Polymer–protein interactions in aqueous two phase systems: fluorescent studies of the partition behaviour of human serum albumin

This study describes the partitioning of fluorescent macromolecules in aqueous two-phase systems (ATPS) comprising phosphate salt and poly(ethylene glycol) of three different molecular masses (i.e. 1000, 1450 and 2000 Da). The impact of system assembly was studied with fluorescent macromolecules introduced in contact with either (i) first salt, then polymer or (ii) first polymer, then salt, or (iii) with both salt and polymer simultaneously. Native human serum albumin (HSA) and derivatives labelled with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid (1,5-IAEDANS) were partitioned using selected ATPS. Partitioning behaviour was characterised by molecular rotational studies of recovered proteins based upon changes of depolarisation. Measurements were undertaken by steady-state fluorescence or time-decay fluorescence using a single-photon counting system. In addition, circular dichroism was used as a tool for the study of macromolecular secondary structure. Two discrete categories of stable molecular structure have been identified that exist irrespective of the phase environment. The findings form the basis for a discussion of polymer–protein interactions and the molecular micro-environment of proteins in ATPS.     

Antioxidant protection of human serum albumin by chitosan

Inhibition of protein oxidation by reactive oxygen species (ROS) would confer benefit to living organisms exposed to oxidative stress, because oxidized proteins are associated with many diseases and can propagate ROS-induced damage. We measured the ability of 2800Da chitosan, D-glucosamine and N-acetyl glucosamine to protect human serum albumin from oxidation by peroxyl radicals derived from 2,2'-azobis(2-amidinopropane)dihydrochloride and N-centered radicals from 1,1'-diphenyl-2-picrylhydrazyl and from 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Comparison with the antioxidant action of vitamin C showed that, on a molar basis, chitosan was equally effective in preventing formation of carbonyl and hydroperoxide groups in human serum albumin exposed to peroxyl radicals. It was also a potent inhibitor of conformational changes in the protein, assessed by absorption spectrum and intrinsic fluorescence. D-glucosamine was much less effective and N-acetyl glucosamine was not a useful antioxidant. Protection of the albumin from peroxyl radicals was achieved by scavenging of peroxyl radical. Chitosan was also a good scavenger of N-centered radicals, with glucosamine and N-acetyl glucosamine much less effective. The results suggest that administration of low molecular weight chitosans may inhibit neutrophil activation and oxidation of serum albumin commonly observed in patients undergoing hemodialysis, resulting in reduction of oxidative stress associated with uremia.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

Use of non-covalent labeling in illustrating ligand binding to human serum albumin via affinity capillary electrophoresis with near-infrared laser induced fluorescence detection

This paper demonstrates the use of a near-infrared (NIR) dye as a non-covalent label for human serum albumin (HSA). The dye is a water soluble, heptamethine cyanine dye. The utility of the dye as a tracer illustrating the binding of various drugs to HSA is demonstrated via affinity capillary electrophoresis with near-infrared laser-induced fluorescence detection (ACE-NIR-LIF). Additionally, the factors affecting the separation of relevant species were investigated. The change in quantum yield of the dye upon complexation with HSA was calculated. Spectrophotometric measurements were conducted to study the stoichiometry of the dye albumin complex.     

Application of aqueous two-phase partition to the production of homogeneous preparations of fluorescently labelled human serum albumin

The development of a traceable molecular probe was investigated for the monitoring of partition behaviour of biomolecules in aqueous two-phase systems. This work was based upon the selective labelling of the free thiol group of human serum albumin (i.e. Cys34) with the fluorophore N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic acid. The preparation of homogeneously labelled protein required purification operations. A succession of five processes was successfully applied, comprising two size-exclusion chromatographic operations by gel filtration and a series of three appropriately manipulated aqueous two-phase systems comprising PEG 1450 and phosphate salt. Aqueous two-phase partitioning is herein presented as an alternative to difficult separation and could be applied for ‘fine’ purifications.     

Studies of phenytoin binding to human serum albumin by high-performance affinity chromatography

High-performance affinity chromatography was used to study the binding of phenytoin to an immobilized human serum albumin (HSA) column. This was accomplished through frontal analysis and competitive binding zonal elution experiments, the latter of which used four probe compounds for the major and minor binding sites of HSA injected into the presence of mobile phases containing known concentrations of phenytoin. It was found that phenytoin can interact with HSA at the warfarin-azapropazone, indole-benzodiazepine, tamoxifen, and digitoxin sites of this protein. The association constants for phenytoin at the indole-benzodiazepine and digitoxin sites were determined to be 1.04 (+/-0.05) x 10(4)M(-1) and 6.5 (+/-0.6) x 10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Both allosteric interactions and direct binding for phenytoin appear to take place at the warfarin-azapropazone and tamoxifen sites. This rather complex binding system indicates the importance of identifying the binding regions on HSA for specific drugs as a means for understanding the transport of such substances in blood and in characterizing their potential for drug-drug interactions.     

Flavonoid binding to human serum albumin

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3 × 10⁻⁶ and 3.9 × 10⁻⁵ M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.     

New mathematical approach for the evaluation of drug binding to human serum albumin by high-performance liquid affinity chromatography

A novel mathematical approach for investigation of drug–human serum albumin (HSA) interactions by means of high-performance liquid affinity chromatography is developed. The model is based on the assumption that two types of competitive binding sites exist on the HSA molecule. The widely used single-site binding equation is extended and a proper mathematical analysis is proposed allowing the determination of the major parameters characterizing the multisite binding (cobinding) process. The utility of the new approach is proved by competitive studies on HSA binding of two model drugs, diazepam and diclofenac.     

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van’t Hoff plots. An enthalpy–entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy–Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.     

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA+cysteine, and HSA+glucose in the ratio approximately 50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA+cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.     

Alteration of redox state of human serum albumin in patients under anesthesia and invasive surgery

Human serum albumin is a mixture of mercapt- (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form). We studied the mercapt↔nonmercapt conversion of human serum albumin, which reflects the redox state of the extracellular fluids, in cardiac and other common surgical patients using high-performance liquid chromatography. Mean values of [(HMA)/(HMA + HNA)] ± standard deviation [f_HMA ± σ], for patients who received common surgery (group 1) and cardiac surgery (group 2) at the start of anesthesia were0.636±0.50(n=83) and 0.615±0.062(n=14), respectively. f_HMA values were markedly lower than those for healthy male adults of 0.750±0.028(n=28). f_HMA values increased at 24 h after the start of anesthesia and decreased on the 4th postoperative day in most of the patients. These postoperative changes were prominent in surgical cardiac patients. Although f_HMA values after the 7th postoperative day recovered to those at the start of anesthesia in almost all of common surgical patients, those in cardiac surgical patients, never recovered even on the 21st postoperative day.     

Enrichment of cysteinyl adducts of human serum albumin

We report a method to enrich cysteinyl adducts of human serum albumin (HSA), representing biomarkers of exposure to systemic electrophiles. Because the major site of HSA adduction is the single free sulfhydryl group at Cys34, we used thiol-affinity resins to remove mercaptalbumin (i.e., unadducted HSA) from the cysteinyl adducts. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment of HSA. Differences in adduct content were detected across samples of freshly isolated, archived, and commercial HSA. Cysteinylated and glycosylated adducts were present in all samples, with abundances decreasing in the following order: commercial HSA > archived HSA > fresh HSA. After enrichment of HSA, mercaptalbumin was no longer observed in mass spectra. The ratios of HSA adducts post-/preenrichment, quantified via the Bradford assay and gel electrophoresis, were 0.029 mg adducts/mg HSA in fresh HSA and 0.323 mg adducts/mg HSA in archived HSA. The apparent elevation of adduct levels in archived samples could be due to differences in specimen preparation and storage rather than to differences in circulating HSA adducts. We conclude that thiol-affinity resins can efficiently remove mercaptalbumin from HSA samples prior to characterization and quantitation of protein adducts of reactive systemic electrophiles.     

Chromatographic studies of changes in binding of sulfonylurea drugs to human serum albumin due to glycation and fatty acids

This report examines the use of high-performance affinity chromatography as a screening tool for studying the change in binding by sulfonylurea drugs to the protein human serum albumin (HSA) during diabetes. The effects of both the non-enzymatic glycation of HSA and the presence of fatty acids on these interactions were considered using a zonal elution format. It was found that there was a significant increase (i.e., 2.7- to 3.6-fold) in the relative retention of several sulfonylurea drugs (i.e., acetohexamide, tolbutamide, glybenclamide and gliclazide) on columns containing normal versus glycated HSA. The addition of various long chain fatty acids to the mobile phase gave the same trend in retention for the tested drugs on both the HSA and glycated HSA columns, generally leading to lower binding. Most of the fatty acids examined produced similar or moderately different relative shifts in retention; however, palmitic acid was found to produce a much larger change in retention on columns containing glycated HSA versus normal HSA under the conditions used in this study.     

Use of an immobilised human serum albumin HPLC column as a probe of drug-protein interactions: the reversible binding of valproate

The reversible binding of valproate to human serum albumin determines a decrease of the binding of ligands that selectively bind to site I, site II, and bilirubin binding site. The binding inhibition was followed by displacement chromatography methodology using increasing concentrations of the competitor, i.e. valproate, in the mobile phase. Significant binding inhibition was observed for drugs binding at site I and site II. The greater displacement was observed for the more retained enantiomer of benzodiazepines and profens. A reduction of the affinity was observed also in the case of phenol red, this compound being selected as representative of bilirubin binding site. Difference circular dichroism spectroscopy was also used to characterise the binding of valproate to human serum albumin. This antiepilectic drug was proved to affect the binding at site I, II, and bilirubin binding site. The data have physiological relevance because significant inhibition of the binding resulted at clinic concentrations of valproate.     

Stable misfolded states of human serum albumin revealed by high-pressure infrared spectroscopic studies

Pressure unfolding-refolding and the subsequent aggregation of human serum albumin (HSA) was investigated by high-pressure Fourier transform infrared measurements. HSA is completely unfolded at 1 GPa pressure, but the unfolding is not cooperative. Hydrogen-deuterium exchange experiments suggest that a molten globule-like conformation is adopted above 0.4 GPa. An intermediate was formed after decompression, which differs from the native state only slightly in terms of the secondary structure, but this intermediate is more stable against the temperature-induced gel formation than the pressure-untreated native protein. This observation can be explained by assuming that the pressure unfolded-refolded protein is in a misfolded state, which is more stable than the native one.     

Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool: Nanomolar human serum albumin quantitation

The commercially available dye, NanoOrange, has been investigated as a potential tool for clinical diagnostics due to its low cost, ease of use, and ability to detect nanomolar concentrations of protein. Virtually non-fluorescent in dilute aqueous solutions, NanoOrange fluorescence is enhanced by at least an order of magnitude upon non-covalent interaction with proteins. These features, coupled with the requirement for high throughput assays in the clinical laboratory has prompted the development of two orthogonal NanoOrange approaches. Human serum albumin (HSA) was used as a model protein for the development of both 96-well microplate and capillary electrophoresis laser-induced fluorescence (CE–LIF) assay formats. Dye performance in five commonly used buffers of various concentrations and pH indicated considerable flexibility in assay buffer selection, with optimal performance at pH 9.0. A salt concentration study indicated that increasing NaCl concentration generally decreases fluorescence emission and can be minimized by pre-diluting biological samples to a final salt concentration of 20–80 mM. Titration of protein with NanoOrange resulted in optimal HSA–NanoOrange complex formation utilizing 1× and 2× NanoOrange in the 96-well microplate and CE–LIF approaches, respectively. A NanoOrange binding model based on rapid signal enhancement and zero order fluorescence emission kinetics is proposed. The utilization of NanoOrange in CE–LIF based human serum analysis results in a signal-to-background ratio improvement of up to two orders of magnitude.     

Pyrazolo[3,4-d]pyrimidines-loaded human serum albumin (HSA) nanoparticles: Preparation,characterization and cytotoxicity evaluation against neuroblastoma cell line

Pyrazolo[3,4-d]pyrimidine derivatives 1–5, active as c-Src inhibitors, have been selected to be formulated as drug-loaded human serum albumin (HSA) nanoparticles, with the aim of improving their solubility and pharmacokinetic properties. The present study includes the optimization of a desolvation method-based procedure for preparing HSA nanoparticles. First, characterization by HPLC-MS and Dynamic Light Scattering (DLS) showed a good entrapment efficacy, a controllable particle size (between 100 and 200 nm) and an optimal stability over time, confirmed by an in vitro drug release assay. Then, 1–4 and the corresponding NPs were tested for their antiproliferative activity against neuroblastoma SH-SY5Y cell line. Notably, 3-NPs and 4-NPs were identified as the most promising formulation showing a profitable balance of stability, small size and a similar activity compared to the free drugs in cell-based assays. In addition, albumin formulations increase the solubility of pyrazolo[3,4-d]pyrimidine avoiding the use of DMSO as solubilizing agent.     

Study the interactions between human serum albumin and two antifungal drugs: Fluconazole and its analogue DTP

Binding affinities of fluconazole and its analogue 2-(2,4-dichlorophenyl)-1,3-di(1H-1,2,4-triazol-yl)-2-propanol (DTP) to human serum albumin (HSA) were investigated under approximately human physiological conditions. The obtained result indicated that HSA could generate fluorescent quenching by fluconazole and DTP because of the formation of non-fluorescent ground-state complexes. Binding parameters calculated from the Stern–Volmer and the Scatchard equations showed that fluconazole and DTP bind to HSA with binding affinities of the order 10⁴ L/mol. The thermodynamic parameters revealed that the binding was characterized by negative enthalpy and positive entropy changes, suggesting that the binding reaction was exothermic. Hydrogen bonds and hydrophobic interaction were found to be the predominant intermolecular forces stabilizing the drug–protein. The effect of metal ions on the binding constants of fluconazole–HSA complex suggested that the presence of Mg²⁺ and Zn²⁺ ions could decrease the free drug level and extend the half-life in the systematic circulation. Docking experiments revealed that fluconazole and DTP binds in HSA mainly by hydrophobic interaction with the possibility of hydrogen bonds formation between the drugs and the residues Arg 222, Lys 199 and Lys 195 in HSA.     

New fluorescence of nonenzymatically glucosylated human serum albumin

Glucosylated human serum albumin (G-HSA) obtained under incubation with glucose at 37 degrees C for 8 days showed a new fluorescence with a maximum at 430 nm, resulting in quenching of the fluorescence of only one tryptophan residue on HSA. The quantum yield of new fluorescence is 0.024 at 25 degrees C. The analysis of the excitation spectra allowed us to conclude the absence of energy transfer. In G-HSA, non-disulfide cross-linking hexamer was confirmed by SDS-PAGE.     

DNA-duplex linker for AFM-SELEX of DNA aptamer against human serum albumin

DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350 pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFM-SELEX.