Biospecific adsorption chromatography of phospholipase A2 from different sources

Methods of biospecific adsorption chromatography of phospholipase A2 obtained from porcine pancreas and Naja naja oxiana, Vipera ursini renardi, Vespa orientalis venoms were developed. Granulated polyamide with covalently linked phosphatidylethanolamine were used as an affinity adsorbent. Chemical inertness of linked phosphatidylethanolamine to the hydrolytic action of phospholipase A2 and its high affinity for biospecific complexes are shown. Forms of phospholipase A2 different in their affinity for an immobilized substrate was isolated by biospecific adsorption chromatography. The role of hydrophobic and electrostatic interactions in formation of enzyme-ligand complexes was studied.     

Quantitative affinity chromatography: increased versatility of the technique for studies of ligand binding

The potential of affinity chromatography for the characterization of strong solute-ligand interactions is explored by studying the NADH-dependent elution of rabbit muscle lactate dehydrogenase from a column of trinitrophenyl-Sepharose in 0.067 M phosphate, pH 7.2. An interesting development is the simplification of the general affinity chromatography theory that emanates from the use of affinity matrices with a high concentration of immobilized reactant groups. The resultant expression allows evaluation of the intrinsic association constant for solute-ligand interactions from a single series of either zonal or frontal affinity chromatographic experiments conducted in the presence of a range of free ligand concentrations. Thus, contrary to previous belief, an affinity matrix designed for solute purification work should prove to be an asset for, rather than an impediment to, the study of solute-ligand interactions by quantitative affinity chromatography.     

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.     

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.     

Influence of the nature of coupling agents on insulin adsorption on supports grafted with sialic acid for high-performance affinity chromatography

Porous silica exhibits excellent mechanical properties for use as a stationary phase for high-performance liquid chromatography. However, negative surface charges make it unusable in its native state. For this reason, silica beads are coated with dextran polymers carrying a calculated amount of diethylaminoethyl groups. Both the minimization of non-specific interactions and the hydrophilic character of such supports allow their functionalization with biospecific ligands and finally their use in high-performance affinity chromatography of biological products. The use of these modified supports in high-performance affinity chromatography requires a better understanding of various characteristics of stationary phases. For this purpose, several techniques were utilized, in particular, size-exclusion chromatography and adsorption of radiolabelled albumin. These methods provided complementary information on the structure of these supports. Coated silica-based supports were functionalized with sialic acid by means of different coupling agents. The affinity of these supports for insulin was determined by the establishment of adsorption isotherms and by high-performance affinity chromatography, to evidence the relationships between structural characteristics of the supports and their separation properties. The study of interactions between these supports and insulin allowed us to show the importance of the coupling method on the performances of supports in affinity chromatography.     

Biospecific adsorption of lysozyme onto monoclonal antibody ligand immobilized on nonporous silica particles

It is very important to understand the equilibrium and dynamic characteristics of biospecific adsorption (affinity chromatography) for both scientific and application purposes. Experimental equilibrium and dynamic column data are presented on the adsorption of lysozyme onto antibody immobilized on nonporous silica particles. The Langmuir model is found to represent the equilibrium experimental data satisfactorily, and the equilibrium association constants and heats of adsorption have been estimated for two systems with different ligand densities. The effects of nonspecific interactions are more pronounced in the system with low-density ligand. The dynamic interaction kinetic parameters are estimated by matching the predictions of a fixed-bed model with the experimental breakthrough curves. The agreement between theory and experiment is good for the initial phases of breakthrough, where the mechanism of biospecific adsorption is dominant. In the later phase (saturation neighborhood) of breakthrough, the effects of nonspecific interactions appear to be greater in the low-density ligand system. The kinetics of the nonspecific interactions were estimated from the data of the later phase of breakthrough and were found to be considerably slower than those attributed to biospecific adsorption.     

Effect of experimental conditions on strong biocomplimentary pairing in high-performance monolithic disk affinity chromatography

The effect of flow-rate on quantitatively determined binding parameters for several biocomplementary pairs in affinity mode high-performance monolithic disk affinity chromatography (HPMDAC) has been investigated using frontal analysis approach. Affinity interactions were evaluated from linearized adsorption isotherms and dynamic dissociation constants of the complexes K(diss.) and the theoretical adsorption capacities Q(max) were calculated. HPMDAC isolation of a typical protein trypsin from both buffered solution and artificial mixture as well as biospecific extraction of antibodies against bovine serum albumin and recombinant protein G from such complex mixtures as blood serum and cellular lysate were examined. Immobilized counterparts soybean trypsin inhibitor, bovine serum albumin, and human immunoglobulin G were used in chromatographic experiments. The maximum adsorption capacities obtained at different flow-rates were compared with those determined at static conditions. The dependence of quantitative parameters on the surface density of immobilized ligands has also been explored. Finally, a series of experiments was carried out to evaluate the dependence of dynamic affinity binding on temperature for two complementary pairs.     

Nanotechnologies in proteomics

Progress in proteomic researches is largely determined by development and implementation of new methods for the revelation and identification of proteins in biological material in a wide concentration range (from 10(-3) M to single molecules). The most perspective approaches to address this problem involve (i) nanotechnological physicochemical procedures for the separation of multicomponent protein mixtures; among these of particular interest are biospecific nanotechnological procedures for selection of proteins from multicomponent protein mixtures with their subsequent concentration on solid support; (ii) identification and counting of single molecules by use of molecular detectors. The prototypes of biospecific nanotechnological procedures, based on the capture of ligand biomolecules by biomolecules of immobilized ligate and the concentration of the captured ligands on appropriate surfaces, are well known; these are affinity chromatography, magnetic biobeads technology, different biosensor methods, etc. Here, we review the most promising nanotechnological approaches for selection of proteins and kinetic characterization of their complexes based on these biospecific methods with subsequent MS/MS identification of proteins and protein complexes. Two major groups of methods for the analysis and identification of individual molecules and their complexes by use of molecular detectors will be reviewed: scanning probe microscopy (SPM) (including atomic-force microscopy) and cryomassdetector technology.     

Versatility of mixed-function adsorbents in biospecific protein desorption: Accidental affinity and an improved purification of aspartate transcarbamoylase from wheat germ

Under appropriate experimental conditions (usually but not invariably including low ionic strength) wheat germ aspartate transcarbamoylase can be specifically desorbed by the substrate, carbamoyl phosphate, from hydroxyapatite, from N-(3-carboxypropionyl)aminooctyl-Sepharose, from 10-carboxydecylamino-Sepharose, from Cibacron Blue F3GA-Sepharose, and from Coomassie Blue R250-Sepharose. Experimental evidence suggests that (a) the enzyme is adsorbed at heterogeneous sites on each column, only some of which are susceptible to substrate-specific desorption; (b) in none of these cases is the initial adsorption essentially biospecific, i.e., these are not cases of classical affinity chromatography; (c) in the case of 10-carboxydecylamino-Sepharose, and therefore presumably also in the other cases, the desorption is biospecific, i.e., involves the formation of the catalytically significant enzyme-carbamoyl phosphate complex. Substrate-specific desorption in these cases appears to derive from “accidental” affinity between, on the one hand, clusters of active (ionic, hydrophobic, aromatic, etc.) groups on the protein and, on the other, complementary clusters on the adsorbent, some of these interactions being perturbed when the ligands binds to the protein. Biospecific desorption from 10-carboxydecylamino-Sepharose has been incorporated as the sole chromatographic step in a new, 8000-fold purification of the enzyme. It is suggested that biospecific desorption from essentially nonbiospecific adsorbents could explain some published purifications currently described as “affinity chromatography.”     

Surface-labelling studies on skeletal-muscle cells in vitro. Heterogeneity of iodinated cell-surface proteins.

Some theoretical aspects of the desorption of a column-bound protein by elution with its biospecific ligand are considered in cases where, in comparison with the unliganded protein, the protein-ligand complex has a diminished but finite affinity for the adsorbent. A quantity termed the biospecific sensitivity, B, is introduced to facilitate comparison between different systems. Biospecific sensitivity may be defined as the fractional change in standard free energy of adsorption on formation of the protein-ligand complex. The effects of a moderate-to-low biospecific sensitivity on theoretical desorption profiles have been examined by using a computer simulation of the classical multiple-plate column model. Desorption was simulated under various boundary conditions involving protein-adsorbent and protein-ligand affinities and the initial concentrations of adsorption sites, protein and ligand. These simulations suggest that, when the biospecific sensitivity is low, desorption is optimized if (a) the unliganded protein is adsorbed as weakly as possible, (b) the column is loaded to near-saturation with the required protein, (c) the free ligand concentration is many times greater than that giving near-saturation of the protein in free solution, and (d) protein contaminants with high affinity for the adsorbent, and present in large amount, are removed in preliminary purification steps.     

蛋白质间相互作用技术的研究近况

蛋白质间相互作用技术的研究近况黄翠芬叶棋浓（军事医学科学院生物工程研究所,北京１００８５０关键词：蛋白质,相互作用,技术ＲｅｃｅｎｔＡｄｖａｎｃｅｓｉｎｔｈｅＴｅｃｈｎｉｑｕｅｓｏｆＰｒｏｔｅｉｎ┐ＰｒｏｔｅｉｎＩｎｔｅｒａｃｔｉｏｎｓＨｕａｎｇＣｕ...     

Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods

The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.     

Affinity chromatographic sorting of carboxypeptidase a and its chemically modified derivatives

Carboxypeptidase A and derivatives obtained by chemical modification of various active center components were subjected to affinity chromatography on a p-aminobenzylsuccinic acid-Sepharose 4B conjugate. Tetardation of the enzyme on the column was dependent on the residue modified when elution was carried out with 0.3 m NaCl at pH 7.0. Both the functional zinc atom and the active site residue Glu-270 are essential for effective adsorption while alteration of residues involved in hydrophobic interaction with substrate or in recognition of its terminal carboxyl group decreased retention on the affinity matrix. Elution of native carboxypeptidase with competing soluble benzylsuccinic acid indicated that only active center binding of the immobilized inhibitor accounts for retardation of the enzyme on the column. Hence, affinity chromatography on this biospecific adsorbent using mild elution conditions (which do not distort protein structure) provides an excellent tool for the rapid isolation and purification of active center modified enzyme even from a complex mixture of reaction products.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Thermodynamic and structural analysis of interactions between peptide ligands and SEB

Staphylococcal enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus and commonly associated with food poisoning. In this study, SEB‐binding peptides were identified by screening a phage displayed peptide library. The binding of peptides to SEB was tested with isothermal titration calorimetry (ITC) and of the five selected peptides, three showed affinity to SEB, with one measured to have the highest affinity constant (10⁵ M^?1). ITC revealed that the interaction of peptide ligands with SEB was driven entropically and the binding was dominated by hydrophobic interactions. Circular dichroism (CD) measurements and molecular dynamics (MD) simulations, together, give a structural insight into the interaction of peptides with SEB. While SEB binding peptides showed random coil structure before binding, after complex formation they had more ordered structures. The peptide with highest affinity to SEB showed stable conformation during MD simulation. Taken together, our approach about thermodynamic and structural characterization of peptide ligands can be used to develop aptamers, with high affinity and selectivity, for biosensor applications. Copyright © 2009 John Wiley & Sons, Ltd.     

In vitro modeling of cell-scaffold interaction

A simple method of controlling the efficiency of surface ligand-cell receptor interaction has been developed in the course of modeling the specific adhesion of cells on a support with their subsequent proliferation and bone tissue formation, using affinity chromatography on macroporous monolithic sorbents. The biospecific peptide GRGDSP played the role of an active ligand on the support, whereas cells were simulated by polymeric (polystyrene) microparticles with the peptide EDYPVDIYYLMDLSYSMKDD immobilized on their surface. The latter peptide is part of the active site of the integrin molecule responsible for binding the RGD sequence. Thus, the monolithic ultrashort column (CIM® disk) represented a simplified model of the support (structural scaffold) possessing biospecific properties. The parameters of the interaction of affinity partners were quantitatively estimated by frontal analysis involving the construction of adsorption isotherms, followed by their linearization and mathematical processing. The data obtained indicate a high specificity of biological pairing, which is supported by the results of cell culture experiments.     

Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its alpha, beta, and gamma(c) receptors

Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the alpha, beta and the "common gamma" (gamma(c)) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor-ligand, and receptor-receptor interactions that are necessary for formation of the high-affinity IL-2/alphabetagamma(c) signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its alpha receptor, a moderately entropically favorable low affinity interaction between IL-2 and its beta receptor, and no interaction between IL-2 and the shared receptor, gamma(c). Formation of the stable intermediate trimolecular complexes of IL-2 with alpha and beta receptors, as well as IL-2 with beta and gamma(c) receptors proceeds through enthalpy-entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor alpha and beta chains, suggesting that a preformed alphabeta complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Ralphabetagamma(c) high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by gamma(c) contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Streaming potential—a general affinity sensor

Electrokinetic phenomena, such as streaming potential or streaming current, have so far been used for studies of unspecific adsorption of ionic compounds on various materials. This paper shows that streaming potential measurements can also be used for studies of biospecific interactions.Several different interactions were studied, e.g. lectin-carbohydrate, IgG-Protein A. Regardless of the type of interaction an increasing change in streaming potential was obtained with increasing concentration of the interacting molecule. The authors also observed a correlation between streaming potential and affinity constants for carbohydrate-induced desorption of Concanavalin A from partially hydrolyzed Sephadex G50. In conclusion, it is shown that streaming potential measurements can be used to study molecular interactions, and to determine concentrations and relative binding constants of interacting molecules.