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.     

Macromolecular assemblies based on coupled inclusion complex and electrostatic interactions

Macromolecular assemblies are elaborated by mixing beta-cyclodextrin-containing polymer (polybetaCD), dextran sulfate polyanion (NaDxS), and cationic amphiphiles which are adamantane derivatives (Ada1 or Ada2) in aqueous medium. These components are assembled via coupled inclusion complex interactions (adamantyl group with cyclodextrin cavity) and electrostatic attractive interactions (positive charges of Ada with negative charge of NaDxS). The structural properties are studied by viscometry and small angle neutron scattering. Ternary aggregates with larger size and lower compacities are observed as the cation concentration is increased, until phase separation occurs. The results are in good agreement with a core-shell association mechanism, the core being made of one polybetaCD chain, the shell of NaDxS chains, and the Ada amphiphiles being distributed more or less homogeneously inside the cyclodextrin cavities. The nature of the Ada counterions has a strong influence on the association as Ada1 with I(-) counterions give smaller and less compact aggregates than Ada2 with Br(-) counterions.     

Insight into the chiral recognition of warfarin enantiomers by epichlorhydrin/beta-cyclodextrin polymer-based supports: determination of stoichiometry and stability of warfarin/beta-cyclodextrin polymer complexes

The complexation of warfarin (W) enantiomers by a hydrosoluble high-molecular-weight beta-cyclodextrin/epichlohydrin polymer (EP/beta-CD polymer) was studied using HPLC with a mobile phase of methanol/0.1 M Na acetate/acetic acid (pH 4) at 22 degrees C. It was found that the complexes (W/beta-CD unit) have a 1:1 stoichiometry. The stability constants of the complex involving each enantiomer and the polymer beta-CD units were determined in the mobile phase, and the highest stability of the complex (S-warfarin/beta-CD unit) was observed. From the chromatographic separations of warfarin enantiomers on different beta-CD or its derivative supports, we have deduced the role of the simultaneous presence of several glyceryl (-O-CH(2)-CHOH-CH(2)-O-) and dihydroxypropyl (-O-CH(2)-CHOH-CH(2)OH) groups on one beta-CD ring in promoting the chiral recognition of warfarin enantiomers.     

Temperature-responsive size-exclusion chromatography using poly(N-isopropylacrylamide) grafted silica

Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase for the High Performance Size-Exclusion Chromatography (HPSEC) of proteins. For this purpose, porous silica beads were coated with hydrophilic polymer gels (dextrans of different molecular weights) carrying a calculated amount of diethylaminoethyl groups (DEAE). Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, temperature responsive polymer of poly(N-isopropylacrylamide) (PIPAAm) was introduced into the surface of dextran-DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Scanning Electronic Microscopy (SEM) and Scanning Force Microscopy (SFM). An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time.     

A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains.

A liquid chromatographic stationary phase was prepared by covalently binding to the surface of microparticulate silica gel functionality (benzylsilane), which mimics the side chain of the amino acid phenylalanine. The chromatographic retentions of the N-acetyl C-(N'-methyl) amides of various hydrophobic and amphiphilic amino acids on this stationary phase were measured using an aqueous mobile phase. A retention order of Gly < Ala < Cys < Val < Met < Pro < Ile < Leu < Tyr < Phe < Trp is seen at room temperature. Chromatographic retentions were used to derive free energies of adsorption of the amino acid derivatives on the chromatographic support relative to that of the glycine derivative. The temperature dependencies of the retention of aromatic and aliphatic amino acid derivatives differ in curvature, indicating a qualitative difference in the absorption mechanism. An adsorption model for retention is proposed, and arguments are made as to the suitability of an adsorption model for describing the contacts between amino acid side chains during the initial steps of protein folding.     

Thermodynamics of interactions between amino acid side chains: experimental differentiation of aromatic-aromatic, aromatic-aliphatic, and aliphatic-aliphatic side-chain interactions in water

A stationary phase for high-pressure liquid chromatography has been prepared by derivatizing microparticulate silica gel with functionality mimicking the side chain of isoleucine. The chromatographic retentions of a series of hydrophobic and amphiphilic amino acid analytes on this stationary phase (Ile MSP) using an aqueous mobile phase were measured as a function of temperature from 273 K to 323 K. Observed temperature dependencies are consistent with a constant change in heat capacity, DeltaC degrees P, upon binding of the analyte to the stationary phase. The curvatures of plots of retention data versus temperature (related to the magnitude of DeltaC degrees P) are distinctly different for retention of aromatic and aliphatic analytes, with retention of aliphatic analytes Val, Ile, and Leu exhibiting the characteristic signature of the hydrophobic effect, i.e., a large negative DeltaC degrees P upon desolvation from water and a maximum of retention around room temperature. Retention of aromatic analytes (Trp, Phe, and Tyr) involves smaller heat capacity changes and pronounced negative enthalpies of interaction with the stationary phase. Estimates of DeltaC degrees P for the interactions of analyte side chains with the Ile side chain were obtained by fitting the temperature dependence of retention to an expression derived from thermodynamic considerations and chromatographic theory. Similar estimates were made for interactions with the Phe side chain, using previously published data for a phenylalanine mimic stationary phase (Phe MSP) (. Protein Sci. 1:786-795). As with the Ile MSP, the retentions of aliphatic analytes show temperature dependencies markedly different from those of aromatic analytes. Data from both phases indicate that a realistic differentiation can be made between the interactions of various types of amino acid side chains tested (i.e., aliphatic/aliphatic, aliphatic/aromatic, and aromatic/aromatic) by comparison of the corresponding thermodynamic functions for pairwise interactions. The retention of leucine on the Phe MSP and that of phenylalanine on the Ile MSP showed similar DeltaC degrees P values, suggesting that the aromatic-aliphatic interaction is reasonably independent of the residue attached to the stationary phase. This result is consistent with a one-to-one interaction and suggests a simple way to estimate the column-dependent phase factor, making it possible to compare entropies and free energies of interaction obtained using different MSPs. The possibilities for using MSP-derived interaction potentials in folding simulations are discussed.     

Investigation of the separation of heterocyclic aromatic amines by reversed phase ion-pair liquid chromatography coupled with tandem mass spectrometry: the role of ion pair reagents on LC-MS/MS sensitivity

Reversed phase ion-pair chromatography (RP-IPC) of seven heterocyclic aromatic amines encompassing quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP) and carboline derivatives (AalphaC, Harman, Norharman) was carried out with formate as counter ion in an aqueous eluent with acetonitrile as organic modifier. TSKgel ODS-80TS was used as the stationary phase. With the aim of acquiring a better insight into the mutual influence of ion-pair reagent and the organic modifier upon solute retention, the study was performed by using an experimental design approach able to evidencing the effect of the simultaneous variation of the two factors. A model for the chromatographic behavior of the amines is proposed that includes classical ion-pair mechanism involving formate in the case of MeIQx, PhIP, Harman and Norharman. A competitive ion-exchange mechanism was hypothesized to govern retention of quinoline compounds, whereas electrostatic interactions and hydrogen bond formation with the silanols of the stationary phase were judged to be responsible for the retention of AalphaC. Further, the chromatographic behavior of the analytes using the formic acid-ammonium formate buffer in the mobile phase was compared with that observed using acetic acid-ammonium acetate buffer. The method based on the use of RP IPC with tandem mass spectrometry when the eluent contained formate buffer at pH 2.8 exhibited higher detectability with respect to that achieved using the acetate buffer.     

Structure/function relationships of several biopolymers as related to invertase stability in dehydrated systems

Structure/function relationships of different biopolymers (alginate, dextran, or beta-cyclodextrin) were analyzed as single excipients or combined with trehalose in relation to their efficiency as enzyme stabilizers in freeze-dried formulations and compared to trehalose. Particularly, a novel synthesized polymer beta-cyclodextrin-branched alginate (beta-CD-A) was employed as excipient. During freeze-drying, the polymers or their mixtures did not confer better protection to invertase compared to trehalose. Beta-CD-A (with or without trehalose), beta-cyclodextrin (beta-CD), or dextran with trehalose were the best protective agents during thermal treatment, while beta-CD and alginate showed a negative effect on invertase activity preservation. The beta-CD linked alginate combined the physical stability provided by alginate with the stabilization of hydrophobic regions of the enzyme provided by cyclodextrin. Beta-CD-A was effective even at conditions at which trehalose lost its protective effect. A relatively simple covalent combination of two biopolymers significantly affected their functionalities and, consequently, their interactions with proteins, modifying enzyme stability patterns.     

Effects of beta-cyclodextrin addition and temperature on the modulation of hydrophobic interactions in aqueous solutions of an associative alginate

Novel information about the effects of beta-cyclodextrin (beta-CD) addition and temperature on structural and rheological features of semidilute solutions of alginate and its hydrophobically modified analogue (HM-alginate) is given. Enhanced turbidity is observed for the HM-alginate solutions at high levels of beta-CD addition and low temperatures. The viscosity results revealed cross-linking of the alginate chains at high beta-CD concentrations and low temperatures. Rheological results for the HM-alginate solutions demonstrated that high levels of beta-CD addition and elevated temperatures promoted decoupling of the hydrophobic polymer-polymer associations via inclusion complex formation between beta-CD cavities and the hydrophobic side chains of the polymer. Analysis of small-angle neutron scattering (SANS) results from HM-alginate solutions in the presence of beta-CD suggested that the polymer chains are locally stretched at all of the considered levels of beta-CD and temperatures. The SANS results revealed association structures. The general picture that emerges is that beta-CD addition and temperature can be combined to tune the intensity of the hydrophobic interactions and to cross-link the unmodified alginate.     

Ucon-benzoyl dextran aqueous two-phase systems: protein purification with phase component recycling

Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride. A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed. The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature. This two-phase system has been used to purify 3-phosphoglycerate kinase from bakers' yeast. The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature. The bottom-phase polymer (benzyol dextran) could be recovered by addition of salt. Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system. After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase. The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na₂SO₄.     

Studies on adsorption of dyes on beta-cyclodextrin polymer

Beta-cyclodextrin (beta-CD) polymers are used for the removal of various dyes from aqueous solutions. Three insoluble polymers with different degrees of beta-CD were used. Results of adsorption experiments showed that these polymers exhibited high sorption capacities toward dyes. The mechanism of adsorption was both physical adsorption and hydrogen bonding due to the polymer and the formation of an inclusion complex due to the beta-CD molecules through host-guest interactions.     

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.     

Effect of the solute molecular structure on its enantioresolution on cellulose tris(3,5-dimethylphenylcarbamate)

The effects of the molecular structures for 13 structurally similar chiral solutes on their HPLC retention and enantioresolutions on a commercially important polysaccharide-based chiral stationary phase, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) are studied. Among these 13 solutes, only methyl ephedrine (MEph) shows significant enantioresolution. The retention factors of these chiral solutes vary significantly from 0.7 to 3.2 in n-hexane/2-propanol (90/10, v/v) at 298 K. The retention factors of some simpler non-chiral solutes having similar but fewer functional groups than their chiral counterparts are also studied under the same conditions and are compared to those of the chiral solutes. The H-bonding interactions between the functional groups of the solute and the C=O and NH functional groups of the polymer are probed with attenuated total reflection-infrared spectroscopy (ATR-IR) for the polymer, for binary sorbent-solute systems. The CDMPC IR amide band wavenumbers change significantly, indicating H-bonding interactions of the polymer C=O and NH groups with the solutes. The elution orders predicted for the enantiomers of these chiral solutes using molecular dynamics (MD) simulations of the polymer-solute binary systems are consistent with the HPLC results. The CDMPC cavity nano-structure and the potential interactions with chiral solutes are proposed based on HPLC data, IR data, and the simulations. The results are consistent with the three-point attachment model and support the hypothesis that significant enantioresolution requires at least three different synergistic interactions which can be a combination of steric hindrance, H-bonding, or pi-pi interactions.     

Modeling and prediction of the mixed-mode retention mechanisms for puerarin and its analogues on n-octylamine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monoliths

n-Octylamine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) monoliths were prepared for the rapid screening and determination of puerarin content of a crude extract Radix puerariae. The mixed-mode retention mechanisms for puerarin and its analogues on n-octylamine modified monoliths were investigated using a variety of solvent systems, chromatographic evaluation and molecular dynamics (MDs) modeling. The equilibrated conformations between cross-linked polymers and target molecules were obtained from MD modeling. Both the polymer skeleton and functional groups played important roles in the recognition process. The cross-linker formed a structural network skeleton, in which recognition cavities were formed surrounded by functional groups. The polymer network structures provided good interaction access for isoflavones. The active groups recognized isoflavones by both intermolecular hydrogen bonding and hydrophobic interaction. The interaction energies and retention factors between polymers and target molecules were also evaluated and compared. A higher value of interaction energy corresponded to a higher value of retention factor. The potential of using modeling technology for predicting the chromatographic performances of target molecules was explored.     

Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.     

Separation of icosenoic acids,monohydroxyicosenoic acids,and prostaglandins by high-pressure liquid chromatography on a silver ion-loaded cation-exchange column

Prostaglandins and monohydroxy fatty acids derived from 8,11,14-icosatrienoic acid and arachidonic acid have been separated by high-pressure liquid chromatography using a cation-exchange column loaded with silver ions. The retention times in a variety of solvent systems have been determined for prostaglandin E₁(PGE₁), PGF_1α, PGD₂, PGE₂, PGF_2α, 6-oxoPGF_1α, 15-hydroxy-8,11,13-icosatrienoic acid, 5-hydroxy-6,8,11,14-icosatetraenoic acid, 8-hydroxy-5,9,11,14,-icosatetraenoic acid, 9-hydroxy-5,7,11,14-icosatetraenoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid, 12-hydroxy-5,8,10,14-icosatetraenoic acid, 15-hydroxy-5,8,11,13-icosatetraenoic acid, 8,11,14,-icosatrienoic acid, and arachidonic acid. The mechanisms involved in the interaction of solutes with the stationary phase have been investigated. Retention times on silver ion columns appear to be determined by a combination of interactions between (a) the silver ions of the stationary phase and double bonds of the solute and (b) polar groups of the stationary phase and polar groups of the solute. The relative contributions of these two types of interactions to the retention of solutes can be varied over a wide range by altering the composition of the solvent. In this way the selectivity of the stationary phase can be controlled in order to optimize the separation of any given group of solutes. The maximum separation of solutes on the basis of the number of double bonds they possess is obtained by using polar solvents containing low concentrations of acetonitrile. As the polarity of the mobile phase is reduced or the concentration of acetonitrile increased, the selectivity of the stationary phase tends to resemble that of normal-phase chromatography on silicic acid.     

Complex retention behavior of pyrimidines on biomembrane-mimic immobilized-artificial-membrane phase

The influence of the chemical substitutions on the interfacial interactions of pyrimidines with the phospholipid-mimic immobilized-artificial-membrane (IAM) chromatographic stationary phase was evaluated. Monocyclic pyrimidine nucleic acid bases (nucleobases) were revealed behaving differently from their bicyclic purine counterparts substantially. The computed electrostatic potential surfaces for both the IAM phase and the interacting nucleobases are intuitive in deconvoluting the retention patterns of pyrimidines molecularly. A structure-retention model has also been derived using quantitative 3D-QSAR methodology pertinent to the IAM-retention of pyrimidines for the potential use in molecular design. IAM phase is found particularly suitable in assessing the retention of pyrimidines with bulky or elongated exocyclic substituents in the biological context than the alkyl-based chromatographic counterparts.     

HPCPC separation of proteins using polyethylene glycol-potassium phosphate aqueous two-phase.

High Performance Centrifugal Partition Chromatography (HPCPC) is a practical and suitable method, particularly on the preparative scale, for the separation of biomolecules such as proteins, enzymes, etc. Aqueous two-phase system is also very attractive for the isolation of biomolecules. Aqueous polymer phase system composed of polyethylene glycol 6000-potassium phosphate has been used for the countercurrent chromatographic separation of bovine serum albumin (BSA) and lysozyme using HPCPC. The separation of BSA and lysozyme under various conditions such as various flow rates, rotational speeds, pH of the solvent, and the retention of stationary phase has been studied in the present investigation. The baseline separation of BSA and lysozyme has been also observed. The results of this study demonstrate that HPCPC is useful for separation of proteins with aqueous two-phase systems.     

Retention behaviour of proteins on poly(vinylimidazole)-copper(II) complexes supported on silica: application to the fractionation of desialylated human α1-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 with 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 α₁-acid glycoprotein.     

Immobilized serum albumin: rapid HPLC probe of stereoselective protein-binding interactions

A human serum albumin-based HPLC chiral stationary phase (HSA-CSP) has been examined as a tool to investigate binding of chiral drugs to HSA and drug-drug protein-binding interactions. Rac-oxazepam hemisuccinate (OXH) was used as a model compound and the chromatographic retention (k') of its enantiomers was determined after addition of displacers to the mobile phase. Compounds known to bind at the same site as OXH and at different sites were tested for their displacing capacities. Competitive binding interactions between the OXH enantiomers and displacers in the mobile phase were reflected by decreases in the k's of (R)- and (S)-OXH. The results indicate that retention on the HSA-CSP accurately reflects binding to native HSA and the technique can determine enantioselective and competitive binding interactions at specific sites on HSA. The HSA-CSP was also able to recognize separate binding areas for (S)- and (R)-OXH.