-S deletions at the time of resection could predict tumor recurrence in HCC patients after curative resection.

One-pot synthesis of Claisen–Schmidt reaction through (E)-chalcone derivatives: Spectral studies in human serum albumin protein binding and molecular docking investigation

ABSTRACTAn efficient and environmentally benign one-pot multicomponent synthesis of E-chalcones was developed using a mild and reusable new boron nitride-sulphonic acid catalyst. The catalyst was prepared by activating the boron nitride surface with nitric acid, followed by a simple reaction with 3-mercaptopropyl trimethoxysilane. The catalyst was characterized and morphological properties were studied by Fourier transform infrared, X-ray diffraction, transmission electron spectroscopy, scanning electron microscopy, Brunauer–Emmett–Teller theory, and Raman spectroscopy techniques. The solid acid catalyst was recycled five times in a Claisen–Schmidt reaction to synthesize new chalcone derivatives, and X-ray crystallography was used to elucidate the structure of (E)-1-(anthracen-9-yl)-3-(2-(4-methylpiperazin-1-yl)quinolin-3-yl)prop-2-en-1-one. A fluorescence quench titration method was used to assess its binding ability with human serum albumin (HSA), while molecular docking was also performed to get a more detailed insight into their interaction at the binding site of HSA.

Binding modes of environmental endocrine disruptors to human serum albumin: insights from STD-NMR, ITC, spectroscopic and molecular docking studies

Given that bisphenols have an endocrine-disrupting effect on human bodies, thoroughly exposing their potential effects at the molecular level is important. Saturation transfer difference (STD) NMR-based binding studies were performed to investigate the binding potential of two bisphenol representatives, namely, bisphenol B (BPB) and bisphenol E (BPE), toward human serum albumin (HSA). The relative STD (%) suggested that BPB and BPE show similar binding modes and orientations, in which the phenolic rings were spatially close to HSA binding site. ITC analysis results showed that BPB and BPE were bound to HSA with moderately strong binding affinity through electrostatic interactions and hydrogen bonds. The order of binding affinity of HSA for two test bisphenols is as follows: BPE > BPB. The results of fluorescence competitive experiments using 5-dimethylaminonaphthalene-1-sulfonamide and dansylsarcosine as competitors, combined with molecular docking indicated that both bisphenols are prone to attach to the binding site II in HSA. Spectroscopic results (FT-IR, CD, synchronous and 3D fluorescence spectra) showed that BPB/BPE induces different degrees of microenvironmental and conformational changes to HSA.

Molecular interaction study of flavonoids with human serum albumin using native mass spectrometry and molecular modeling.

Noncovalent interactions between proteins and small-molecule ligands widely exist in biological bodies and play significant roles in many physiological and pathological processes. Native mass spectrometry (MS) has emerged as a new powerful tool to study noncovalent interactions by directly analyzing the ligand-protein complexes. In this work, an ultrahigh-resolution native MS method based on a 15-T SolariX XR Fourier transform ion cyclotron resonance mass spectrometer was firstly used to investigate the interaction between human serum albumin (HSA) and flavonoids. Various flavonoids with similar structure were selected to unravel the relationship between the structure of flavonoids and their binding affinity for HSA. It was found that the position of the hydroxyl groups and double bond of flavonoids could influence the noncovalent interaction. Through a competitive experiment between HSA binding site markers and apigenin, the subdomain IIA (site 1) of HSA was determined as the binding site for flavonoids. Moreover, a cooperative allosteric interaction between apigenin and ibuprofen was found from their different HSA binding sites, which was further verified by circular dichroism spectroscopy and molecular docking studies. These results show that native MS is a useful tool to investigate the molecular interaction between a protein and its ligands. Graphical abstract Unravel the relationship between the structure of flavonoids and their binding affinity to HSA by native MS.

In silico investigation of propofol binding sites in human serum albumin using explicit and implicit solvation models

All-atom molecular dynamics (MD) simulations are presented on general anesthetic propofol bound to human serum albumin (HSA) due to the drug pharmacokinetics and pharmacodynamics in the circulatory system. We implemented the explicit and implicit solvation models to compare the binding affinity of propofol at the different binding sites (PR1 and PR2) in the HSA protein. Only the implicit solvation models provided the evidence in accordance with the experimental data indicating that the HSA-ligand interactions are dominanted by hydrophobic forces due to the higher drug affinity at the PR1 position with a ΔGMM-PB/SA value of −23.44kcalmol−1. Overall, this study provides important information on the accuracy of explicit and implicit solvation models to characterize the propofol interaction with different HSA binding sites.

Human Serum Albumin as Chiral Selector in Enantioselective High-Performance Liquid Chromatography.

Enantioselective high-performance liquid chromatography (eHPLC) using chiral stationary phases (CSPs) is surely the most used technique for the determination of the enantiomeric excess (e.e.) of chiral drugs, a fundamental parameter for reliable studies on the relationship between stereochemistry and pharmacological activity. A key aspect of this enantioseparation technique is the efficiency of the chiral selector, which can be optimized to obtain higher selectivity and a wider applicability. Thus, the determination of the mechanisms behind chiral recognition is very important to predict and improve the enantioselectivity of CSPs. The present review deals with the preparation and use of CSPs for eHPLC with human serum albumin (HSA) as chiral selector, with particular emphasis on the modulation of the chromatographic performance. HSA-based CSPs allow a relatively easy prediction of the binding sites involved in the retention of analytes and the possibility to improve the selectivity of enantioresolution by modulating the binding process, using either reversible or covalent modifications of the protein. Significant improvements of the chromatographic parameters, such as reduction of analysis time and increase of enantioselectivity, have been obtained for selected analytes by using competitors for a particular binding site of HSA dissolved in the mobile phase or by selectively modifying the protein structure at single amino acid residues.

Albumin binding-site alteration in melancholic depression under pharmacotherapy: Recording with the use of subnanosecond fluorescence spectroscopy

It is known that the conformation of human serum albumin binding sites is sensitive to pathological processes. In this work, changes in the physical–chemical properties of albumin binding sites in melancholic depression were studied. The K-35 fluorescent probe (dimethylaminonaphthalic acid N-carboxyphenylimide, CAPIDAN) was used as a reporter of these changes. It is shown that the fluorescence decay of K-35 depends on the state of the drug-binding sites of albumin. The kinetics of fluorescence decay were measured with a time resolution of approximately 30–50 ps. The parameters that characterize the fluorescence decay of K-35 in serum were reliably responsive to melancholic depression and the dynamics of its treatment. With melancholic depression, a decrease in the concentration of nonesterified long-chain fatty acids that are capable of affecting binding sites of serum albumin was observed. However, variations in the concentration of NEFA cannot be considered as a cause of the alterations of albumin binding sites. In addition to NEFA, other factors are likely to affect structural and physical-chemical properties of albumin in depression patients.