Antigen-antibody interactions in capillary electrophoresis

Immunoreactions in combination with separations by capillary electrophoresis (CE) are increasingly being used to quantitate specific analytes in biological fluids. Both competitive and non-competitive approaches have been used for the purpose and, in selected cases, now compare favorably with conventional quantitative immunoassays with respect to concentration limits of detection. CE is also a useful method to evaluate antigen-antibody binding on-line and offers unique possibilities for binding constant estimates, also for weakly binding antibodies and antibody fragments. In this review we cover recent developments in the use of antigen-antibody interactions in conjunction with CE and conclude that continued development of miniaturization, on-line preconcentration and more sensitive detection schemes will contribute to the further dissemination of CE-based immunoassays building on already established affinity CE approaches.     

Capillary electrophoresis for the study of affinity interactions

Molecular recognition may be characterized both qualitatively and quantitatively by electrophoretic methods if complexed molecules differ in electrophoretic mobility from unbound ones. The use of capillary zone electrophoresis (CE) for the characterization of affinity interactions is advantageous because of the high resolution, reproducibility and wide applicability of the technique and because of the mild conditions, i.e., physiological buffers without additions of organics or detergents, that are often sufficient for highly efficient separations. CE gives the ability to characterize binding between small amounts of unlabelled reactants in solution, has few requirements for special characteristics of the interacting molecules and is also applicable to the study of interactions of individual components in mixtures, as detection of binding and analytical separation are achieved in one step. This is unique compared with other techniques for the study of non-covalent interactions. The advantages and disadvantages of using CE to demonstrate molecular interactions, to screen for specific ligand binding in complex mixtures and to calculate binding constants will be discussed.     

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis

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.     

Quantitative affinity chromatography.

The objective of this review is to summarize developments in the use of quantitative affinity chromatography to determine equilibrium constants for solute interactions of biological interest. Affinity chromatography is an extremely versatile method for characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reacting solutes. Adoption of different experimental strategies, such as column chromatography, simple partition equilibrium experiments, solid-phase immunoassay, and biosensor technology, has led to a situation whereby affinity chromatography affords a means of characterizing interactions governed by an extremely broad range of binding affinities--relatively weak interactions (binding constants below 10(3) M(-1)) through to interactions with binding constants in excess of 10(9) M(-1). In addition to its important role in solute separation and purification, affinity chromatography thus also possesses considerable potential for investigating the functional roles of the reactants thereby purified.     

Separation methods for pharmacologically active xanthones

Xanthones, as a kind of polyphenolic natural products with many strong bioactivities, are attractive for separation scientists due to the similarity and diversity of their structures resulting in difficult separation by chromatographic methods. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are traditional methods to separate xanthones. Recently, capillary electrophoresis (CE), as a micro-column technique driven by electroosmotic flow (EOF), with its high efficiency and high-speed separation, has been employed to separate xanthones and determine their physicochemical properties such as binding constants with cyclodextrin (CD) and ionization constants. Since xanthones have been used in clinic treatment, the development of chromatographic and CE methods for the separation and determination of xanthones plays an essential role in the quality control of some herbal medicines containing xanthones. This article reviewed the separation of xanthones by HPLC, TLC and CE, citing 72 literatures. This review focused on the CE separation for xanthones due to its unique advantages compared to chromatographic methods. The comparison of separation selectivity of different CE modes including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC) and capillary electrochromatography (CEC) was discussed. Compared with traditional chromatographic methods such as HPLC and TLC, CE has higher separation efficiency, faster separation, lower cost and more flexible modes. However, because of low sensitivity of UV detector and low contents of xanthones in herbal medicines, CE methods have seldom been applied to the analysis of real samples although CE showed great potential for xanthone separation. The determination of xanthones in herbal medicines has been often achieved by HPLC. Hence, how to enhance CE detection sensitivity for real sample analysis, e.g. by on-line preconcentration and CE-MS, would be a key to achieve the quantitation of xanthones.     

Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions

Isothermal titration calorimetry (ITC) is a well established technique for the study of biological interactions. The strength of ITC is that it directly measures enthalpy changes associated with interactions. Experiments can also yield binding isotherms allowing quantification of equilibrium binding constants, hence an almost complete thermodynamic profile can be established. Principles and application of ITC have been well documented over recent years, experimentally the technique is simple to use and in ideal scenarios data analysis is trivial. However, ITC experiments can be designed such that previously inaccessible parameters can be evaluated. We outline some of these advances, including (1) exploiting different experimental conditions; (2) low affinity systems; (3) high affinity systems and displacement assays. In addition we ask the question: What if data cannot be fit using the fitting functions incorporated in the data-analysis software that came with your ITC? Examples where such data might be generated include systems following non 1:n binding patterns and systems where binding is coupled to other events such as ligand dissociation. Models dealing with such data are now appearing in literature and we summarise examples relevant for the study of ligand-DNA interactions.     

Insulin and IGF-1 manifest differential effects in a clonal capillary endothelial cell line.

125I-insulin (10 fmoles) binding plus internalization (BI) to a clonal capillary endothelial (CE) cell line reached to a steady state after 20 min. Acid-washed fraction accounted for nearly half of the total specifically-bound hormone. Dissociation constants (Kd) for insulin-surface receptor in acid-extractable fraction were 0.04 nM (high affinity) and 4.7 nM (low affinity) with a total number of 210,000 high affinity receptors per cell. When 125I-labeled IGF-1 (15 fmoles) was incubated similarly, BI reached only a quasi-equilibrium by 6 min and continued to increase thereafter. 2-Deoxyglucose transport in these cells was stimulated by insulin whereas IGF-1 inhibited its entry.     

Models of the cooperative mechanism for Rho effector recognition: implications for RhoA-mediated effector activation

Activated GTPases of the Rho family regulate a spectrum of functionally diverse downstream effectors, initiating a network of signal transduction pathways by interaction and activation of effector proteins. Although effectors are defined as proteins that selectively bind the GTP-bound state of the small GTPases, there have been also several indications for a nucleotide-independent binding mode. By characterizing the molecular mechanism of RhoA interaction with its effectors, we have determined the equilibrium dissociation constants of several Rho-binding domains of three different effector proteins (Rhotekin, ROCKI/ROK beta/p160ROCK, PRK1/PKNalpha where ROK is RhoA-binding kinase) for both RhoA.GDP and RhoA.GTP using fluorescence spectroscopy. In addition, we have identified two novel Rho-interacting domains in ROCKI, which bind RhoA with high affinity but not Cdc42 or Rac1. Our results, together with recent structural data, support the notion of multiple effector-binding sites in RhoA and strongly indicate a cooperative binding mechanism for PRK1 and ROCKI that may be the molecular basis of Rho-mediated effector activation.     

Conformational aspects of inhibitor design: enzyme-substrate interactions in the transition state.

The theory of absolute reaction rates suggests that enzymes, like other catalysts, can enhance the rate of a reaction only to the extent that they bind the altered substrate in the transition state (S++) more tightly than they bind the substrate in the ground state (S). ES dissociation constants commonly fall in the physiological range, but recent kinetic studies indicate that formal ES++ dissociation constants of less than 10(-20) M are achieved by enzymes of several classes. Studies with stable analogues suggest that these remarkable powers of discrimination involve a tendency of the enzyme to close around S++ in such a way as to maximize binding contacts; that several parts of the substrate contribute to S++ binding; and that their contributions to binding affinity can be strongly synergistic.     

Ligand binding affinity determined by temperature-dependent circular dichroism: cyclin-dependent kinase 2 inhibitors

To support drug discovery efforts for cyclin-dependent kinase 2 (CDK2), a moderate-throughput binding assay that can rank order or estimate the affinity of lead inhibitors has been developed. The method referred to as temperature-dependent circular dichroism (TdCD) uses the classical temperature-dependent unfolding of proteins by circular dichroism (CD) to measure the degree of protein unfolding in the absence and presence of potential inhibitors. The midpoint of unfolding is the Tm value. Rank ordering the affinity and predictions of the dissociation constant of compounds is obtained by measuring the increase in Tm for different protein-inhibitor complexes. This is the first time an extensive characterization of the TdCD method has been described for characterizing lead inhibitors in a drug discovery mode. The method has several favorable properties. Using the new six-cell Peltier temperature controller for the Jasco 810 spectropolarimeter, one can determine the affinity of 12-18 compounds per day. The method also requires only 20-40 microg protein per sample and can be used to estimate the affinity of compounds with dissociation constants of picomolar to micromolar. An important property of the method for lead discovery is that dissociation constants of approximately 5 microM can be estimated from a single experiment using a low concentration of compound such as 20 microM, which is generally low enough for most small molecules to be soluble for testing. In addition, the method does not require labeling the compound or protein. Although other methods such as isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of binding, ITC requires 1-2 mg protein per sample, cannot readily determine binding constants below nanomolar values, is most versatile with soluble compounds, and has a throughput of two to three experiments per day. The ITC method is not usually used in a high-throughput drug discovery mode; however, using the thermodynamic information from several ITC experiments can make the TdCD method very robust in determining reliable binding constants. Using the kinase inhibitors BMS-250595, purvalanol B, AG-12275, flavopiridol, and several other compounds, it is demonstrated that one can obtain excellent comparisons between the Kd values of binding to CDK2 obtained by TdCD and ITC.     

Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5' splice site selection in the hnRNP A1 mRNA precursor

In the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 pre-mRNA, different regions in the introns flanking alternative exon 7B have been implicated in the production of the A1 and A1B mRNA splice isoforms. Among these, the CE1a and CE4 elements, located downstream of common exon 7 and alternative exon 7B, respectively, are bound by hnRNP A1 to promote skipping of exon 7B in vivo and distal 5' splice site selection in vitro. Here, we report that CE1a is flanked by an additional high affinity A1 binding site (CE1d). In a manner similar to CE1a, CE1d affects 5' splice site selection in vitro. Consistent with a role for hnRNP A1 in the activity of CE1d, a mutation that abrogates A1 binding abolishes distal 5' splice site activation. Moreover, the ability of CE1d to stimulate distal 5' splice site usage is lost in an HeLa extract depleted of hnRNP A/B proteins, and the addition of recombinant A1 restores the activity of CE1d. Notably, distal 5' splice site selection mediated by A1 binding sites is not compromised in an extract prepared from mouse cells that are severely deficient in hnRNP A1 proteins. In this case, we show that hnRNP A2 compensates for the A1 deficiency. Further studies with the CE4 element reveal that it also consists of two distinct portions (CE4m and CE4p), each one capable of promoting distal 5' splice site use in an hnRNP A1-dependent manner. The presence of multiple A1/A2 binding sites downstream of common exon 7 and alternative exon 7B probably plays an important role in maximizing the activity of hnRNP A1/A2 proteins.     

Quadruplex-coupled kinetics distinguishes ligand binding between G4 DNA motifs

G-quadruplex (or G4 DNA) specific ligands are important potential anticancer molecules as telomerase inhibitors. On the other hand, emerging evidence implicates G4 DNA in regulation of several oncogenes making telomerase inhibitors amenable to undesired effects (Borman, S. (2007) Chem. Eng. News 85 (22), 12-17). Therefore molecules which can discriminate between G4 DNA are of interest, both as telomerase inhibitors and for selective intervention of gene expression. Design of selective molecules requires resolution of the coupled equilibria between intramolecular quadruplex-formation and bimolecular ligand-binding. Several previous studies have reported G4-ligand binding kinetics, however the primary equilibrium of intramolecular G4 DNA folding/unfolding was not considered. Here, we quantitatively assess the linked equilibrium in G4-ligand complexes using a novel real time surface plasmon resonance-based technique. Kinetic constants for G4 folding/unfolding and ligand binding were simultaneously determined, for the first time, from a single reaction by resolving the coupled equilibrium. We demonstrate the coupled model by showing that affinity of TMPyP4 (a well-established anticancer telomerase inhibitor) for the human telomere quadruplex is only 3-fold more than the c-MYC promoter G4, which is known to repress c-MYC. This provides quantitative rationale to poor selectivity of TMPyP4 in recently observed cell-based assays. In the light of recent advances indicating G4's regulatory potential in several important genes, quantitative evaluation of selectivity vis-à-vis affinity as presented here will augment design and preliminary screening of new molecules.     

Identification of a male-specific RNA binding protein that regulates sex-specific splicing of Bmdsx by increasing RNA binding activity of BmPSI

Bmdsx is a sex-determining gene in the silkworm and is alternatively spliced in males and females. CE1 is a splicing silencer element responsible for the sex-specific splicing of Bmdsx. To identify sex-specific factors implicated in the sex-specific splicing of Bmdsx, we performed RNA affinity chromatography using CE1 RNA as a ligand. We have identified BmIMP, a Bombyx homolog of IGF-II mRNA binding protein (IMP), as a male-specific factor that specifically binds to CE1. The gene encoding BmIMP is localized on the Z chromosome and is male-specifically expressed in various tissues. Antisense inhibition of BmIMP expression increased female-specific splicing of Bmdsx pre-mRNA. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown analyses demonstrated that BmIMP physically interacts with BmPSI, which has been identified as a factor implicated in the sex-specific splicing of Bmdsx, through the KH domains of BmIMP. The functional consequence of this interaction was examined using RNA mobility shift analysis. BmIMP increased BmPSI-CE1 RNA binding activity by decreasing the rate of BmPSI dissociation from CE1 RNA. Truncation analysis of BmIMP suggested that the KH domains are responsible for enhancing BmPSI-CE1 RNA binding activity. These results suggest that BmIMP may enhance the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI.     

Emerging challenges in ligand discovery: new opportunities for chromatographic assay

Ligand discovery initiatives are facing interesting challenges as ever-increasing numbers of proteins are entering screening programs. As an answer to steady pressure to improve performance in drug discovery, ligand discovery can expect to play an expanded role in generating small molecules as probes to help uncover the function of novel proteins. Chromatographic assay formats can offer new entry points into standard interaction characterization (binding and rate constants) as well as powerful, scaleable methods for compound screening. This review presents recent advancements in chromatographic assay technology, with a particular focus on frontal affinity chromatography as a platform technology for interaction analysis.     

Affinity capillary electrophoresis: important application areas and some recent developments

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting (`plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen–antibody, hapten–antibody, lectin–sugar, drug–protein, and enzyme–substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.     

From gel filtration to biosensor technology: the development of chromatography for the characterization of protein interactions

The objective of this review is to summarize the development of chromatographic techniques for the determination of reaction stoichiometries and equilibrium constants for solute interactions of biological importance. Gel chromatography is shown to offer a convenient means of characterizing solute self-association as well as solute-ligand interactions. Affinity chromatography is an even more versatile method of characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reactants. Adoption of different experimental strategies such as column chromatography, simple partition equilibrium experiments and biosensor technology has created a situation wherein affinity chromatography affords a means of characterizing the whole range of reaction affinities-from relatively weak interactions (binding constants less that 10(3)M (-1)) to tight interactions with binding constants greater than 10(9)M (-1). In addition to its established prowess as a means of solute separation and purification, chromatography thus also possesses considerable potential for investigation of the functional roles of the purified reactants-an endeavour that requires characterization as well as identification of the interactions responsible for a physiological phenomenon.     

Emerging challenges in ligand discovery: new opportunities for chromatographic assay

Ligand discovery initiatives are facing interesting challenges as ever-increasing numbers of proteins are entering screening programs. As an answer to steady pressure to improve performance in drug discovery, ligand discovery can expect to play an expanded role in generating small molecules as probes to help uncover the function of novel proteins. Chromatographic assay formats can offer new entry points into standard interaction characterization (binding and rate constants) as well as powerful, scaleable methods for compound screening. This review presents recent advancements in chromatographic assay technology, with a particular focus on frontal affinity chromatography as a platform technology for interaction analysis.     

Application of surface plasmon resonance toward studies of low-molecular-weight antigen-antibody binding interactions

Methods for studying low-molecular-weight antigen-antibody binding interactions using surface plasmon resonance detection are presented. The experimental parameters most relevant to studies of low-molecular-weight antigen-antibody binding interactions are discussed. Direct kinetic analysis of the binding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be calculated. Equilibrium analysis, including steady-state and solution affinity studies, offers an alternative approach to direct kinetic analysis when knowledge of the individual kinetic rate constants is not required or difficult to determine. The various methods are illustrated by studies of an anti-T(4) Fab fragment binding interaction with several thyroxine analogs. The methods utilized were dependent on the affinity of the interaction. The high-affinity anti-T(4) Fab fragment/l-T(4) binding interaction was evaluated using direct kinetic analysis. An intermediate affinity anti-T(4) Fab fragment/l-T(3) binding interaction was evaluated using a combination of direct kinetic analysis, steady-state analysis, and solution affinity analysis. The relatively weak anti-T(4) Fab fragment/l-T(2) binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyroxine tracers that could not be immobilized to a biosensor surface were also evaluated via the solution affinity format. In cases where a given binding interaction was examined using multiple methods the results were comparable.