The reversible binding of anti-human serum albumin to poly beta-cyclodextrin-coated porous silica supports.

A supramolecular system involving host-guest interactions between immobilized beta-cyclodextrin (beta-CD) cavities and adamantyl groups was evaluated for the preparation of immunosorbents which can be regenerated after use. First a dextran layer bearing both adamantyl groups and carboxylic functions is immobilized onto beta-CD-modified porous silica particles (400 nm) by formation of inclusion complexes. Then, antibody molecules are grafted to the polymer layer. The stationary phases can be prepared in batch or directly in the column. They are stable in aqueous media and are able to trap specifically the corresponding antigen. In case of alteration of the antibody layer, it is possible to remove it by passing a SDS solution through the column. The feasibility of the procedure was evaluated, using the anti-HSA/HSA system.

Binding of human serum albumin to silica particles by means of polymers: a liquid chromatographic study of the selectivity of resulting chiral stationary phases.

Chiral stationary phases obtained by immobilization of human serum albumin (HSA) on various polymer-coated silicas were tested to resolve DL-tryptophan, DL-NBP, RS-oxazepam and RS-warfarin racemic mixtures. HSA immobilized on anion exchangers [quaternized poly(vinylimidazole)-coated silica] was highly selective. Stable and selective chiral stationary phases were also prepared by covalent binding of HSA to silica particles via reactive-polymers. Poly(acryloyl chloride), poly(methacryloyl chloride) and poly(vinyl chloroformate) derivatives were compared. Parameters that govern the selectivity of resulting chiral supports were evaluated, especially the orientation of HSA after immobilization, the mobility of polymer chains and the number of covalent linkages between the protein and the polymer.

Structural changes of human serum albumin immobilized on chromatographic supports: a high-performance liquid chromatography and Fourier-transform infrared spectroscopy study.

Chiral stationary phases obtained by immobilization of HSA on [C8] and [C18] reversed-phases and on poly(1-vinylimidazole)-coated silica were tested to resolve DL-tryptophan, N-benzoyl-DL-phenylalanine, RS-oxazepam and RS-warfarin racemic mixtures. Parameters of enantioselectivity measured in HPLC are correlated to structural and solvation states for adsorbed HSA, evaluated by FTIR spectroscopy. HSA immobilized on [PVI]-anion-exchangers is highly selective. HSA molecules are not self-associated, only unfolded for a small hydrophobic helix. The HSA-coated reversed-phases have a lower selectivity. Unfolding is larger but the indole-benzodiazepine chiral site is preserved and remains accessible.

Study of the retention properties of warfarin enantiomers on a beta-cyclodextrin polymeric support.

High-performance liquid chromatography was used to study the retention properties of (R)- and (S)-warfarins on a silica support coated with a beta-cyclodextrin polymer. The influence of the methanol content of the acetate buffer eluent was investigated at pH 4. The measure of the variations of retention time with temperature enables one to determine the enthalpy and the entropy of adsorption. The plot of the two thermodynamic functions shows a minimum around 30% (v/v) methanol. At low methanol contents, the decrease of the hydrophobic interactions with increasing methanol content explains the decrease of the enthalpic and entropic terms. Above 40% (v/v) methanol, the decrease of the adsorption enthalpy absolute value is due to the solvation by the organic component. From the analysis of peak shape in mass-overload conditions, the column capacity toward each enantiomer was determined. A lower capacity was found toward (S)-warfarin, the more retained enantiomer. Peak shape analysis in mass-overload conditions was used to determine the adsorption isotherm. A Langmuir-type adsorption isotherm accounts well for the experimental data.

High-performance liquid chromatographic study of the interactions between immobilized beta-cyclodextrin polymers and hydrophobically end-capped polyethylene glycols.

The formation of inclusion complexes between polyethylene glycols (PEGs) bearing hydrophobic ends (naphtyl and phenyladamantyl) and beta-cyclodextrin polymers (poly beta-CD) immobilized onto silica particles was studied by high-performance liquid chromatography (HPLC). It was shown that hydrophobic interactions were involved in the retention mechanism of these compounds, since retention volumes decreased when organic solvents were added to the mobile phase while it was the contrary in the presence of salts. Moreover, the association could be reversed by adding a competitor (hydroxypropyl beta-cyclodextrin) to the mobile phase. A theoretical model permitted the evaluation of affinity constants of 1:1 complexes formed between the modified PEGs and the immobilized poly beta-CD which depended on the type of hydrophobic groups grafted to the PEG.

A simple chiral high-performance liquid chromatographic method to study the enantiomer-differentiating action of microorganisms. An assay with DL-tryptophan.

To investigate the 'enantiomer-differentiating' action of the microorganisms colonizing a phosphate-buffered DL-tryptophan solution, a novel chiral high-performance liquid chromatographic (HPLC) arrangement was developed and established. As the HPLC stationary phase, bovine serum albumin (BSA) bonded silica gel was used. In the function of the mobile phase, phosphate-buffered DL-tryptophan solution was applied. The composition of the eluate was monitored by an HPLC spectrophotometric detector. After injecting the assayed sample into the eluent stream, the content of each tryptophan enantiomer was evaluated either from the positive or negative responses of the HPLC detector. The validity and the performance of this novel approach were confirmed by applying another chiral HPLC device working with human serum albumin (HSA) bonded silica gel as the stationary phase and with 1-propanol containing phosphate buffer as the eluent.

A study of strontium binding to albumins, by a chromatographic method involving atomic emission spectrometric detection.

A chromatographic method involving ICP-AES (inductively coupled plasma atomic emission spectrometry) detection has been successfully applied for the study of strontium-protein complexes. The chromatographic step involves the use of gel filtration-a large-zone Hummel and Dreyer method-which allows to dissociate the bound metallic ions and the free ones. This step is followed by an ICP-AES analysis of fractions collected throughout the chromatographic experiment: the concentration of ionic metallic species in solution can therefore be calculated. Two proteins have been tested: bovine serum albumin, which showed only weak interactions with Sr2+ ions, and bovine alpha-lactalbumin: this protein, well-known for its calcium binding capacity, proved to interact strongly with strontium. The influence of various parameters on the formation of strontium-lactalbumin complexes were determined, namely temperature, pH. Competition experiments between Sr2+ ions and, respectively Na+ and Ca2+ ions were also performed, by varying ionic strength of the medium, and by using both apo and native forms of bovine alpha-lactalbumin.

Capillary electrophoretic behavior of milk proteins in the presence of non-ionic surfactants.

The electrophoretic behavior of alpha-lactalbumin and beta-lactoglobulins (A and B) in the presence of non-ionic surfactants was studied by capillary electrophoresis (CE), using a poly(ethylene glycol) coated capillary column. The surfactants (Tween 20, Brij 35 and 78) were used as buffer additives. The separation is based on the difference in the strength of protein-surfactant association complexes, which results in a change of the effective electrophoretic mobility. The modification of the electrophoretic mobilities of proteins was observed and this variation permitted the estimation of the interaction between protein and surfactant. The effect of surfactant type and concentration on the migration behavior of protein in CE is discussed. It is found that the retention behavior of the milk proteins (the alpha-lactalbumin and the beta-lactoglobulins) in CE is very different. The pH of the buffer and the surfactant type influence significantly the protein-surfactant interactions.

Effective one/two step purification of various materials by solid-phase extraction.

Simple one/two step purification procedures based on the solid-phase extraction technique were effectively exploited to clean up radiolabelled drugs represented by dihydrochloride of [6-3H]-stobadine and hydrochloride of [4-3H]-pentacaine, derivatization agents such as 4-nitrobenzoyl chloride or 3,5-dinitrobenzoyl chloride, as well as the aqueous phosphate or triethylamine acetate buffer solutions.

Enantiomeric properties of human albumin immobilized on porous silica supports coated with polymethacryloyl chloride.

Human serum albumin (HSA) was bound to porous silica, using a reactive polymer derived from polymethacryloyl chloride. Two different procedures were used for coating silica with the polymer. In the first method, the polymer was deposited onto amino-silica by reaction between its reactive functions and NH2 groups on silica. In the second method, the monomer was first linked to the amino-silica and copolymerization with the excess of monomer initiated thereafter. The enantiomeric properties of the resulting supports after the coupling of HSA were compared using different mobile phases. The higher amount of HSA bound using the later method, resulted in higher retention of the enantiomers and better enantioselectivity.

Enantioselectivity properties of human serum albumin immobilized on anion-exchangers based on polyvinylimidazole-coated silica. Effect of protein loading on separation properties.

Chiral chromatographic supports were obtained by continuously applying solutions contained HSA to ion-exchange columns. The columns were packed with silica modified with polyvinylimidazole and a copolymer polyvinylpyrrolidone-polyvinylimidazole (75:25) respectively, quaternized and crosslinked. Small changes in the concentration of NaCl during immobilization of HSA lead to variations in the amount of HSA bound to the supports. These variations have consequences in terms of chromatographic retention (k'), selectivity (alpha) and resolution (Rs) of enantiomers. The effects of varying the pH and organic modifier of the mobile phase on the chromatographic properties were also examined.

Chromatographic method involving inductively coupled plasma atomic emission spectrometric detection for the study of metal-protein complexes.

A chromatographic method has been used to study metal ion-protein complexes. It involves successively a gel filtration technique to separate and distinguish the complexed from the free metallic ions, and a spectrometric technique, inductively coupled plasma atomic emission spectrometry (ICP-AES), which allows us to calculate accurately the concentration of ionic metallic species in solution. In the chromatographic step, we applied a large-zone Hummel and Dreyer method. Thus, fractions can be collected throughout the chromatographic experiment and their metal concentration measured by ICP-AES, at constant and known protein concentration. This method has been tested on the copper complex of bovine serum albumin. Results of our study are in good agreement with previous studies on this complex.

Reversible binding interactions between the tryptophan enantiomers and albumins of different animal species as determined by novel high performance liquid chromatographic methods: an attempt to localize the D- and L-tryptophan binding sites on the human serum albumin polypeptide chain by using protein fragments.

The stereoselectivity of the reversible binding interactions between the D- and L-tryptophan enantiomers and serum albumins of different animal species and fragments of human serum albumin (HSA) was investigated by applying three novel high performance liquid chromatographic (HPLC) arrangements. The separations were performed by means of 1) an achiral (diol-bond), 2) a chiral (bovine serum albumin-bond) silica gel sorbent, and 3) a column switching technique which uses both the diol- and HSA-bond HPLC stationary phases. A polarimetric detector and/or an ultraviolet (UV) spectrophotometer were used to monitor the separation process. HPLC arrangement 3 allowed the evaluation of enantioselective binding for D- and L-tryptophan to different albumins and albumin fragments. At present, column switching can be considered the technique of the broadest applicability for investigating the reversible binding interactions between a protein and drug enantiomers.

Insight into the distribution of molecular weights and higher-order structure of hyaluronans and some beta-(1 --> 3)-glucans by size exclusion chromatography.

The effects of high energy ultrasound and slightly raised temperature combined with the denaturing action of dimethylsulphoxide on the molecular weight and higher-order structure of hyaluronans and some beta-(1 --> 3)-glucans were studied by means of size exclusion chromatography (SEC) technique. Some experimental conditions connected with the (bio-)polymer sample preparation prior to its SEC analysis are overviewed in the light of informational relevance of studies where the action of a physical and/or chemical agent changes the hydrodynamic size of the m omolecule.

Study of conformational effects of recombinant interferon gamma adsorbed on a non-porous reversed-phase silica support.

Reversed-phase chromatography is a powerful method for separating recombinant interferon gamma and one of its analogues differing only by a single amino acid residue. Structural differences of the proteins explain this separation ability as demonstrated from adsorption studies on a non-porous reversed-phase support. To reveal the structural differences occurring in the adsorbed state, two different and independent methods were employed. The variation of the retention with the slope of the linear gradient gave information about the molecular contact area of the protein with the support. For different experimental conditions, these data were correlated with the adsorbent capacities measured on an n-octadecyl-modified non-porous silica support. These supports are useful for these types of experiments because the protein is adsorbed exclusively at the external surface of the beads. Moreover, a small amount of protein is necessary to saturate the column, owing to its low capacity.

Molecular approach to protein-polymer interactions in ion-exchange chromatography.

A model was developed and implemented to aid in understanding and predicting the retention behaviour of proteins in ion-exchange chromatography. The model structures chosen were calcium-loaded and -depleted alpha-lactalbumin (ALC) and hen egg white lysozyme (HEWL) and a comparison was made with chromatographic measurements. A characteristic charge of -3.4 was found under the experimental conditions applied for both forms of ALC, and HEWL was not retained. The model explicitly considers all of the atoms, each being assigned a set of force field parameters. Because of the computational time necessary to include them, water molecules were not taken into account, but a sigmoidal function of the dielectric permittivity was introduced in the calculations. Interaction potential energies from bulk down to the contact were evaluated for each protein. The results were in qualitative agreement with those of the chromatographic experiments. It was possible to reproduce the difference in retention between both forms of ALC and also the behaviour of HEWL.

Retention behaviour of proteins on poly(vinylimidazole)-copper(II) complexes supported on silica: application to the fractionation of desialylated human alpha 1-acid glycoprotein variants.

The retention behaviour of various amino acids, peptides and proteins on poly(vinylimidazole)-Cu(II) complexes supported on silica was investigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary structure were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results obtained the proteins containing metal binding groups suggested that, in addition to the presence of donor-acceptor interactions between the macromolecules and the immobilized metal, other additional (essentially ionic and/or hydrophobic) interactions took place between the proteins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary phase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the proteins and the supports via donor-acceptor interactions was less favourable, proteins were eluted from the columns without the addition of a competing agent in the mobile phase. With respect to the binding of these proteins, ionic and/or hydrophobic interactions were no longer negligible during the chromatographic process and the retention of the macromolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractionation of the three main genetic variants of desialylated alpha 1-acid glycoprotein.

Chromatographic kinetic measurements of human serum albumin adsorption on monoclonal antibodies.

A chromatographic method was employed to study the kinetics of human serum albumin (HSA) adsorbed on immobilized monoclonal antibodies. The antibodies of various specificities were covalently bound to a high-performance liquid chromatography (HPLC) silica support. For very low desorption rates, successive amounts of the reacting protein were injected until column saturation. The analysis of the increase of the non-retained fraction calculated from peak area measurements gives the capacity of the support and the rate of the biospecific adsorption process. The model is based on a second-order Langmuir kinetic law and assumes a global mass transfer for the adsorption process. The use of a silica support of small pore size permits the reduction of the contribution for mass transfer in the stagnant fluid and the decrease in the column capacity: due to its large size, the reacting molecule is adsorbed on the external surface of the particle. The adsorption rate constants of HSA on five monoclonal anti-HSA antibodies of different specificities were determined. For all the immuno-adsorbents studied, the adsorption rate constant is significantly lower than that found on immobilized polyclonal antibodies. Measurements at different flow rates reveal that the mass transfer due to the transport to the adsorbent surface is small and can be estimated.

Propafenone binding interaction with human alpha 1-acid glycoprotein: assessing experimental design and data evaluation.

Binding data on racemic RS-propafenone as well as individual R- and S-drug enantiomers interacting reversibly with human alpha 1-acid glycoprotein, as obtained by a high-performance liquid chromatographic method, are evaluated according to three different approaches introduced, respectively, by Scatchard, Bjerrum, and by Tobler and Engel. A non-linear curve-fitting procedure was applied to compute the binding parameters exclusively for the binary system comprising the examined protein and R- and S-propafenone, individually. The exactness of the study design rather than the numerical values were the focus of attention in the evaluation of the data found.

Reactivity of 42 disulfides with thiol group of human haemoglobin and human serum albumin.

The reactivities of disulfides of different compound families towards thiol groups of human haemoglobin and human serum albumin were determined at physiological pH 7.4 by anion-exchange liquid chromatography. The apparent second-order kinetic rate constants, K1, were calculated for the reaction of these disulfides with each protein. The results show that the studied heterocyclic disulfides are the most reactive compounds with both proteins. The lipophilic properties of these disulfides were evaluated by reversed-phase high performance liquid chromatography, using the percentage of acetonitrile (PAC) required for eluting each compound of the chromatographic column in a water-acetonitrile gradient. The structure-reactivity correlations between log K1 and log PAC are stated for each protein and compared. They fit a parabolic curve which permits one to define a lipophilic domain corresponding to a quantitative reaction of disulfides towards these proteins. The studied disulfides present a similar optimum of reactivity for both proteins.