Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation have been prepared. They were obtained by a “ post-magnetization” procedure involving a simple treatment of the various affinity gels with magnetic ferrofluid. The magnetic biospecific adsorbents tested include magnetic protein A-Sepharose for isolation of IgG antibodies, magnetic human serum albumin (HSA)-Sepharose for anti-HSA isolation, and magnetic 2′,5′-ADP for isolation of glucose-6-phosphate dehydrogenase from baker’s yeast and hemolyzates of human red blood cells. For the latter enzyme, a 11,000-fold purification was achieved in one step.     

分析型色谱饼对人血清白蛋白的快速纯化

采用分析型色谱饼对标准蛋白混合物进行了分离,结果表明装填有小颗粒填料的色谱饼在高流速条件下仍然具有良好的分离能力。在较大流速(5 mL/min)条件下,在10 min内对人血清白蛋白样品进行了快速纯化,其纯化后的人血清白蛋白的纯度大于85%,回收率为65%,说明分析型色谱饼可以用于快速分离纯化生物大分子。     

Evidence of changes in hydrophilic/hydrophobic balance and in chemical activity of HSA induced by thermal treatments

Samples of human serum albumin (HSA) obtained as a result of heat denaturation followed by refolding controlled by a cooling of the protein solution were studied by several methods: chromatographic measurements, kinetic of the reaction with a water soluble free radical and by electron paramagnetic resonance (EPR) spectroscopy. In this context the interaction of this protein with β-cyclodextrin (β-CD) and sodium dodecyl sulfate (SDS) was also investigated. Reversed phase thin layer chromatography (RP-TLC) showed changes in lipophylicity of HSA, which are related with the existence of different ensembles of conformers. The UV-Vis absorption spectra had shown the broadening of absorption band of the protein and a hyperchrom effect in the presence of SDS; β-CD reduces the effect of SDS on protein UV-Vis spectra.     

Calorimetric characterization of 2′,3′-dideoxyinosine water solution

A study of 2′,3′-dideoxyinosine (ddI) stability and its interaction with human serum albumin (HSA) was carried out by differential scanning microcalorimetry DSC. Scan rate dependent and irreversible endothermic thermal degradation of ddI was analyzed with use of kinetic approach. Observed process could be interpreted in terms of simple first-order one step kinetic model. Moreover it was shown that ddI bound weakly to the human serum albumin and stabilized this protein.     

Fluorescence of 1,3-Di(1-pyrenyl)propane probe incorporated into human serum albumin protein enforced conformations of the probe

 Steady-state and time-resolved fluorescence spectra of 1,3-di(1-pyrenyl)propane (1Py-(3)-1Py) incorporated into macromolecules of human serum albumin (HSA), into micelles of dodecyltrimethylammo-nium chloride (DTAC), and dissolved in 1,4-dioxane were compared. The steady-state fluorescence spectra indicated that in all the mentioned environments, upon excitation of 1Py-(3)-1Py, light was emitted from the single pyrene chromophores (1Py^*) and from the 1Py, 1Py^* excimers. The time-resolved fluo-rescence emission registered at 480 nm (excimer emission) for 1Py-(3)-1Py in the DTAC micelles and dissolved in 1,4-dioxane allowed to monitor formation of excimer with time constant τ₁=40.0 ns and 9.6 ns, for 1Py-(3)-1Py in the DTAC micelles and in 1,4-dioxane, respectively. However, when the 1Py-(3)-1Py probe was located inside of the macromolecules of HSA, only the decay of emission was observed for excimer with our set-up (t>2 ns after excitation). The instantaneous formation of excimer, unrelated to the decay of monomer excitation, indicates that the considerable fraction of 1Py-(3)-1Py in the hydrophobic pockets of HSA is present as the ground state dimer. The red shift (Δλ=8 nm) and broadening of UV absorption for 1Py-(3)-1Py in HSA (when compared with absorption 1Py-(3)-1Py in 1,4-dioxane) and comparison of exci-tation spectra of 1Py-(3)-1Py in HSA and in 1,4-dioxane also indicate that label molecules bound to some sites of HSA are in the ground state in the dimer conformation. Moreover, the close values of the ratios of intensities of monomer emission to excimer emission, registered 2 ns (5 ns gate) after excitation pulse with duration 300 ps and at the steady-state conditions, indicate that the interconversion between conformers of 1Py-(3)-1Py inside of the macro-molecules of HSA is slow in comparison with the decay time of Py chromophore in the excited state in HSA (two-exponential decay with decay times τ₁=2.41 ns, τ₂=69.0 ns). Thus, ratios of the intensities of monomer and excimer emissions of 1Py-(3)-1Py in HSA do not allow to obtain any information on the local microfluidity inside of the protein macromolecules but could be used for discrimination between different conformations of the probe, possibly located in different protein pockets. Received: 29 April 1996 Accepted: 15 August 1996     

Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes

Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation.     

光谱法研究噻菁染料与血清蛋白的相互作用

本文通过吸收和荧光光谱法研究了一种噻菁染料与人血清蛋白及牛血清蛋白的相互作用。吸收光谱数据表明,与血清蛋白结合后,噻菁染料单体的吸收峰发生红移,同时强度也有很大变化;还通过吸收光谱计算确定了噻菁染料与血清蛋白的结合位点数（ n ）。与人血清蛋白或牛血清蛋白结合后,噻菁染料的荧光量子产率增加。分析噻菁染料的荧光强度随溶液中血清蛋白浓度的变化得到了二者反应的表观结合常数（ K _a）和自由能变化（ ΔG ）。根据表观结合常数（ K _a）可以判断,人血清蛋白比牛血清蛋白与噻菁染料的结合更强。     

Probing the dynamics of domain III of human serum albumin entrapped in sol–gel derived silica using a Sudlow’s site II specific fluorescent ligand

Previous studies from our lab reported on the use of time-resolved fluorescence anisotropy (TRFA) to probe the dynamics of domains I and II within the model protein, human serum albumin (HSA), in solution and when entrapped into sol–gel derived silica. In order to further our understanding of the dynamics within this multi-domain protein, TRFA was used to measure the dynamics of domain III of the protein. For this purpose, the fluorescence ligand dansylsarcosine (DS), which has a 400-fold higher emission intensity in the bound state relative to the free state and an emission lifetime of >22 ns when bound to Sudlow’s site II (domain III) in HSA, was selected. This probe is able to accurately report on slow rotational motions (up to 300 ns correlation time) and the bound form of the probe can be selectively measured at 475 nm, ensuring that the dynamics reflect only the properly folded form of the protein. The mobility of HSA with bound dansylsarcosine (HSA–DS) was evaluated in solution and after entrapment in sol–gel derived silica prepared from sodium silicate under varying ionic strength and pH conditions. The results here show that (1) the 43 ns global rotational correlation time of HSA in buffered solution can be accurately measured via labeling with DS with no interference from faster local or segmental motions; (2) the global motion of HSA in silica is greatly hindered immediately after encapsulation, with no correlation time faster than 300 ns discernable, indicative of strong templating of the silica around domain III of the native protein; and (3) the addition of salt and variation of pH have essentially no effect on HSA mobility, ruling out electrostatics as the primary interaction restricting HSA motion. The results from this study are compared to past studies using intrinsic tryptophan fluorescence (domain II) or fluorescein-labeled HSA (domain I), and demonstrate that motion observed using such probes likely reflects differential mobility of the three domains, consistent with domain III of HSA adsorbing to or templating with silica upon entrapment while the other domains protrude into the pore. Restricted motion of domain III of HSA was also observed in silica materials derived from diglycerylsilane or tetraethylorthosilicate, showing that templating is not dependent on the silica precursor or processing conditions.     

一种转基因猪血中重组人血清白蛋白分离纯化新方法

基因工程技术已经成为研究和生产重组人血清白蛋白(rHSA)替代人血清白蛋白(HSA)的重点技术,而白蛋白的纯化则是该技术的关键。本文主要介绍了从转基因猪血中纯化rHSA的一种新方法,即热乙醇沉淀与多级色谱分离相结合的rHSA纯化方法。热乙醇沉淀法可从猪血浆中获得rHSA粗提取液,此时rHSA的纯度可达69.5%,回收率达51.3%。进一步采用多级色谱分离法,即阴离子交换色谱和反相色谱法进一步纯化,得到rHSA的最终纯度约为100.0%,总回收率为41.1%。该方法为从转基因猪血浆中大规模纯化用于临床和生化研究的高纯度rHSA提供可能,同时也为rHSA替代HSA奠定了基础。     

Free flow electrophoresis for the purification of proteins: I. Zone electrophoresis and isotachophoresis

The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97% alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.     

Determination of trace proteins with pyronine Y and SDS by resonance light scattering

A new resonance light scattering (RLS) probe for determining proteins is presented. The weak RLS of pyronine Y–SDS can be enhanced substantially by adding proteins in the presence of H₂SO₄, resulting in a strong and wide RLS band in the region 310–425 nm. The interaction of pyronine Y–SDS with proteins was studied on the basis of this behavior and a new quantitative method was developed for determining proteins. The enhanced RLS intensity is proportional to the concentration of proteins in the range 0.15–3.6 μg mL⁻¹ for bovine serum albumin (BSA) and 0.06–4.8 μg mL⁻¹ for human serum albumin (HSA), with detection limits of 21.0 and 12.0 ng mL⁻¹, respectively. This method is characterized by high sensitivity, rapidity of reaction, and simplicity. Four synthetic samples were determined satisfactorily and recovery was 99.5–101.5%. Results for human serum and urine samples were in agreement with those obtained by the Bradford method, with relative standard deviations (RSD) of 1.5–3.1%.     

Preparation and chromatographic behavior of acetate-fiber filter rods with dye affinity ligands

Summary The dyes cibacron blue F3GA and reactive red 120 have been immobilized on acetate fiber filter rods to produce potential affinity matrixes. The isothermal adsorption of bovine serum albumin or human serum albumin on these matrixes was investigated and proved to conform to the Freundlich equation. In the static adsorption of human plasma the adsorption capacity for human serum albumin was 12.5 mg g⁻¹ fiber filter and one band was observed in SDS-PAGE electrophoresis of human serum albumin isolated by the immobilized cibacron blue F3GA filter rod. Ligand utilization efficiencies and breakthrough volumes were obtained for adsorption of HSA on the cibacron blue F3GA filter rod when the feed-rates were from 0.5 to 6 mL min⁻¹. In the affinity chromatography of human plasma the yield of human serum albumin was 1.27 g per 100 mL plasma.     

Polarographic immunoassay coupled with catalysis of non-radioactive multiple iodine label

A now polarographic immunoassay was developed In this assay,human serum albumin (HSA) as the model antigen was covalently labeled with organic compound erythrosin B(EB) containing four non-radioactive iodides through Ⅰ step chemical reaction The labeling procedure is simple and the conditions needed are moderate.The molar labeling ratio of KB HSA was 12 Ⅰ The content of iodine in the conjugate obtained by the proposed procedure is ninth higher than that by the other existing methods.A heterogeneous competitive immunoassay was established by compling the catalysis of the conjugate to substrate As(Ⅲ)-Ce(Ⅳ) reaction with the linear-sweep polarographic detec-tion of As(Ⅲ) amount HSA can be determined in the HSA concentration range from 1 to 200μg/mL,with the de-tection hum of 0 66μg/ml.     

Chiral discrimination ofN-(dansyl)-dl-amino acids on human serum albumin stationary phase: Effect of a mobile phase modifier

Summary The effect of perchlorate anion as mobile phase modifier on the separation factor, α, forN-(dansyl)-dl-norvaline andN-(dansyl)-dl-tryptophan on a human serum albumin (HSA) column was studied by varying the concentration,c, of the chaotropic agent and the column temperatureT. Gibbs-Helmholtz parameters Δ(ΔH) and Δ(ΔS) between thed andl enantiomers were determined from linear van't Hoff plots of lnα against 1/T. Thermodynamic results indicated that the enhancement of the separation factor observed asc was increased was enthalpically controlled owing to stereoselective H-bonding interactions. Such behavior was used to optimize the chromatographic conditions for separation ofN-(dansyl)-amino acids on HSA.     

Characterization of interaction property of multicomponents in Chinese Herb with protein by microdialysis combined with HPLC

Interaction of traditional Chinese Herb Rhizoma Chuanxiong and protein was studied by microdialysis coupled with high performance liquid chromatography. Compounds in Rhizoma Chuanxiong, such as ferulic acid, senkyunolide A and 3-butylphthalide, were identified by HPLC, HPLC-MS and UV-vis. Microdialysis recoveries and binding degrees of compounds in Rhizoma Chuanxiong with human serum albumin (HSA) and other human plasma protein were determined: recoveries of microdialysis sampling ranged from 36.7 to 98.4% with R.S.D. below 3.1%; while binding to HSA ranged from 0 to 91.5% (0.3 mM HSA) and from 0 to 93.5% (0.6 mM HSA), respectively. Compared with HSA, most of compounds bound to human blood serum more extensively and the results showed that binding of these compounds in Rhizoma Chuanxiong was influenced by pH. Two compounds were found to bind to HSA and human blood serum, their binding degrees were consistent with ferulic acid and 3-butylphthalide, the active compounds in Rhizoma Chuangxiong.     

Selective on-line serum peptide extraction and multidimensional separation by coupling a restricted-access material-based capillary trap column with nanoliquid chromatography–tandem mass spectrometry

As the serum peptidome gets increasing attention for biomarker discovery, one of the important issues is how to efficiently extract the peptides from highly complex human serum for peptidome analysis. Here we developed a fully automated platform for direct injection, on-line extraction, multidimensional separation and MS detection of peptides present in human serum. A capillary SPE column packed with a novel mix mode restricted access material (RAM) exhibiting strong cation exchange and size exclusion chromatography (SCX/SEC) properties were coupled with a nanoliquid chromatography–mass spectrometry (nanoLC-MS) system. The capillary SPE column excludes the high abundant serum proteins such as HSA by size exclusion chromatography and simultaneously extracts the low molecular weight peptides by binding to sulfonic acid residues. Subsequently, the trapped peptides are eluted to a capillary LC column packed with a RP-C18 stationary phase. After injection of only 2 μL human serum to the one-dimensional nanoLC-MS system around 400 peptides could be identified. When conducting a multidimensional separation, the described SCX/SEC/RP-MS platform allows the separation and identification of 1286 peptides present in human serum by the injection and on-line processing of 20 μL human serum sample.     

Determination of drug-protein interactions by combined microdialysis and high-performance liquid chromatography

Summary A simple and rapid method for determination of the parameters of the interaction between drugs and protein, including the association constant and the number of binding sites, has been developed by use of a microdialysis sampling technique combined with high-performance liquid chromatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazepine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as examples) were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. The microdialysis probe was the used to sample the mixed CBZ-HSA solution at a perfusion rate of 1 μL min⁻¹. The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance liquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; theRSD ofR was approximately 1.85%. The estimated association constant (K) and the number of the binding sites,n, on one molecule of HSA were 1.06×10⁴ M⁻¹ and 0.880, respectively, which is in good agreement with the literature values determined by high-performance frontal analysis. The potential use of microdialysis is also discussed.     

High-performance affinity chromatography and the analysis of drug interactions with modified proteins: binding of gliclazide with glycated human serum albumin

This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (K _a) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high-affinity sites (average K _a, 7.1–10 × 10⁴ M⁻¹) and a group of lower-affinity sites (average K _a, 5.7–8.9 × 10³ M⁻¹) at pH 7.4 and 37 °C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the K _a values for gliclazide at these sites being 1.9 × 10⁴ and 6.0 × 10⁴ M⁻¹, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification.     

A Crosslinked Carboxylic Acid Containing Cation Exchange Monolithic Cryogel for Human Serum Albumin Separation

For this work, we synthesized poly(N-isopropylacrylamide-acrylamide)-acrylic acid (poly(NIPAM-Am)-AAc) monolithic cryogel for a human serum albumin separation (HSA) from a protein mixture (human serum immunoglobulin, human serum albumin and lysozyme) and performed HSA adsorption studies using the cryogel to do continuous system experiments in a syringe column connected by a peristaltic pump. Poly(NIPAM-Am)-AAc with a pore size of 10–100 μm was produced by free radical polymerization that proceeded in an aqueous solution of monomers frozen inside a syringe column. The monolithic poly(NIPAM-Am)-AAc cryogel was characterized by performing swelling studies, FTIR and SEM that showed a swelling ratio of 6.2 g H₂O/g dry cryogel. The maximum HSA adsorption by the cryogel was 42.5 mg/g polymer at pH 4.0 in a 50 mM acetate buffer. We also studied the effect of two different temperatures (25 and 40°C). The higher temperature increased the adsorption capacity of the cryogel. HSA molecules could be reversibly adsorbed and desorbed five times with the same poly(NIPAM-Am)-AAc cryogel without a noticeable loss of their HSA adsorption capacity. The synthesized cryogel was used to separate albumin from the protein mixture. Adsorbed albumin was eluted by changing the pH of the buffer (pH 7.0 and 25°C). Poly(NIPAM-Am)-AAc monolithic cryogel behaved as a cation exchange column because of its functional carboxylic group.     

Capillary electrophoresis frontal analysis for the study of flavonoid interactions with human serum albumin

Capillary electrophoresis based on the principles of frontal analysis (CE-FA) was used to characterize the binding of flavonoids to human serum albumin (HSA) at near-physiological conditions: 67 mM phosphate buffer (pH 7.4), temperature 36.5 °C. The studied flavonoids (flavone, rutin, quercitrin) displayed moderate affinities toward the human serum albumin with binding constants in the range 10³−10⁴ M⁻¹. The binding of the flavonoids to the protein noticeably depended on their lipophilicity and decreased in the case of glycosylation. The corresponding thermodynamic parameters characterized the acting forces between the HSA and flavonoids as mainly hydrophobic forces and electrostatic interactions. Based on the results of the displacement experiments, the binding of the flavonoids took place at site I of the HSA molecule. The results demonstrated by CE-FA were similar to those obtained by fluorescence spectroscopy. The developed method proved to be a reliable alternative to conventional methods, providing a lot of useful parameters for characterization of ligand–protein interactions.