Binding Capacity Of Human Serum Albumin Research Articles

Bisphenol a modification and how its structure influences human serum albumin binding force

Bisphenol A (BPA) is an environmental hormone with substantial physiological toxicity to the human body. Three electron-withdrawing derivatives of BPA are synthesized in this study to reduce its toxicity. The interaction mechanism between BPA and its three derivatives with human serum albumin (HSA) is investigated using molecular docking, electrochemistry, circular dichroism spectrum analysis, and fluorescence spectroscopy. Temperature-dependent tests and time-resolved fluorescence spectra reveal that all four compounds dynamically quench the internal fluorescence of HSA. Based on molecular docking and site competition outcomes, four compounds are primarily bound to site I's active pocket. Thermodynamic parameters indicate that the compounds’ interaction with HSA is spontaneous, and introducing weak electron-withdrawing groups changes the action force. Circular dichroism and synchronous fluorescence measurements suggest four compounds impact HSA's structure. The substituents' electron attraction capacity determines their impact. Electrochemical studies examine the effect of electron-absorbing groups on the BPA and HSA binding capacity. Pharmacokinetics showed that nitro-substituted BPA may be a potential superior substitute for BPA. These results indicate crucial practical value, providing theoretical support for future research.

Effects of Oxidation of Human Serum Albumin on the Binding of Aripiprazole.

Aripiprazole (ARP) is one of antipsychotics and binds to human serum albumin (HSA) with a high affinity. In this study, we investigated the binding characteristics of ARP to oxidized HSA as observed in chronic disease conditions. Oxidized HSAs were prepared using chloramine-T (CT-HSA) or metal-catalyzed oxidation system (MCO-HSA) in vitro, respectively. An increase in the carbonyl content was confirmed in oxidized HSAs. From the results of circular dichroism (CD) and tryptophan fluorescence spectra, no significant structural change of oxidized HSAs was observed. These results indicate that prepared HSAs are mildly oxidized and well reflects the status of HSA during chronic diseases. However, oxidized HSAs were observed to have a significant decrease in binding to ARP. The results of the induced CD spectrum suggested that ARP bound to oxidized HSAs with a similar orientation. These results suggest that oxidation of HSA during chronic disease state significantly affected the microenvironment of the binding site for ARP and binding capacity of HSA to ARP.

Effect of Cu2+ and Zn2+ ions on human serum albumin interaction with plasma unsaturated fatty acids.

Human serum albumin (HSA) serves as a depot and carrier of multiple unrelated ligands including several participants of the pathogenesis of Alzheimer's disease (AD), such as amyloid β peptide (Aβ), Zn2+/Cu2+ ions, docosahexaenoic (DHA), linoleic (LA), and oleic (OA) acids. To explore the interplay between HSA interaction with Zn2+/Cu2+ and the plasma unsaturated fatty acids (DHA, LA, OA, and arachidonic acid (ArA)), we have studied the metal dependence of the fatty acid (FA) binding capacity of HSA (nmax) and structural consequences of the HSA-FA interactions. HSA loading with Zn2+ decreases nmax value by 0.3-1.5, while its saturation with Cu2+ causes the FA-dependent nmax changes by up to 0.9. The Cu2+-induced decline in nmax value for DHA is due to conformational rearrangements in HSA molecule. In other cases, the changes in nmax are attributed to steric hindarance/facilitation of the HSA-FA interaction because of the protein multimerization/monomerization, as confirmed by chemical crosslinking. The surface hydrophobicity of HSA is Cu2+-, Zn2+-, and FA-dependent and decreases upon the FA binding, according to bis-ANS fluorescence data. Overall, Zn2+ or Cu2+ binding selectively affect HSA interaction with the FAs studied, in part due to changes in quaternary structure of the protein.

New insights into the altered binding capacity of pharmaceutical-grade human serum albumin: site-specific binding studies by induced circular dichroism spectroscopy

The ADMET profile of drugs is strongly affected by human serum albumin (HSA), due to its leading role as carrier of poorly soluble compounds in plasma; a critical assessment of the binding capacity of HSA and the evaluation of binding competition between drugs are therefore pivotal for a reliable pharmacokinetic and pharmacodynamic characterization. In clinical practice, a potential source of impairment in the binding properties of HSA is the use of octanoate and N-acetyltryptophan as stabilizers during the production of pharmaceutical-grade HSA for infusion (i-HSA), which is currently administered in the treatment of a growing range of pathological conditions. The peculiar sensitivity of circular dichroism (CD) spectroscopy towards the stereochemical features of high-affinity binding events is herein exploited to achieve a site-specific assessment of the effect of stabilizers on the binding properties of i-HSA. The binding affinity and capacity of fatty-acid-free HSA towards site-selective induced circular dichroism (ICD) markers for the three high-affinity binding sites of HSA was compared to that of i-HSA submitted to ultrafiltration and dialysis to remove both stabilizers. Results showed a considerable impairment of the binding capacity of i-HSA at site II and a relatively lower influence on the binding properties of site I. Ultrafiltration proved to be ineffective in depleting octanoate, while the proposed dialysis protocol, which involves a pH-induced reversible unfolding of the protein, resulted in a total clearance of both stabilizers, confirmed by the full restoration of the binding properties of HSA at all binding sites. The outcomes of this study proved that CD spectroscopy is a suitable technique to evaluate the binding properties of i-HSA, ensuring an assessment of the availability of the binding sites and the possibility of monitoring the clearance of stabilizers. Eventually, the proposed method for their depletion might constitute a connection bridge between albumin in vitro studies and its clinical applications.

Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid conjugation

Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the conjugation yield likely due to the enhanced solubility of palmitic acid analog.

Non-enzymatic glycation enhances human serum albumin binding capacity to sodium fluorescein at room temperature: A spectroscopic analysis

BackgroundSodium fluorescein (SF) is a fluorescent tracer dye used extensively in diagnostic tools in the field of Ophthalmology, particularly in intravenous fluorescein angiography (IVFA). The binding of SF to human serum albumin (HSA) has been predicted by molecular docking and investigated by circular dichroism (CD) and fluorescence spectroscopy with or without glycation at temperatures 296, 301, and 310K. MethodsThe binding parameters were calculated by quenching of emission spectrum of a constant concentration of SF (2μmol/l) at 513nm against increasing concentrations of glycated or unmodified HSA as quencher starting from stoichiometry ratio of 1:1. ResultsThe HSA-SF interaction found to be a static binding. The Stern-Volmer constants (Ksv) were in the range of ~104M−1 and other thermodynamic parameters like enthalpy (ΔH°), free energy (ΔG°) and entropy (ΔS°) are similar to albumin ligand bindings reported by previous workers. ConclusionsThe interactions were found to be spontaneous, irrespective of temperature or glycation. Glycated HSA is clinically used to monitor unstable glycemic controls in diabetic patients. A 39% increase in binding affinity (log K) and free energy (ΔG°) is reported on glycation at 310K (room temperature), which may be important in the SF based angiographies. On glycation HSA-SF binding appears to change from an enthalpy-driven to an entropy-driven reaction. SF shows best binding to FA binding site III of HSA, which also overlaps with drug binding site II of subdomain IIIA. Leu430 seems to play a pivotal role in the interaction.

Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding

The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp–HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment–HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA).

In vitro hemocompatibility and toxic mechanism of graphene oxide on human peripheral blood T lymphocytes and serum albumin.

Graphene oxide (GO) has shown tremendous application potential as a biomedical material. However, its interactions with blood components are not yet well understood. In this work, we assess the toxicity of pristine GO (p-GO) and functionalized GO (GO-COOH and GO-PEI) to primary human peripheral blood T lymphocytes and human serum albumin (HSA), and also study the underlying toxic mechanism. Our results indicate that p-GO and GO-COOH have good biocompatibility to T lymphocytes at the concentration below 25 μg mL(-1), but notable cytotoxicity above 50 μg mL(-1). By contrast, GO-PEI exhibits significant toxicity even at 1.6 μg mL(-1). Further investigations show that although p-GO does not enter into the cell or damage the membrane, its presence leads to the increase in reactive oxygen species (ROS), moderate DNA damage, and T lymphocyte apoptosis. Interestingly, little effect on T lymphocyte immune response suppression is observed in this process despite p-GO inflicting cell apoptosis. The toxic mechanism is that p-GO interacts directly with the protein receptors to inhibit their ligand-binding ability, leading to ROS-dependent passive apoptosis through the B-cell lymphoma-2 (Bcl-2) pathway. Compared with p-GO, GO-COOH exhibits a similar toxic effect on T lymphocytes except keeping a normal ROS level. A proposed toxic mechanism is that GO-COOH inhibits protein receptor-ligand binding, and passes the passive apoptosis signal to nucleus DNA through a ROS-independent mechanism. On the other hand, GO-PEI shows severe hematotoxicity to T lymphocytes by inducing membrane damage. For plasma protein HSA, the binding of GO-COOH results in minimal conformational change and HSA's binding capacity to bilirubin remains unaffected, while the binding of p-GO and GO-PEI exhibits strong toxicity on HSA. These findings on the interactions of two-dimensional nanomaterials and biological systems, along with the enquiry of the mechanisms, would provide essential support for further safety evaluation of the biomedical applications of GO.

Immobilization of HSA on polyamidoamine-dendronized magnetic microspheres for application in direct chiral separation of racemates.

Magnetic microspheres containing dendrimers and chiral selectivity show great performance for applications in pharmacology and biomedicine. In this study, a novel chiral magnetic nano-selector was prepared by immobilizing human serum albumin (HSA) on polyamidoamine dendrimer (PAMAM)-modified magnetic silica microspheres (PMSMs). The functionalized magnetic microspheres had a core-shell structure, with an average diameter of 600 nm. They exhibited strong magnetic response, with a high magnetization saturation of 46 emu g-1. UV-vis spectrophotometry and confocal laser scanning microscopy indicated that the HSA binding capacity on PMSM increased with increasing PAMAM generation. An immunoaffinity assay indicated that HSA retained a high level of activity, after immobilization on PMSM. HSA-immobilized PMSM (HSA-PMSM) was applied in the direct chiral separation of three biological racemates. The separation results show that HSA-PMSM selectively interacted with the racemates, and exhibited different activity toward each enantiomer. The results obtained by CD and CE indicated that HSA immobilized on third-generation PMSM possessed excellent chiral separation capability and biocompatibility. The chiral recognition capability of HSA-PMSM increased with increasing PAMAM generation. The chiral magnetic nano-selector is effective for the enantiomeric separation of chiral drugs, and has potential application in pharmacological and biomedical research.

Effects of nanoparticle surface ligands on protein adsorption and subsequent cytotoxicity.

With the widespread use of nanoparticles (NPs) such as quantum dots (QDs) in biomedical applications, and the growing concerns about nanotoxicity of these engineered nanoparticles, the importance of nanoparticle-protein interaction has not been well emphasized. In order to better understand the physical basis of the biological activity of nanoparticles in nanomedicine applications or under conditions of environmental exposure, the interaction of CdSe/ZnS QDs having different surface ligands as a model with human serum albumin (HSA) has been investigated in detail by various spectroscopic techniques including UV-vis absorption, fluorescence, circular dichroism, and Fourier transform infrared (FTIR) spectroscopies. We find that QDs coated with zwitterionic d-penicillamine (DPA-QDs) or anionic mercaptosuccinic acid (MSA-QDs) bind to the same site of serum albumin, domain II A, site I, and the differences of the Stern-Volmer quenching constant KSV and the binding constant K are about sixfold and sevenfold after 4 h of mixing, respectively. We also find tentative evidence that the model proteins undergo conformational changes upon association with the QDs that have different surface ligands. Additional cellular cytotoxicity assays, with HeLa cells, reveal that the stronger adsorption capacity of HSA on the surface of MSA-QDs results in more reduced cytotoxicity for the protein-coated QDs, while the weaker binding capacity of HSA on DPA-QDs has less effect on the interaction of QDs with cells. These findings have shed light on the design and application of QDs nanomaterials to nanomedicine by a comprehensive preconsideration of their interaction with human serum proteins.

Regulation of Amantadine Hydrochloride Binding with IIA Subdomain of Human Serum Albumin by Fatty Acid Chains

Human serum albumin (HSA) is a major protein component of blood plasma that has been exploited to bind and transport a wide variety of endogenous and exogenous organic compounds. Although anionic drugs readily associate with the IIA subdomain of HSA, most cationic drugs poorly associate with HSA at this subdomain. In this study, we propose to improve the association between cationic drugs and HSA by modifying HSA with fatty acid chains. For our experiments, we tested amantadine hydrochloride, a cationic drug with antiviral and antiparkinsonian effects. Our results suggest that extensive myristoylation of HSA can help stabilize the interaction between amantadine and HSA in vitro. Our X-ray crystallography data further elucidate the structural basis of this regulation. Additionally, our crystallography data suggest that anionic drugs, with a functional carboxylate group, may enhance the association between amantadine and HSA by a mechanism similar to myristoylation. Ultimately, our results provide critical structural insight into this novel association between cationic drugs and the HSA IIA subdomain, raising the tempting possibility to fully exploit the unique binding capacity of HSA's IIA subdomain to achieve simultaneous delivery of anionic and cationic drugs.

Purification of human IgG by negative chromatography on ω-aminohexyl-agarose

The ω-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69–76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir–Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL −1 of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing ω-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.

Albumin stabilizes (–)‐epigallocatechin gallate in human serum: Binding capacity and antioxidant property

(-)-Epigallocatechin gallate (EGCg) is the major component of green tea and is known to show strong biological activity, although it can be easily oxidized under physiological conditions. In this study, we indicate that EGCg is stable in human serum and that human serum albumin (HSA) stabilizes EGCg under aerobic condition. Although EGCg is usually decomposed within 1 h in aqueous solution at neutral pH, EGCg in serum and phosphate buffer (pH 7.4) containing HSA was stable over 1 h, even at neutral and slightly alkaline pH. Under these conditions, EGCg binds to HSA non-covalently. The sulfhydryl group acts as an antioxidant for EGCg oxidation. Incubation of EGCg with HSA is accompanied by the oxidation of a free sulfhydryl group in HSA. These results suggest that the antioxidant property and the binding capacity of HSA contribute to the stabilization of EGCg in human serum.

Separation of human plasma proteins HSA and HIgG using high-capacity macroporous gel-filled membranes

This paper discusses the separation of human serum albumin (HSA), the most abundant protein present in plasma from human immunoglobulin G (HIgG) by membrane chromatography using a novel macroporous gel-filled membrane (designated Q Type 2). The membrane was prepared by anchoring a quaternary ammonium salt macroporous gel within the pores of a non-woven, polypropylene fabric. Factors affecting HSA binding were examined and operating conditions suitable for separating it from human plasma were identified. At an optimized condition, the HSA binding capacity of this novel membrane under saturating conditions was in the range of 290–300 mg/ml. This was not only significantly higher than binding capacities reported for other chromatographic membranes, but also higher than binding capacities of conventional gel based chromatographic media. The protein binding capacity was also largely insensitive to the superficial velocity, indicating the dominance of convective protein transport to and from the binding sites. The suitability of using this membrane for plasma fractionation was demonstrated by the separation of a simulated feed solution consisting of HSA and HIgG.

Location of High and Low Affinity Fatty Acid Binding Sites on Human Serum Albumin Revealed by NMR Drug-competition Analysis

Human serum albumin (HSA) is an abundant plasma protein that transports a wide variety of drugs and endogenous compounds. The complex binding capacity of HSA has made it a challenging system to study in detail but in order to develop our understanding of the interactions between ligands for HSA, the locations and relative affinities of different ligand binding sites must be determined. Albumin possesses multiple binding sites for its primary physiological ligand, non-esterified fatty acids (FA). Previously, titration of BSA with 13C-labeled FA revealed multiple chemical shifts and allowed identification of a subset of three chemical shifts that were associated with high affinity FA binding. Recent crystallographic studies of HSA have mapped at least seven FA binding sites for long-chain FA and delineated the overlap with binding sites for drugs and other endogenous compounds. We aim to correlate NMR and structural data for FA to provide a more complete description of the binding capacity of HSA. Our recent mutagenesis studies allowed us to identify two high affinity binding sites in domain III of HSA. Here, we use NMR to study the binding of 13C-carboxyl labeled palmitate to HSA in the presence and absence of competitor ligands to complete the correlation of NMR chemical shifts with specific structural binding sites. We carefully selected ligands with specific binding sites identified by crystallography and used them, either singly or in combination, to compete with [ 13C]palmitate for binding to HSA. We show that FA sites 2, 4 and 5 bind FA with high affinity, while sites 1, 3, 6 and 7 exhibit low affinity for FA, thus providing the first complete determination of relative affinities of all seven long-chain FA sites on HSA. Our results also yield direct insights into the interactions between FA and other ligands.

Binding of vanadate to human albumin in infusion solutions, to proteins in human fresh frozen plasma, and to transferrin

The binding of vanadate (V) to human serum albumin (HSA) in infusion solutions, to human fresh frozen plasma (FFP), and to human transferrin (TF) was investigated over a wide concentration range. Free V concentrations were obtained by ultrafiltration. Total and free V concentrations were determined using electrothermal atomic absorption spectrometry (ETAAS). Binding parameters were obtained by non-linear regression. V only bound appreciably to HSA at low concentrations (<1 μM). The binding capacity of HSA was about 1000-fold lower than that of FFP and TF per mole of protein. Binding to FFP and TF in the concentration range investigated could be described by a combination of saturable and additional non-saturable binding. The respective maximal binding capacities ( B max, μM), dissociation constants ( k D, μM), and proportionality constants ( C) for the non-saturable, linear binding were B max=27, k D=2.5, C=0.19 for FFP and B max=47, k D=0.47, C=0.38 for TF. The results suggest that V is predominantly bound to transferrin in FFP. It is concluded that HSA in infusion solutions represents a reservoir of readily accessible V. Nevertheless, given the high binding capacity of transferrin in plasma, the amount of vanadate delivered via the brief administration of HSA solutions is unlikely to be of major importance.