Journal of Chromatography B (v.797, #1-2)

Foreword by Ivan Miksik; Zdenek Deyl (1).

Separation methods for the indirect estimation of the octanol–water partition coefficient (log  P) are reviewed with an emphasis on high throughput methods with a wide application range. The solvation parameter model is used to identify suitable separation systems for estimating log  P in an efficient manner that negates the need for empirical trial and error experiments. With a few exceptions, systems based on reversed-phase chromatography employing chemically bonded phases are shown to be unsuitable for estimating log  P for compounds of diverse structure. This is because the fundamental properties responsible for chromatographic retention tend to be different to those responsible for partition between octanol and water, especially the contribution from hydrogen bonding interactions. On the other hand, retention in several micellar and microemulsion electrokinetic chromatography systems is shown to be highly correlated with the octanol–water partition coefficient. These systems are suitable for the rapid, high throughput determination of log  P for neutral, weakly acidic, and weakly basic compounds. For compounds with a permanent charge, electrophoretic migration and electrostatic interactions with the stationary phase results in inaccurate estimation of partition coefficients. The experimental determination of solute descriptors offers an alternative approach for estimating log  P, and other biopartitioning properties. A distinct advantage of this approach is that once the solute descriptors are known, solute properties can be estimated for any distribution or transport system for which a solvation parameter model has been established.
Keywords: Reviews; Octanol–water partition coefficient;

Biopartitioning micellar separation methods: modelling drug absorption by L Escuder-Gilabert; J.J Martı́nez-Pla; S Sagrado; R.M Villanueva-Camañas; M.J Medina-Hernández (21-35).
The search for new pharmacologically active compounds in drug discovery programmes often neglects biopharmaceutical properties as drug absorption. As a result, poor biopharmaceutical characteristics constitute a major reason for the low success rate for candidates in clinical development. Since the cost of drug development is many times larger than the cost of drug discovery, predictive methodologies aiding the selection of bioavailable drug candidates are of profound significance. This paper has been focussed on recent developments and applications of chromatographic systems, particularly those systems based on amphiphilic structures, in the frame of alternative approaches for estimating the transport properties of new drugs. The aim of this review is to take a critical look at the separations methods proposed for describing and predicting drug passive permeability across gastrointestinal tract and the skin.
Keywords: Reviews; Biopartitioning; Drug absorption;

Drug delivery systems: polymers and drugs monitored by capillary electromigration methods by Carolina Simó; Alejandro Cifuentes; Alberto Gallardo (37-49).
In this paper, different electromigration methods used to monitor drugs and polymers released from drug delivery systems are reviewed. First, an introduction to the most typical arrangements used as drug delivery systems (e.g., polymer–drug covalent conjugates, membrane or matrix-based devices) is presented. Next, the principles of different capillary electromigration procedures are discussed, followed by a revision on the different procedures employed to monitor the release of drugs and the degradation or solubilization of the polymeric matrices from drug delivery systems during both in vitro and in vivo assays. A critical comparison between these capillary electrophoretic methods and the more common chromatographic methods employed to analyze drugs and polymers from drug delivery systems is presented. Finally, future outlooks of these electromigration procedures in the controlled release field are discussed.
Keywords: Polymers; Drugs;

The use of the Hummel and Dreyer method to measure binding parameters of ligand–macromolecule associations is reviewed. The possibility to determine the number of binding sites and their association constants, even in the case of low affinity, and to control the free ligand concentration as an independent variable are the main advantages of the method. The conditions of the validity are rapid equilibrium kinetics, independence between ligand binding and macromolecule association, and identical retention rates between free and bound macromolecules. Initially developed on soft gels, the method has been applied to high-performance chromatography and capillary zone electrophoresis. Technical progress such as increase in resolution, detection sensitivity, and automation have improved its utilization. The binding parameters given by the Hummel and Dreyer method are in general similar to those obtained by other techniques, in comparable experimental conditions (equilibrium dialysis, ultrafiltration, frontal elution, vacancy peak method, vacancy affinity capillary electrophoresis, retention analysis, affinity chromatography and affinity capillary electrophoresis, physical methods). The choice between these methods is directed by material availability and practical constraints. Separation by new types of chromatographic columns or by capillary zone electrophoresis would enable the study of the simultaneous binding of different drugs on the same macromolecule and their competition.
Keywords: Reviews; Macromolecule–ligand binding; Hummel and Dreyer method;

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug–protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug–protein adducts. The methods used for drug–protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), two-dimensional SDS–PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug–protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.
Keywords: Reviews; Drug–protein adducts;

The review article covers advances of chiral drugs analysis by high-performance liquid chromatography (HPLC) methods achieved during last 10 years. Emphasis is given to various aspects of influence of biological matrix in pharmacodynamics, pharmacokinetics, HPLC analysis. Discussed is composition of main biological matrices from the point of view of potential interferences to above-mentioned fields of study. Beside typical analytical approaches to chiral recognition in HPLC, sample pretreatment and/or clean-up by conventional extraction procedures, column switching (CSW) techniques using restricted access materials (RAMs), microdialysis (MCD) is discussed. Measurement of unbound drug concentration and discussion of column maintenance and remedy is an additional source of information and field where knowledge on complex properties and interactions of biological matrix is usefully applicable.
Keywords: Reviews; Biological matrices; Pharmacodynamics; Pharmacokinetics; Chiral drugs;

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs–bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs’ binding parameters to immobilized bio-polymers is examined.
Keywords: Reviews; Drug-binding constants; High-throughput screening; Bio-polymers, immobilized;

Proteins display interesting chiral discrimination properties owing to multiple possibilities of intermolecular interactions with chiral compounds. This review deals with proteins which have been used as immobilized chiral selectors for the enantioseparation of drugs in liquid chromatography and capillary electrophoresis. The main procedures allowing the immobilization of proteins onto matrices, such as silica and zirconia particles, membranes and capillaries are first presented. Then the factors affecting the enantioseparation of drugs in liquid chromatography, using various protein-based chiral stationary phases (CSPs), are reviewed and discussed. Last, chiral separations already achieved using immobilized protein selectors in affinity capillary electrochromatography (ACEC) are presented and compared in terms of efficiency, stability and reproducibility.
Keywords: Reviews; Enantiomer separation; Chiral selectors; Proteins;

Compared with traditional sampling methods, microdialysis is a technique for protein unbound drug sampling without withdrawal of biological fluids and involving minimal disturbance of physiological function. Conventional total drug sample consists of unbound drugs and protein bound drugs, which are loosely bound to plasma proteins such as albumin and alpha-1 acid glycoprotein, forming an equilibrium ratio between bound and unbound drugs. However, only the unbound fraction of drug is available for absorption, distribution, metabolism and elimination, and delivery to the target sites for pharmacodynamic actions. Although several techniques have been used to determine protein unbound drugs from biological fluids, including ultrafiltration, equilibrium dialysis and microdialysis, only microdialysis allows simultaneous sampling of protein unbound chemicals from plasma, tissues and body fluids such as the bile juice and cerebral spinal fluid for pharmacokinetic and pharmacodynamic studies. This review article describes the technique of microdialysis and its application in pharmacokinetic studies. Furthermore, the advantages and limitations of microdialysis are discussed, including the detailed surgical techniques in animal experiments from rat blood, brain, liver, bile duct and in vitro cell culture for unbound drug analysis.
Keywords: Reviews; Microdialysis techniques; Proteins; Unbound drugs;

Enzymes catalyze a rich variety of metabolic transformations, and do so with very high catalytic rates under mild conditions, and with high reaction regioselectivity and stereospecificity. These characteristics make biocatalysis highly attractive from the perspectives of biotechnology, analytical chemistry, and organic synthesis. This review, containing 128 references, focuses on the use of separation techniques in the elucidation of enzyme–inhibitor and enzyme–substrate interactions. While coverage of the literature is selective, a broad perspective is maintained. Topics considered include chromatographic methods with soluble or immobilized enzymes, capillary electrophoresis, biomolecular interaction analysis tandem mass spectrometry (BIA–MS), phage and ribosomal display, and immobilized enzyme reactors (IMERs). Examples were selected to demonstrate the relevance and application of these methods for determining enzyme kinetic parameters, ranking of enzyme inhibitors, and stereoselective synthesis and separation of chiral entities.
Keywords: Reviews; Enzyme–inhibitor interactions; Enzyme–substrate interactions; Enzymes;

The separation of membrane protein complexes can be divided into two categories. One category, which is operated on a relatively large scale, aims to purify the membrane protein complex from membrane fractions while retaining its native form, mainly to characterize its nature. The other category aims to analyze the constituents of the membrane protein complex, usually on a small scale. Both of these face the difficulty of isolating the membrane protein complex without interference originating from the hydrophobic nature of membrane proteins or from the close association with membrane lipids. To overcome this difficulty, many methods have been employed. Crystallized membrane protein complexes are the most successful example of the former category. In these purification methods, special efforts are made in the steps prior to the column chromatography to enrich the target membrane protein complexes. Although there are specific aspects for each complex, the most popular method for isolating these membrane protein complexes is anion-exchange column chromatography, especially using weak anion-exchange columns. Another remarkable trend is metal affinity column chromatography, which purifies the membrane protein complex as an intact complex in one step. Such protein complexes contain subunit proteins which are genetically engineered so as to include multiple-histidine tags at carboxyl- or amino-termini. The key to these successes for multi-subunit complex isolation is the idea of keeping the expression at its physiological level, rather than overexpression. On the other hand, affinity purification using the Fv fragment, in which a Strep tag is genetically introduced, is ideal because this method does not introduce any change to the target protein. These purification methods supported by affinity interaction can be applied to minor membrane protein complexes in the membrane system. Isoelectric focusing (IEF) and blue native (BN) electrophoresis have also been employed to prepare membrane protein complexes. Generally, a combination of two or more chromatographic and/or electrophoretic methods is conducted to separate membrane protein complexes. IEF or BN electrophoresis followed by 2nd dimension electrophoresis serve as useful tools for analytical demand. However, some problems still exist in the 2D electrophoresis using IEF. To resolve such problems, many attempts have been made, e.g. introduction of new chaotropes, surfactants, reductants or supporting matrices. This review will focus in particular on two topics: the preparative methods that achieved purification of membrane protein complexes in the native (intact) form, and the analytical methods oriented to resolve the membrane proteins. The characteristics of these purification and analytical methods will be discussed along with plausible future developments taking into account the nature of membrane protein complexes.
Keywords: Membrane protein complexes;

Capillary electrophoresis-based immunoassay by W.S.B. Yeung; G.A. Luo; Q.G. Wang; J.P. Ou (217-228).
Capillary electrophoresis-based immunoassay (CEIA) is a developing analytical technique with a number of advantages over conventional immunoassay, such as reduced sample consumption, simpler procedure, easy simultaneous determination of multiple analytes, and short analysis time. However, there are still a number of technical issues that researchers on CEIA have to solve before the assay can be more widely used. These issues include method to improve the concentration sensitivity of the assay, requirement for robust separation strategy for different analytes, and method to increase the throughput of the assay. The approaches to solve these issues are reviewed. Several studies have been devoted to develop general separation strategies for CEIA, and to enhance the sensitivity of detection. The recent development of microchip-based CEIA is encouraging and is likely to address more drawbacks of CEIA, particularly on the throughput issue.
Keywords: Review; Capillary electrophoresis; Immunoassays;

Protein and antibody microarray technology by Jörn Glökler; Philipp Angenendt (229-240).
Following the age of genomics having sequenced the human genome, interest is shifted towards the function of genes. This new age of proteomics brings about a change of methods to study the properties of gene products on a large scale. Protein separation technologies are now applied to allow high-throughput purification and characterisation of proteins. Two-dimensional-gel electrophoresis (2DE) and mass spectrometry (MS) have become widely used tools in the field of proteomics. At the same time, protein and antibody microarrays have been developed as successor of DNA microarrays to soon allow the proteome-wide screening of protein function in parallel. This review is aimed to introduce this new technology and to highlight its current prospects and limitations.
Keywords: Review; Microarray technologies; Proteomics; Proteins; Antibodies;

The objective of this review is to emphasize the application of separation science in evaluating the blood–brain barrier (BBB) permeability to drugs and bioactive agents. Several techniques have been utilized to quantitate the BBB permeability. These methods can be classified into two major categories: in vitro or in vivo. The in vivo methods used include brain homogenization, cerebrospinal fluid (CSF) sampling, voltametry, autoradiography, nuclear magnetic resonance (NMR) spectroscopy, positron emission tomography (PET), intracerebral microdialysis, and brain uptake index (BUI) determination. The in vitro methods include tissue culture and immobilized artificial membrane (IAM) technology. Separation methods have always played an important role as adjunct methods to the methods outlined above for the quantitation of BBB permeability and have been utilized the most with brain homogenization, in situ brain perfusion, CSF sampling, intracerebral microdialysis, in vitro tissue culture and IAM chromatography. However, the literature published to date indicates that the separation method has been used the most in conjunction with intracerebral microdialysis and CSF sampling methods. The major advantages of microdialysis sampling in BBB permeability studies is the possibility of online separation and quantitation as well as the need for only a small sample volume for such an analysis. Separation methods are preferred over non-separation methods in BBB permeability evaluation for two main reasons. First, when the selectivity of a determination method is insufficient, interfering substances must be separated from the analyte of interest prior to determination. Secondly, when large number of analytes is to be detected and quantitated by a single analytical procedure, the mixture must be separated to each individual component prior to determination. Chiral separation in particular can be essential to evaluate the stereo-selective permeation and distribution of agents into the brain. In conclusion, the usefulness of separation methods during BBB permeability evaluation is immense and more application of these methods is foreseen in the future.
Keywords: Reviews; Blood–brain barrier;

Survey of binding properties of fatty acid-binding proteins by G. Massolini; E. Calleri (255-268).
Fatty acid-binding proteins (FABPs) are members of a super family of lipid-binding proteins, and occur intracellularly in vertebrates and invertebrates. This review briefly addresses the structural and molecular properties of the fatty acid binding proteins, together with their potential physiological role. Special attention is paid to the methods used to study the binding characteristics of FABPs. An overview of the conventional (Lipidex, the ADIFAB and ITC) and innovative separation-based techniques (chromatographic and electrophoretic methods) for the study of ligand–protein interactions is presented along with a discussion of their strengths, weak points and potential applications. The best conventional approaches with natural fatty acids have generally revealed only limited information about the interactions of fatty acid proteins. In contrast, high-performance affinity chromatography (HPAC) studies of several proteins provide full information on the binding characteristics. The review uses, as an example, the application of immobilized liver basic FABP as a probe for the study of ligand–protein binding by high-performance affinity chromatography. The FABP from chicken liver has been immobilized on aminopropyl silica and the developed stationary phase was used to examine the enantioselective properties of this protein and to study the binding of drugs to FABP. In order to clarify the retention mechanism, competitive displacement studies were also carried out by adding short chain fatty acids to the mobile phase as displacing agents and preliminary quantitative structure–retention relationship (QSRRs) correlations were developed to describe the nature of the interactions between the chemical structures of the analytes and the observed chromatographic results.The results of these studies may shed light on the proposed roles of these proteins in biological systems and may find applications in medicine and medicinal chemistry. This knowledge will yield a deeper insight into the mechanism of fatty acid binding in order to indisputably show the central role played by FABPs in cellular FA transport and utilization for a proper lipid metabolism.
Keywords: Reviews; Fatty acid-binding proteins;

Methods for transcription factor separation by Robert A Moxley; Harry W Jarrett; Suchareeta Mitra (269-288).
Recent advances in the separation of transcription factors (TFs) are reviewed in this article. An overview of the transcription factor families and their structure is discussed and a computer analysis of their sequences reveals that while they do not differ from other proteins in molecular mass or isoelectric pH, they do differ from other proteins in the abundance of certain amino acids. The chromatographic and electrophoretic methods which have been successfully used for purification and analysis are discussed and recent advances in stationary and mobile phase composition is discussed.
Keywords: Reviews; Transcription factors;

Affinity chromatography is one of the powerful techniques in selective purification and isolation of a great number of compounds. New challenges in scientific research, such as high-throughput systems, isolation procedures that allow to obtain a single substance from a complex matrix in high degree of purity, low costs and wide availability, have led to the discovery of new tailor-made synthetic recognition systems. In this review the design, synthesis, purification and characterisation of peptides with recognition properties are discussed. Applications of peptide ligands are described and analytical tools mentioned.
Keywords: Reviews; Immobilisation; Specific binding capacity; Peptides;

Increasing reports regarding the isolation or purification of biospecies for therapeutic purpose using the immobilized metal affinity chromatography have been presented in recent years. At the same time, membrane chromatography technique has also gained more and more attention for their advantage in speeding the separation process. The immobilized metal affinity membrane technique developed by combining these two techniques may provide an alternative potential tool for separating the therapeutically relevant biospecies. In this review paper, the features of the immobilized metal affinity membranes are discussed and concentrated on three subtopics: membrane matrices, immobilized metal affinity method, and membrane module designs. Several examples of practically applying the immobilized metal affinity membranes on the purification of potential therapeutics reported in the literature are subsequently presented. Lastly, this review also provides an overall evaluation on the possible advantages and problems existing in this technique to point out opportunities and further improvements for more applied development of the immobilized metal affinity membranes.
Keywords: Reviews; Immobilized metal affinity membranes; Purification;

Separation procedures capable of revealing DNA adducts by Yukihiro Esaka; Shinsuke Inagaki; Masashi Goto (321-329).
Detection and quantification of DNA adducts are very important in relation to diseases such as cancer. Both high sensitivity and high selectivity are required for the detection of DNA adducts because the content of adducts in DNA is very small compared with those of normal bases and only small amounts of DNA samples are available for analysis in general cases. In this paper are described separation procedures such as liquid chromatography, gas chromatography and capillary electrophoresis combined with a detection and identification method such as 32 P -postlabeling, mass spectrometry, electrochemical detection, fluorescence detection and immunoassay. The merits and demerits of the procedures are also discussed.
Keywords: Review; DNA adducts;

Small molecular ion adsorption on proteins and DNAs revealed by separation techniques by Murielle Rabiller-Baudry; Bernard Chaufer (331-345).
Ion binding is a term that assumes that the ion is included in the solvation sphere characterising the biomolecule. The binding forces are not clearly stated except for electrostatic attraction; weak forces (hydrogen bonds and Van der Waals forces) are likely involved. Many publications have dealt with ion binding to proteins and the consequences over the past 10 years, but only a few studies were performed using high-performance liquid chromatography (HPLC: ion exchange, reversed phase without the well-identified immobilised metal affinity chromatography) and capillary zone electrophoresis (CZE). This review focuses on the binding of proteins and DNAs mainly to the oxyanions (phosphate, borate, citrate) and amines used as buffers for both the HPLC eluent and the background electrolyte of CZE. Such specific ion adsorption on biomolecules is evidenced by physico-chemical characteristics such as the mobility or retention volume, closely associated with the net charge, which differ from the expected or experimental data obtained under the conditions of an indifferent electrolyte. It is shown that ion binding to proteins is a key parameter in the electrostatic repulsion between the free protein and a fouled membrane in the ultrafiltration separation of a protein mixture.
Keywords: Reviews; Small molecular ion adsorption; Proteins; DNA;

Hyaluronic acid: separation and biological implications by Kazuaki Kakehi; Mitsuhiro Kinoshita; Shin-ichi Yasueda (347-355).
Hyaluronic acid (hyaluronan) is a ubiquitous extracellular matrix component, and present at high concentrations in skin, joints and cornea. In the skin, it is synthesized primarily by dermal fibroblasts and by epidermal keratinocytes. Hyaluronic acid usually exists as a high molecular mass (600 000–1 000 000) and non-sulfated glycosaminoglycan composed of a disaccharide unit of –3GlcNAcβ1–4GlcAβ1–. Hyaluronic acid has been widely used not only for osteoarthritis and ophthalmology but also for cosmetics for skin care. To examine the biological activities of hyaluronic acid, we have to accurately determine the quantity and molecular masses in biological samples. We review recent development in the analysis of hyaluronic acid having various molecular sizes using electrophoretic and chromatographic techniques. Recently, interactions between hyaluronic acid oligomers and hyaluronic acid-binding proteins have attracted the interest for understanding the biological functions. We show some interesting reports on biological interactions of hyaluronic acid and its oligomers with some proteins.
Keywords: Reviews; Hyaluronic acid;

Electrophoretic investigation of interactions of sanguinarine and chelerythrine with molecules containing mercapto group by Radim Vespalec; Petr Barták; Vilı́m Šimánek; Markéta Vlčková (357-366).
Using mercaptoethanol and (l)-cysteine as representatives of mercapto compounds and capillary zone electrophoresis as experimental technique, it was evidenced that sanguinarine and chelerythrine do not react with the mercapto group of organic compounds at pH 7.4. Their interaction is fast and reversible complexation based on non-bonding intermolecular interaction in which enter uncharged forms of sanguinarine or chelerythrine. A negatively charged group, either bound to the mercapto ligand or supplied from solution, participates in the complexation. Simple 1:1 interaction scheme reported in literature holds therefore only for mercapto compounds bearing anionic group. Stoichiometric binding constants corrected for the abundance of the uncharged alkaloid form at pH 7.4 are of the order of magnitude of 104 l/mol and depend on both cations and anions of the background electrolyte. Interaction of sanguinarine and chelerythrine with human or bovine serum albumins does not qualitatively differ from their interaction with simple mercapto compounds. Stoichiometric binding constants for the binding of sanguinarine with human and bovine serum albumins in sodium phosphate buffer pH 7.4, corrected for the abundance of the interacting uncharged form, are 332 000±38 400 and 141 000±14 400 l/mol, respectively. The former agrees well with the value K=385 000 (or log  K=5.59) from static photometric experiments. Constants for the complexation of uncharged chelerythrine with human and bovine serum albumins are 2 970 000±360 000 and 1 380 000±22 600 l/mol, respectively.
Keywords: Sanguinarine; Chelerythrine; Benzo[c]phenanthridine alkaloids; Albumins;

New purification method for glucocorticoid receptors by Kazuki Okamoto; Fumihide Isohashi (367-371).
In this report, we describe a new purification method for activated recombinant glucocorticoid receptor (GR) utilizing a cation-exchanger (Mono S) at pH 8.4. This method is based upon a new finding that activated GR binds to both Mono Q and Mono S columns at the same pH. This method enables us to purify recombinant GR within 3 h. The purified GR represents more than 97% of the eluted proteins. Purified recombinant GR is able to bind specifically to a DNA fragment containing the glucocorticoid response element. Recombinant GR has no tag sequence that can be utilized for purification. Thus, this separation method is also applicable to purification of native GR.
Keywords: Reviews; Purification; Glucocorticoid receptor;

The enantioselectivity of the interaction of dextromethorphan (DM) and levomethorphan (LM) with an immobilized α3β4 subtype of the nicotinic acetylcholine receptor (nAChR) liquid chromatographic stationary phase has been compared to DM- and LM-induced non-competitive blockade of nicotine-stimulated 86 Rb + efflux from cells expressing the α3β4-nAChR. The association rate constants (k on) and dissociation rate constants (k off) for the formation of the DM and LM complexes with the nAChR were determined using non-linear chromatographic techniques and the k off value for DM (1.01±0.01 s−1) was significantly lower than the k off for LM (1.55±0.002 s−1) while the k on values did not significantly differ (23.66±0.61 and 18.61±0.36 μM−1  s−1, respectively). In thermodynamic studies using the van’t Hoff approach, the enthalpy change (ΔH°) of the DM–nAChR complex was 330 cal mol−1 more stable than the LM–nAChR complex, while there was no significant difference in the entropy change (ΔS°). In the functional in vitro cell-based studies, there was no significant difference in the observed IC50 values for DM (10.1±1.01 μM) and LM (10.9±1.08 μM), but the recovery from the DM-induced blockade was slower than the recovery from LM-induced blockade; after 7 min: 38.25±15.46% recovery from DM blockade, 63.30±16.08% from LM blockade; after 4 h: 76.20±4.51% recovery from DM blockade and 93.12±8.76% from LM blockade. The enantioselective differences in the functional effects are consistent with the chromatographic and thermodynamic data and indicate that this difference is due to increased stability of the DM–nAChR complex. The results suggest that the chromatographic approach can be used to probe the interaction of non-competitive inhibitors (NCIs) with nAChRs and to predict relative duration of functional blockades.
Keywords: Reviews; Enantiomer separation; Dextromethorphan; Levomethorphan; Nicotinic acetylcholine receptor;