Methacrylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of human serum albumin

Different biologands carrying synthetic adsorbents have been reported in the literature for protein separation. We have developed a novel and new approach to obtain high protein adsorption capacity utilizing 2-methacrylamidohistidine (MAH) as a bioligand. MAH was synthesized by reacting methacrylochloride and histidine. Spherical beads with an average size of 150–200 μm were obtained by the radical suspension polymerization of MAH and 2-hydroxyethyl-methacrylate (HEMA) conducted in an aqueous dispersion medium. p(HEMA-co-MAH) beads had a specific surface area of 17.6 m²/g. Synthesized MAH monomer was characterized by NMR. p(HEMA-co-MAH) beads were characterized by swelling test, FTIR and elemental analysis. Then, Cu(II) ions were incorporated onto the beads and Cu(II) loading was found to be 0.96 mmol/g. These affinity beads with a swelling ratio of 65%, and containing 1.6 mmol. MAH/g were used in the adsorption/desorption of human serum albumin (HSA) from both aqueous solutions and human serum. The adsorption of HSA onto p(HEMA-co-MAH) was low (8.8 mg/g). Cu(II) chelation onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA adsorption was observed at pH 3.0 Higher HSA adsorption was observed from human plasma (94.6 mg HSA/g). Adsorption of other serum proteins were obtained as 3.7 mg/g for fibrinogen and 8.5 mg/g for γ-globulin. The total protein adsorption was determined as 107.1 mg/g. Desorption of HSA was obtained using 0.1 M Tris/HCl buffer containing 0.5M NaSCN. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cu(II) chelated-p(HEMA-co-MAH) beads without significant decreases in the adsorption capacities.     

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.     

Effective removal of albumin from serum

The protein constituents of serum can range from grams to picograms per liter, making it technically difficult to achieve in-depth proteomic analysis. Removal of highly abundant proteins, such as albumin, coupled to powerful protein separation methods is required for increased sample load, thus facilitating detection and identification of low-abundant proteins. We report here a chemical-based extraction method for the effective and specific removal of albumin from serum.     

Stereoselective binding of ketoprofen enantiomers to human serum albumin studied by high-performance liquid affinity chromatography

A chiral stationary phase based on immobilized human serum albumin (HSA) was used to study the stereoselective binding of ketoprofen enantiomers by means of high-performance liquid affinity chromatography. The technique of zonal elution was applied together with a novel mathematical approach describing attachment to more than one type of binding site. Phenylbutazon (PBZ) and diazepam (DAZ) were used as markers for the major believed binding regions on HSA. Both R- and S-ketoprofen (KTR and KTS) display high affinity to the primary PBZ- and DAZ-binding sites and low-affinity to the secondary DAZ sites. The binding to high-affinity regions is accepted to be a stepwise process initiated by the binding to the primary DAZ sites and followed by the attachment to the primary PBZ sites. The chiral recognition is attributed to the high-affinity PBZ-binding sites and to the low-affinity DAZ-binding sites.     

Human serum albumin chromatography by Cibacron Blue F3GA-derived microporous polyamide hollow-fiber affinity membranes

An affinity dye ligand, Cibacron Blue F3GA was covalently attached onto commercially available microporous polyamide hollow-fibre membranes for human serum albumin (HSA) adsorption from both aqueous solutions and human plasma. Different amounts of Cibacron Blue F3GA were incorporated on the polyamide hollow-fibres by changing the dye attachment conditions, i.e. initial dye concentration, addition of sodium carbonate and sodium chloride. The maximum amount of Cibacron Blue F3GA attachment was obtained at 42.5 μmol g⁻¹ when the hollow-fibres were treated with 3 M HCl for 30 min before performing the dye attachment. HSA adsorption onto unmodified and Cibacron Blue F3GA-derived polyamide hollow-fibre membranes was investigated batchwise. The non-specific adsorption of HSA was very low (6.0 mg g⁻¹ hollow-fibre). Cibacron Blue F3GA attachment onto the hollow-fibres significantly increased the HSA adsorption (147 mg g⁻¹ hollow-fibre). The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma (230 mg HSA g⁻¹ hollow-fibre). Desorption of HSA from Cibacron Blue F3GA derived hollow-fibres was obtained using 0.1 M Tris–HCl buffer containing 0.5 M NaSCN or 1.0 M NaCl. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cibacron Blue F3GA derived polyamide hollow-fibre without significant decreases in the adsorption capacities.     

Efficient removal of albumin from human serum by monosize dye-affinity beads

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for removal of human serum albumin (HSA) from human serum. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by dispersion polymerization. Cibacron Blue F3GA loading was 1.73 mol/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA was low (0.8 mg/g polymer). Dye attachment onto the monosize beads significantly increased the HSA adsorption (189.8 mg/g). The maximum HSA adsorption was observed at pH 5.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption capacity decreased drastically. The equilibrium adsorption of HSA significantly decreased with increasing temperature. The elution studies were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the adsorption of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 87% for all studied concentrations. To test the efficiency of HSA removal from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. Eluted proteins include mainly albumin, and a small number of nonalbumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin were bound by the dye-affinity beads. IgA was not identified in eluted fraction.     

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.     

Characterisation of the binding of digitoxin and acetyldigitoxin to human serum albumin by high-performance affinity chromatography

Zonal elution and high-performance affinity chromatography were used to examine interactions of the drugs digitoxin and acetyldigitoxin with the protein human serum albumin (HSA). This was done by injecting small amounts of digitoxin and acetyldigitoxin onto an immobilized HSA column in the presence of mobile phases that contained various concentrations of digitoxin, acetyldigitoxin or other solutes as competing agents. A fixed concentration of β-cyclodextrin was also present in the mobile phase as a solubilising agent. It was found that digitoxin and acetyldigitoxin each had strong interactions at a single common binding site on HSA, but with slightly different equilibrium constants for this region. Neither compound showed any competition with warfarin or L-tryptophan, which were used as probes for binding at the warfarin-azapropazone and indole-benzodiazepine sites of HSA. These results confirmed the presence of a separate binding region on HSA for digitoxin-related compounds.     

Changes of serum albumin affinity for aspirin induced by fatty acid

Saturated fatty acids such as myristic acid play an important role in the pathogenesis of cardiovascular disorders.

Using the quenching fluorescence method we examined the influence of myristate on the changes of transporting protein affinity towards aspirin—the most popular anticoagulant.

Our results showed that the presence of the myristic acid alters the stability of the anticoagulant–albumin complex. The ranges of [myristate]/[albumin] molar ratio at which the stability of drug–protein complex increases or decreases were determined. The differences in interaction between ligands and human or bovine serum albumins were identified. The competition in binding of ligands with these albumins was also described.     

Removal of fatty acids from serum albumin by Lipidex 1000 chromatography

Fatty acids can be effectively removed from serum albumin preparations by a single passage through a column of Lipidex 1000 at 37 degrees C. The procedure is easier and milder and shows a better (nearly quantitative) recovery of protein than charcoal treatment. The ability for fatty acid binding by the protein is not affected by either procedure.     

Identification of high affinity fatty acid binding sites on human serum albumin by MM-PBSA method

Human serum albumin (HSA) has seven common fatty acid (FA) binding sites. In this study, we used the molecular mechanics Poisson-Boltzmann surface area method to identify high affinity FA binding sites on HSA in terms of binding free energy. Using multiple HSA-FA (myristate, palmitate) complex models constructed by molecular dynamics simulations, two methods were performed in molecular mechanics Poisson-Boltzmann surface area, the “three-trajectory method” and the “single-trajectory method”. The former, which is less precise than the latter but may be more accurate as it includes the effects of conformational change upon binding, was used to classify high and low affinity sites. As a result, Sites 2, 4, and 5 were identified as high affinity sites for both FAs. The latter method, which is precise because energies are calculated from snapshots of the same trajectory for HSA-FAcomplex, was performed to compare the magnitude of binding free energy for these sites. The order of magnitude was 5 > 4 > 2, identical to that of a previous publication by others. In this way, a combination of the two methods was effectively used to identify high affinity sites. This study therefore provides an insight into the quantitative identification of high affinity FA binding sites on HSA.     

The electrophoretic desorption of human serum albumin from an affinity chromatography matrix using a preparative desorption cell.

Preparative amounts of human serum albumin (10 mg) have been electrophoretically desorbed from Cibacron Blue F3G-A-Sepharose 4B in 85% yield. For this purpose an electrophoretic desorption cell was constructed that was reusable and capable of containing larger quantities of affinity matrix.     

Efficient and specific removal of albumin from human serum samples

Patient serum or plasma is frequently monitored for biochemical markers of disease or physiological status. Many of the rapidly evolving technologies of proteome analysis are being used to find additional clinically informative protein markers. The unusually high abundance of albumin in serum can interfere with the resolution and sensitivity of many proteome profiling techniques. We have used monoclonal antibodies against human serum albumin (HSA) to develop an immunoaffinity resin that is effective in the removal of both full-length HSA and many of the HSA fragments present in serum. This resin shows markedly better performance than dye-based resins in terms of both the efficiency and specificity of albumin removal. Immunoglobulins are another class of highly abundant serum protein. When protein G resin is used together with our immunoaffinity resin, Ig proteins and HSA can be removed in a single step. This strategy could be extended to the removal of any protein for which specific antibodies or affinity reagents are available.