Investigation of binding site density: effects on the interaction between cibacron blue-dextran conjugates and lysozyme

Cibacron-blue-dextran conjugates have been produced with a range of ligand loadings using a dextran preparation of average molecular weight of 2 x 10(6). The equilibrium binding capacity of these ligand conjugates for lysozyme was determined using a gel permeation procedure to separate bound from free protein. The results obtained give clear evidence for at least two types of binding showing a marked difference in affinity. For the higher-affinity interaction the half-saturation constant decreases with increasing ligand loading. The number of dye molecules participating in binding is proportional to loading up to 154 mol dye/mol dextran but is reduced at the highest loading used (315 mol dye/mol dextran). This may be due to steric interference or to dye stacking reducing the number of dye molecules available for binding.     

Protein interaction with immobilized ligands: quantitative analyses of equilibrium partition data and comparison with analytical chromatographic approaches using immobilized metal affinity adsorbents

Quantitative or analytical affinity chromatography has been successful primarily for the analysis of biologically determined macromolecular affinity relationships. Quantitative approaches are also needed to better characterize simpler, chemically defined immobilized ligands with potential for selective interaction with specific, predetermined protein surface groups. Protein interaction with immobilized metal is a rather selective and versatile, high-affinity adsorption technique for which there is little quantitative information. Using model protein interactions with immobilized Cu2+ ions, we have compared analytical frontal affinity chromatographic methods to a simple, nonchromatographic protocol for the rapid determination of quantitative affinity relationships. Values obtained for the equilibrium dissociation constant (Kd) and binding capacity (Lt) characterizing the interaction of lysozyme with immobilized Cu2+ were quite similar by frontal analysis (Kd = 37-42 X 10(-6) M; Lt = 6.8-7.4 X 10(-6) mol protein/ml gel) and by equilibrium binding analyses (Kd = 33 +/- 4.7 X 10(-6) M; Lt = 5.8-6.1 X 10(-6) mol protein/ml gel; 14 determinations). The interaction of ovalbumin with immobilized Cu2+ was characterized by an affinity (Kd = 4.2-4.8 X 10(-6) M) and capacity (Lt = 1.5-2.1 X 10(-6) mol protein/ml gel) which were also the same regardless of the method for affinity analysis. These values indicate that the total protein bound at saturation corresponds to as much as 17% of the total immobilized Cu2+ ions (approximately 40 X 10(-6) mol/ml gel). Thus, depending on the fraction of total immobilized Cu2+ available for interaction with a given protein (e.g., lysozyme), the number of individual immobilized ligands actively participating as well as those rendered unavailable upon individual protein binding events may be greater than 1. Linear Scatchard plots obtained for both lysozyme and ovalbumin (purified) suggest the presence of only a single type of immobilized Cu2+-protein interaction operative under the experimental conditions employed. However, Scatchard analyses of data obtained by the nonchromatographic equilibrium binding method also demonstrated the ability to simultaneously resolve the contribution of two components whose presence was predicted by frontal chromatography. Our results support the validity and utility of equilibrium binding data analyzed according to the equations outlined by Scatchard and others as an alternative to analytical chromatographic methods.     

Chromatography of proteins on columns of polyvinylpolypyrrolidone using adsorbed textile dyes as affinity ligands

A simple and inexpensive chromatography system for proteins is introduced. When the amino derivatives of chlorotriazine dyes or other azo dyes were added to an aqueous slurry of the crosslinked polymer polyvinylpolypyrrolidone they were adsorbed, thus forming an immobilized dye chromatographic matrix. The association of the textile dyes with polyvinylpolypyrrolidone did not prevent them from acting as affinity ligands for proteins. Parameters such as ionic strength, dye concentration, and column size modulated the affinity effect exerted by the immobilized dyes. Lysozyme present in an egg white protein mixture bound to a column onto which the amino derivative of Procion Brown H-A was adsorbed and was eluted with a linear gradient of KCl. The resulting purification of the enzyme was 37-fold with 80% of the original activity being recovered. Free dye eluting with the lysozyme was removed on a column of polyvinylpolypyrrolidone equilibrated with 0.5 M KCl. After chromatography, the dye column was regenerated with 0.5 M NaOH and recharged with dye. The system presented here allows one to initially screen large numbers of potentially useful protein ligands to optimize a protein separation, followed by scaleup to a system size determined by the user.     

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.     

NMR screening of new carbocyanine dyes as ligands for affinity chromatography

Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N‐carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α‐chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un‐substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α‐chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α‐chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD‐NMR technique was successfully used to screen cyanine–protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

A new method for the screening of solid-phase combinatorial libraries for affinity chromatography

A new methodology for the rapid assessment of affinity ligands synthesized by combinatorial solid-phase chemistry is reported. This screening strategy utilizes the target protein conjugated to FITC, and represents an almost universal technique for the preliminary screening of solid-phase combinatorial libraries. The assessment of a triazine-scaffolded solid-phase combinatorial library of ligands, designed to bind to human IgG, was performed with FITC-human IgG, and the results compared with those obtained by conventional affinity chromatographic screening assays. The effect of different molar conjugation ratios of FITC-IgG (F/P) was evaluated. Independently of the F/P ratio, no false negative results were observed, although lower F/P ratios diminished non-specific interactions and the number of false positives. The nature of the substituents on the triazine scaffold was not related to the number of false positive IgG-binding ligands. The reproducibility of the FITC technique, using FITC-human IgG conjugates with low F/P ratio (F/P=2), was also evaluated. The FITC-based technique proved to be efficient and accurate in the identification of strongly binding ligands (binding >50% of loaded protein, by standard affinity chromatographic assays), and is envisaged as a versatile and cost-effective method to screen other systems, and evaluate several binding/elution conditions at small-scale, prior to scale-up to standard affinity chromatography.     

Affinity purification of proteins using ligands derived from peptide libraries

Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Leu, as a ligand for the purification of S-protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphazetrade mark gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S-protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.(c) 1995 John Wiley & Sons, Inc.     

Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive adsorption of labeled and native protein in confocal laser scanning microscopy

Confocal laser scanning microscopy has been previously applied to the study of protein uptake in porous chromatography resins. This method requires labeling the protein with a fluorescent probe. The labeled protein is then diluted with a large quantity of native protein so that the fluorescence intensity is a linear function of the labeled protein concentration. Ideally, the attachment of a fluorescent probe should not affect the affinity of the protein for the stationary phase; however, recent experimental work has shown that this assumption is difficult to satisfy. In the present study, we present a mathematical model of protein diffusion and adsorption in a single adsorbent particle. The differences in adsorption behavior of labeled and native protein are accounted for by treating the system as a two-component system (labeled and native protein) described by the steric mass action isotherm (SMA). SMA parameters are regressed from experimental linear gradient elution data for lysozyme and lysozyme-dye conjugates (for the fluorescent dyes Cy3, Cy5, Bodipy FL, and Atto635). When the regressed parameters are employed in the model, an overshoot in the labeled lysozyme concentration is predicted for Cy5- and Bodipy-labeled lysozyme, but not for Atto635-labeled lysozyme. The model predictions agree qualitatively well with recent work showing the dependence of the concentration overshoot on the identity of the attached dye and provide further evidence that the overshoot is likely caused by the change of binding characteristics due to the fluorescent label.     

Molecular characterization of monovalent and multivalent hapten-protein conjugates for analysis of the antigen--antibody interaction

We prepared a hapten-protein conjugate using (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten and hen egg lysozyme (HEL) or bovine serum albumin (BSA) and defined hapten modification sites on the former protein based on results of reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometric analyses performed after enzymatic digestion. The most reactive residue for aminoacetylation in HEL was found to be Lys33, and the second was Lys96 or Lys97. The homogeneous NP-HEL conjugates were purified by HPLC and used for examining the effect of hapten valence on the antigen-antibody interaction. We also examined the molecular nature of NP conjugates of BSA. Analysis using mass spectroscopy showed that the mass distribution of NP-BSA conjugates was limited, although it became broader with an increase in NP valence. Surface plasmon resonance biosensor measurements were employed in measuring antigen-antibody interactions. The results showed that the apparent binding avidity depends on hapten valence, hapten density, size of carrier proteins, and intrinsic binding affinity of the antibody.     

Biochemical and physiological properties of the DNA binding domain of AraC protein

Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.     

The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.     

Aqueous two-phase protein partitioning using textile dyes as affinity ligands.

A simple and inexpensive aqueous two-phase system for the affinity partitioning of proteins is introduced. An aqueous solution consisting of maltodextrin (M100; molecular mass, 1800) and polyvinylpyrrolidone (PVP360; molecular mass, 360,000) formed two phases at 4 degrees C when the concentration of the polymers was 22.5% (w/w) and 4.0% (w/w), respectively. When the amino derivatives of chlorotriazine textile dyes or other azo textile dyes were added to the two-phase system they partitioned asymmetrically, favoring the upper, less dense, PVP360-rich phase. The association of the textile dyes with PVP360 did not prevent them from acting as affinity ligands for proteins. Three of the dyes screened increased the partition coefficient of purified lysozyme nearly 50-fold over a control containing no dye. Parameters such as pH, ionic strength, and dye concentration modulated the affinity-partitioning effect of the system. The partition coefficient of lysozyme in an egg white protein mixture increased severalfold as the total protein content of the system approached 4% (w/w), indicating that protein concentration is also important in determining the partitioning characteristics of this two-phase system. Proteins were efficiently freed of PVP360 and textile dye by recovery in a high-salt solution when another two-phase system was formed upon the addition of a solution of concentrated potassium phosphate to the isolated upper phase of a PVP360/M100/textile dye two-phase system. The affinity-partitioning system presented here allows one to screen large numbers of potentially useful protein ligands to optimize protein separation, followed by direct scaleup to a system size determined by the user.     

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.     

Hormone binding properties of estrophilic oxysteroid dehydrogenase in the rabbit liver

The estrophilic fraction of hydroxysteroid dehydrogenase (HSD) from the soluble fraction of rabbit liver was purified by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and affinity chromatography on estradiol-Sepharose. The isolated protein preparation expresses the 3 alpha-, 3 beta-, 17 beta- and 20 alpha-HSD activities on androgen and progestogen substrates. Some characteristics of 3H-estradiol, 3H-testosterone and 3H-progesterone interaction with the protein, the mutual competition of these hormones and the competitive efficiency of 73 steroids and their analogs in experiments with 3H-progesterone were investigated. It was found that sex steroids of all the three groups interact with a moderate affinity with the isolated HSD (K alpha approximately 10(7) M-1 at 0-4 degrees C). This interaction is characterized by relatively high association and dissociation rates. The main structural determinants of steroid ligands which provide for their affinity for the protein were established. The experimental results suggest that androgens and progestogens interact with the same binding site of the protein, whereas estrogens interact with a different site. It is supposed that by virtue of its high affinity for steroids and high concentration in the cells, the isolated protein can accomplish not only the enzymatic, but also a steromodulin function via reversible interactions with steroid ligands.     

Characterization of HIV-1 p24 self-association using analytical affinity chromatography.

Analytical affinity chromatography (AAC) was used to detect and quantitate the self-association of p24gag, the major structural capsid protein of human immunodeficiency virus (HIV-1). p24gag was immobilized on a hydrophilic polymer (methacrylate) chromatographic support. The resulting affinity column was able to interact with soluble p24, as judged by the chromatographic retardation of the soluble protein upon isocratic elution under nonchaotropic binding conditions. The variation of elution volume with soluble protein concentration fit to a monomer-dimer model for self-association. The soluble p24-immobilized p24 association process was observed using both frontal and zonal elution AAC at varying pH values; the dissociation constant was 3-4 x 10(-5) M at pH 7. That p24 monomer associates to dimers was determined in solution using analytical ultracentrifugation. The solution Kd was 1.3 x 10(-5) M at pH 7. AAC in the zonal elution mode provides a simple and rapid means to screen for other HIV-1 macromolecules that may interact with p24 as well as for modulators, including antagonists, of HIV p24 protein assembly.     

A flash photolysis method to characterize hexacoordinate hemoglobin kinetics

A flash photolysis method is described for analyzing ligand binding to the new and growing group of hemoglobins which are hexacoordinate in the unligated, ferrous state. Simple analysis of a two exponential fit to time courses for CO rebinding at varying CO concentrations yields rate constants for formation and dissociation of the hexacoordinate complex, and the bimolecular rate constant for CO binding. This method was tested with a nonsymbiotic plant hemoglobin from rice for which these values had not previously been determined. For this protein, dissociation and rebinding of the hexacoordinating amino acid side chain, His(73), is rapid and similar to the rate of CO binding at high CO concentrations. These results indicate that hexacoordination must be taken into account when evaluating the affinity of hexacoordinate hemoglobins for ligands.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose

We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)