Theoretical and experimental studies on zone-interference chromatography as a new method for determining macromolecular kinetic constants

Zone-interference chromatography is a new method for studying macromolecular interactions (S. Endo and A. Wada, Anal. Biochem. 124 (1982) 372). This method is a new style of affinity chromatography which requires no preparation of affinity-column materials but utilizes the velocity difference in a column between interacting molecular species. Using the stochastic theory on the behavior of solute molecules, both the association and the dissociation rate constants can be analytically obtained from the degree of deformation of elution patterns, i.e., the change of the first and second moments. In order to verify the present theory, computer simulation of elution profiles by the extended plate theory and a binding experiment between glutamate dehydrogenase and ADP have been carried out.     

Prediction of affinity and kinetics in biomolecular interactions by affinity chromatography

Computer simulation of affinity chromatography is a valuable tool for accurate prediction of column performance. In our study affinity pairs based on lectin and antibody interactions with carbohydrates have been used as model systems. In this well-characterized system we have demonstrated the usefulness of the simulation approach for determination of affinity and kinetics. These properties are typically difficult to obtain for many weakly interacting molecular species (i.e., when dissociation constants (K(D)) are greater than 10(-5) M). The influence of affinity and kinetics on peak broadening in affinity chromatography has also been investigated.     

Stability of asymmetrical hybrid hemoglobins. Determination by anaerobic high performance liquid chromatography

Asymmetrical hybrid hemoglobins formed in mixtures of Hb A and Hb S, Hb F and Hb S, Hb S and Hb York(beta 146 His----Pro), and Hb A and Hb York were separated by high performance liquid chromatography on cation and anion exchange columns under anaerobic conditions. The ratio of the hybrid hemoglobin to the total mixture was consistently lower than that theoretically expected and decreased with longer elution times. The hybrid tetramer appears to be unstable even under anaerobic conditions and dissociates into alpha beta dimers. The time course of dissociation of the hybrid hemoglobins was determined by varying the separation programs and thus separating the hybrid hemoglobin at different elution times. The rate of the dissociation of the hybrid hemoglobins studied follows first order kinetics. The lines representing the time course of dissociation of hybrid hemoglobins were extrapolated to time 0 to determine the fraction of the hybrid hemoglobin in the mixture prior to separation. The values obtained for equimolar mixtures of Hb A and Hb S and Hb York and Hb S or Hb A were in agreement with the expected theoretical value (50%). In contrast, the value obtained for hybrid hemoglobin FS was slightly less (about 40%). AY and SY hybrid hemoglobins dissociated into dimers at a considerably faster rate than did AS and FS hybrid hemoglobins, possibly because of the mutation at the beta 146-position in hybrid hemoglobins containing alpha beta Y dimers. This mutation hinders the formation of salt bridges that normally stabilize the "T" quaternary conformation. Since such hybrid hemoglobins have a partial "R" conformation even when deoxygenated, their rate of dissociation to dimers is expected to increase.     

Analysis of protein-protein interaction by simulation of small-zone size-exclusion chromatography: application to an antibody-antigen association

The association of two or more macromolecules results in the formation of a complex characterized by a larger Stokes radius than that of its components. Therefore, analytical procedures such as ultracentrifugation and size-exclusion gel chromatography that resolve molecules on the basis of size have been used to characterize the association. In this paper we describe an iterative computer simulation of small-zone size-exclusion gel filtration. The simulation describes univalent and bivalent interactions of proteins of equal and nonequal molecular weight and appears to have both qualitative and quantitative application to the evaluation of protein-protein interaction as revealed by alteration of chromatographic elution profiles. To test the validity of the simulation, the model was applied to an antibody-antigen interaction by determining the association constant (Ka) for the interaction between the binding fragment derived from a human immunoglobulin A rheumatoid factor and the antigenic fragment obtained from a human myeloma immunoglobulin G. The self-consistency of the estimated Ka values obtained with a valence value of 2 in contrast to the lack of self-consistency if an antigenic valence of 1 was assumed was taken to support the ability of the algorithm to reasonably emulate the chromatographic processes of interacting proteins. In conjunction with the computer simulation, a sensitive microcomputer-interfaced chromatography system was assembled, which is capable of analyzing 300 ng of protein in less than 1 h. This combination of rapid reagent-conservative chromatography and simulation analysis may contribute to the usefulness of small-zone gel filtration in studies of protein-protein interaction.     

Analysis of ribonuclease-nucleotide interactions by quantitative affinity chromatography.

Quantitative affinity chromatography on uridine-5'-(Sepharose-4-aminophenylphosphoryl)-2'(3')-phosphate was developed for the study of binding of ribonuclease species to nucleotide ligands. Elution of the native species ribonuclease-A and -S on the afffinity matrix in 0.4 M ammonium acetate, pH 5.2, containing various amounts of the soluble competing ligand 2'-cytidine monophosphate, reveals an inverse response of elution volume to concentration of soluble ligand. This response conforms to behavior expected for the competing binding equilibria enzyme-soluble ligand and enzyme-insoluble ligand. A-NALYSIS OF ELUTION DATA ALLOWS CALCULATION OF KI and KIM, the dissociation constants, respectively, for the soluble and insoluble protein-ligand complexes. The values of these chromatographically derived constants are similar to values of dissocation constants determined in solution by kinetics of inhibition by 2'-cytidine monophosphate and uridine-5'-(j-aminophenylphosphoryl)-2'(3')-phosphate. Successful competitive elution experiments with [p-F-Phe8]semisynthetic ribonuclease-S' and individual elution trials for [4-F-His12]semisynthetic ribonuclease-S' indicate the utility of the quantitative affinity chromatographic technique for determination of ligand binding properties of ribonuclease derivatives, including inactive species. Nonbiospecific aspects of the interaction of ribonuclease with the affinity matrix in ammonium acetate buffers of concentrations 0.1 M and below were noted, delinating limits of conditions allowing the biospecificity needed for ligand-binding analyses by competitive elution. The dependence of ribonuclease competitive elution behavior on the amount of protein eluted also was examined and related to theoretical considerations in the quantitative application of affinity chromatography.     

Separation of asymmetrical hybrid containing hemoglobin F by anaerobic anion-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of oxyhemoglobins S and F and A and F were separated by high-performance liquid chromatography on a 4.6 X 250 mm wide-pore polyethyleneimine-silica gel column under anaerobic conditions. The resulting HPLC chromatogram showed three peaks, with the middle peak representing the hybrid hemoglobin. The areas of these three peaks were quantified and the amount of hybrids formed was less than that predicted theoretically. We found that the deviation was due to the equilibrium constant of the FS hybrid hemoglobin differing from that of the parent hemoglobins. In this report, we introduce the anaerobic recycle ion-exchange HPLC method to determine the rate of dissociation of AS and FS hybrid hemoglobins at constant pH buffer conditions. The results obtained by this method demonstrate that FS hybrid hemoglobin is more unstable than AS hybrid hemoglobin. The free energy of association for asymmetrical hybrids containing hemoglobin F is approximately 0.6 Kcal/mol greater than that of the symmetrical parent hemoglobins.     

Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.     

Elution-band relaxation method. A method to analyze isomerization kinetics by HPLC and application to protein denaturation-renaturation

We present a novel method, the 'elution-band relaxation method', to analyze quantitatively reversible isomerization kinetics by elution chromatography, taking advantage of the high resolution and speed of high-performance liquid chromatography (HPLC). The kinetic information is obtained by measuring the first temporal moments of chromatograms of molecules undergoing isomerization and analyzing their dependence on the column length or flow rate. The major advantage of this method is that it is applicable to reactions as fast as the time of elution in HPLC, a speed which has not been attained previously in analysis of isomerization reactions based on the chromatographic property of molecules. We describe the method and report an experimental application to the denaturation-renaturation kinetics of bovine pancreatic ribonuclease A as an example.     

Expression of multivalency in the affinity chromatography of antibodies. Appendix: Derivation and evaluation of equations for independent bivalent interacting systems in quantitative affinity chromatography.

The expression of multivalency in the interaction of antibody with immobilized antigen was evaluated by quantitative affinity chromatography. Zones of radioisotopically labeled bivalent immunoglobulin A monomer derived from the myeloma protein TEPC 15 were eluted from columns of phosphorylcholine-Sepharose both in the absence and presence of competing soluble phosphorylcholine. At sufficient immobilized phosphorylcholine concentration, the variation of elution volume of bivalent monomer with soluble ligand was found to deviate from that observed for the univalent binding of the corresponding Fab fragment. In addition, the apparent binding affinity of the bivalent monomer increased with immobilized antigen density. Use of equations relating the variation of elution volume with free ligand concentration for a bivalent binding protein allowed calculation of microscopic single-site binding parameters for the bivalent monomeric antibody to both immobilized and soluble phosphorylcholine. The chromatographic data not only demonstrate the effect of multivalency on apparent binding affinity but also offer a relatively simple means to measure microscopic dissociation constants for proteins participating in bivalent interactions with their ligands.     

Stability of antibody attachment in immunosorbent chromatography

Detachment of immobilized antibody from its support matrix in an immunosorbent system prepared by the cyanogen bromide activation route was demonstrated. The immunosorbent system, however, was stable under slightly basic conditions. Detachment of antibody from the support material occurred mainly during the elution of the antigen complexed with the immobilized antibody. The antibody was detached from the matrix by different elution buffers. The detachment pattern of antibody was independent of the number of cycles used and also independent of the support materials. A change in the molecular structure of the detached antibody occurred as revealed by an alteration in the ultraviolet absorption spectra of the released antibody. The antibody detachment from the support matrix occurred in more than one antigen-antibody system suggesting that the leakage phenomenon may be a widespread disadvantage associated with the cyanogen bromide activation procedure. Detachment of the antibody could be reduced to < 10 ng ml⁻¹ by immobilizing antibody on the properly oxidized polysaccharide support material or on the N-hydroxysuccinimide activated ester gel. Antibody dissociation from the matrix did not occur when antibodies were immobilized by either amine or amide attachment, thus, immunosorbents prepared by such strategies are suitable for the immunochromatographic purification of proteins from complex mixtures.     

Solvent modulation in hydrophobic interaction chromatography

Solvents play a critical role in hydrophobic interaction chromatography (HIC), since the separation of proteins by HIC is based on the hydrophobicity of the proteins presented to the solvents. This review first describes the solvent properties which determine the effect of cosolvents on the binding and elution of proteins in HIC; i.e., the protein solvent interactions and the surface tension of water/cosolvent mixture. Second are presented the various cosolvents which have been tested as facilitating binding or elution of the proteins. Last, some examples of solvent manipulation which resolved complex mixtures of proteins by HIC are reviewed.     

Predicting the elution behavior of proteins in affinity chromatography on non-porous particles.

Affinity chromatography on non-porous particles of microsize is particularly useful for the rapid analysis and micropreparative separation of proteins. The elution behavior of proteins in an affinity column packed with non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate was investigated both theoretically and experimentally, using the lysozyme-Cibacron Blue 3G-A affinity system. Equations used to predict the elution profiles, resulting from the elution by increasing the ionic strength (NaCl concentration) in the mobile phase, were obtained. The maximum adsorbate concentration, desorption rate constant and equilibrium constant under elution conditions were determined by matching experimental data with predicted elution profiles. Based on the parameters determined at a flow-rate of 0.5 ml/min and with 1 M NaCl in the elution buffer, the model equations could predict the elution profiles for other experimental runs, where different flow-rates and sodium chloride concentrations were used. Both the experimental and predicted results revealed that the affinity interaction kinetics are not significantly influenced by the flow-rate and, hence, the film mass transfer. To elute bound lysozyme from immobilized dye ligand, a higher value of the ionic strength leads to a faster elution and a sharper elution peak. The influence of elution conditions on the kinetic and thermodynamic parameters and, consequently, on the elution peak profiles was evaluated. The model equations can also predict the behavior of protein elution from an affinity column by changing the pH of the mobile phase, according to a previous study.     

Progress in packed column supercritical fluid chromatography: materials and methods

This article summarizes recent developments in packed column supercritical fluid chromatography. Silica-based chemically bonded sorbents, similar to those used for HPLC, are widely used with solvent-modified fluids containing additives to suppress undesirable solute-sorbent interactions that lead to poor peak shapes. Composition programming is the most useful approach to gradient elution separations since solvent-modified fluids have low compressibility. Packed column SFC is most useful for the separation of mixtures usually separated by normal-phase HPLC. Compared to normal-phase HPLC it offers faster separations, higher efficiencies, faster column re-equilibration, and a wider range of experimental variables for optimization. Packed column SFC is being increasingly selected for the analytical and preparative separation of racemic mixtures using enantiomer-selective sorbents.     

Frontal gel chromatography of interacting systems: theoretical and experimental evaluation of the shapes of elution profiles for systems of the type A + B in equilibrium C

A theoretical and experimental study has been made of the advancing elution profile in frontal gel chromatography of interacting systems for which the elution volume of the complex is smaller than that of the larger reactant. First, Gilbert-Jenkins theory is used to delineate the form of the elution profile from the magnitudes of the elution volumes and concentrations of reacting species. This procedure resulted in the detection of a misinterpretation of certain patterns obtained in a gel chromatographic study of the interaction between myoglobin and ovalbumin. Second, a numerical computational procedure, which incorporates both axial dispersion and concentration-dependence of species elution volumes, is used to establish the influence of these two factors on boundary shapes for such systems. Third, frontal gel chromatography on Sephadex G-75 is used to compare experimental behavior with theoretical profiles predicted for the electrostatic interaction between cytochrome c and soybean trypsin inhibitor (pH 6.8, I 0.01). Results of these experiments serve as a guide for future conduct of experiments aimed at characterization of biologically important, reversible complex formation between proteins and/or other macromolecules.     

Immunoaffinity chromatography of enzymes.

Immunoaffinity chromatography of enzymes represents an attractive purification technique suitable for one-step and large-scale purification of enzymes to homogeneity. Monoclonal and polyclonal antibodies can be used equally well. The broad use of the technique is restricted by the harsh elution conditions which are often required. The efforts to overcome these limitations and to optimize the method are reviewed, viz. proenzyme purification, purification of enzymes as part of multienzyme complexes carried out by a mild dissociation step, specific elution by substrates and effectors, enzyme stabilization, electrophoretical desorption and negative elution by adsorbing impurities from the crude extract, and hypotonic elution. The current practice is discussed considering antibody and enzyme selection, optimization of elution conditions, and washing steps using different media. Representative examples are given for various approaches.     

The active site titration of proteinases by using alpha 2-macroglobulin and high-performance liquid chromatography.

The active site titration for various proteinases relies on the development of optimal enzyme titrants for each proteinase, but these titrants are only available for a limited number of proteinases. We have described a new active site titration method applicable to various kinds of endoproteinases using small quantities of the enzymes. This method was carried out by using alpha 2-macroglobulin (alpha 2M) as a titrant and a high-performance liquid chromatography (HPLC) system. When the proteinase solution was treated with alpha 2M, the active proteinase was trapped by alpha 2M. In this reaction alpha 2M does not usually complex with inactive proteinase. After the reaction of proteinase with an excess of alpha 2M, the reaction mixture is applied to an HPLC gel column to separate the uncomplexed enzyme from the one complexed with alpha 2M. The active proteinase is complexed and eluted with alpha 2M, but the inactive proteinase is eluted at the original elution volume. The same amount of the enzyme was also applied to the column. From the decrease of the peak height at the elution position of the uncomplexed proteinase, we can estimate the ratio between enzymatically active proteinases and total proteinases. To test the usefulness of this method, we applied this method to chymotrypsin and trypsin whose activities were predetermined by conventional active site titration, and there was good agreement between both results. With this new method, we can estimate a proteinase activity with as little as 200 ng of the enzyme, a very small amount compared with those required in conventional methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

A structural basis for four distinct elution profiles on concanavalin A--Sepharose affinity chromatography of glycopeptides.

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanvalin A (Con A)--Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains: (abstract:see text). Such glycopeptides have only a single mannose residue capable of interacting with Con A--Sepharose; an interacting mannose residue is either an alpha-linked nonreducing terminal residue or an alpha-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl alpha-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures: (abstract: see text) where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A--Sepharose and elution as a sharp peak with 0.1 M methyl alpha-D-glucopyranoside; glycopeptides giving this pattern had the structures: (abstract: see text) where R2 is either H, glcNAc, Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the mimimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl alpha-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures: (abstract: see text) where R3 is either GlcNAc,Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc.Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A--Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.     

High-performance liquid chromatography analysis of spectrin oligomerization

Gel filtration chromatography has been used to analyze the oligomerization of human erythrocyte spectrin. By applying an exponentially modified Gaussian function we have been able to resolve overlapping elution peaks. From these peaks it was possible to calculate the equilibrium composition of each spectrin concentration and thus also the dissociation constants describing the oligomeric process. The determined dissociation constants for tetramer formation (1.3 microM) and for hexamer formation (24 microM) agree well with other measurements.     

Frontal affinity chromatography of ovalbumin glycoasparagines on a concanavalin A-sepharose column. A quantitative study of the binding specificity of the lectin

The interactions of Sepharose 4B-immobilized concanavalin A (ConA) with 10 glycoasparagines derived from ovalbumin were investigated quantitatively by frontal affinity chromatography. In this method, a carbohydrate solution is applied continuously to a ConA-Sepharose column and the retardation of the elution front is measured as a parameter of the strength of the interaction. The dissociation constant (Kd) for each saccharide with ConA can be determined. An analysis of the binding of p-nitrophenyl-alpha,D-mannoside has shown that the binding properties of ConA do not change essentially after immobilization on Sepharose 4B. Each of the ovalbumin glycoasparagines was labeled with tritium by the reductive methylation method for analysis. A comparison of the Kd values obtained showed that the binding of ConA varies considerably with very slight structural differences of the glycosyl chain. The results suggest that ConA recognizes a specific glycosyl chain structure, Man alpha 1-6(Man alpha 1-3)Man, in which at least one hydroxyl group at the C-3 position of C-6-linked mannose should be free. The glycoasparagines containing this structure bound strongly to ConA-Sepharose with dissociation constants below 3.4 X 10(-7) M.     

Polymer versus monomer as displacer in immobilized metal affinity chromatography

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu²⁺-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48°C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)₆-tag [(His)₆-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu²⁺ ion. Complete elution of bound (His)₆-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)₆-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.