Measurement Of Binding Constants Research Articles

The P1 plasmid partition complex at parS. The influence of Escherichia coli integration host factor and of substrate topology

The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage. ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS. In this report the role of IHF in the formation of the partition complex has been explored. First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution. Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions). IHF induced a strong bend in the DNA at its binding site. Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo. Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold. Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence. These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.

Models of cytochromes b. Attempts to control axial ligand orientation with a "hindered" porphyrin system

The interaction of imidazole ligands with the sterically hindered porphyrin derivative (perchlorato) (tetrakis(2,6-dichlorophenyl)porphinato) iron(III), [Fe(T2,6Cl 2 PP)(OClO 3 )], has been investigated. Measurements of stepwise ligand binding constants for 1-vinyl-, 1-methyl-, and 2-methylimidazole yield derived values of log β' 2 of 7.41, 5.16, and 7.12, respectively. These values clearly show that the eight o-chloro groups do not hinder the binding of the axial imidazole ligands. The preparation of the bis(1-vinylimidazole)iron(III) derivative is reported

The interaction of cations with lipopolysaccharide from Escherichia coli C as shown by measurement of binding constants and aggregation reactions.

Lipopolysaccharide from Escherichia coli C interacts with polyvalent cations at low ionic strength at more than one site. The first site has high affinity with a KD value of 10(-8) M for Ca2+ and even stronger binding for [(NH3)5CoNH2Co(NH3)5]5+ and La3+. The high-affinity site for the latter cations is beyond the sensitivity of the assay method. The second, low-affinity, site for bivalent cations has a Km of 10(-3) M, whereas, for tervalent and quinquevalent metal cations and spermine and hexacyclen (1,4,7,10,13,16-hexa-azacyclo-octadecane), this constant has a value of 10(-5) M. Binding of cations to the high-affinity site does not alter the aggregation state of the lipopolysaccharide, but combination with the low-affinity site gives particles twice the size of those of the sodium salt. Very high Ca2+ concentrations (30 mM) give particles eight times the size of those of the sodium salt.

S4-16 S ribosomal RNA complex: Binding constant measurements and specific recognition of a 460-nucleotide region

The region of the Escherichia coli 16 S ribosomal RNA recognized by the ribosomal protein S4 has been defined by assaying a set of 13 16 S rRNA fragments for S4 binding. The fragments were prepared by transcription in vitro, and binding constants were measured in three ways: retention of labeled RNA fragments on nitrocellulose filters by S4; cosedimentation of labeled S4 with RNA fragments in sucrose gradients; and the distribution of labeled S4 between two RNAs of different sizes in a sucrose gradient. All three methods gave similar relative binding strengths for a variety of 16 S rRNA and non-specific (23 S rRNA) sequences, with the exception of two of the largest 16 S rRNA fragments; these gave smaller association constants in the filter retention assay than in the other methods. We found that specific complexes of S4 with these larger RNAs do not bind well to filters, leaving non-specific complexes to dominate the assay. Specific complexes with RNAs ≤ 891 nucleotides were retained efficiently by S4 on filters, and gave reliable binding constants. All 16 S rRNA fragments containing nucleotides 39 to 500 bound S4 with the same affinity as intact 16 S rRNA, while all fragments with endpoints within 39 to 500 bound at least tenfold more weakly. This sequence must be able to fold independently of the rest of the rRNA. Comparison of this minimal 462-nucleotide S4 binding site with S4 footprinting results suggests that S4 binding might alter the conformations of RNA neighboring the 39 to 500 region in the intact 16 S rRNA. Specific S4-rRNA binding is not sensitive to KCl concentration, but a more normal salt dependence is seen in K 2SO 4 ( ∂ logK ∂ log[K +] ≈ − 3.3). This duplicates the behavior of the specific S4-α mRNA translational repression complex, arguing that S4 recognizes both the mRNA and rRNA substrates by the same mechanism. Mg 2+ is not required to form the specific rRNA complex, at least under conditions which stabilize RNA structure (0.35 m-KCl, 5 °C).

Rapid measurement of binding constants and heats of binding using a new titration calorimeter

A new titration calorimeter is described and results are presented for the binding of cytidine 2′-monophosphate (2′CMP) to the active site of ribonuclease A. The instrument characteristics include very high sensitivity, rapid calorimetric response, and fast thermal equilibration. Convenient software is available for instrument operation, data collection, data reduction, and deconvolution to obtain least-squares estimates of binding parameters n, δH o , ΔS o , and the binding constant K. Sample through-put for the instrument is high, and under favorable conditions binding constants as large as 10 8 m −1 can be measured. The bovine ribonuclease A (RNase)/2′CMP system was studied over a 50-fold range of RNase concentration and at two different temperatures. The binding constants were in the 10 5 to 10 6 m −1 range, depending on conditions, and heats of binding ca. −15,000 cal/mol. Repeat determinations suggested errors of only a few percent in n, ΔH o , and K values over the most favorable concentration range.

Improved method for multisample evaluation of ligand binding to cells: Application to IL-2 receptor measurement

A newly developed assay using microtitre plates with removable wells permits rapid measurements of IL-2 receptors. Triplicate or quadruplicate samples of several ligand dilutions are easily handled, thus giving more reliable results. In addition, measurement of binding parameters may be accomplished simultaneously on several cell lines. Results are presented which show that the binding assay may also be used for measuring binding constants of antibodies.

Evidence for a charge-transfer interaction between dissolved humic materials and organic molecules: I. Study of the binding interaction between humic materials and chloranil

Recent evidence indicates that organic environmental contaminants associate with or bind to dissolved humic materials. Binding constants for many systems have been determined, but little is known about the nature of these interactions. Using UV difference spectroscopy we have established the electron donating ability of humic materials and introduced a simple, direct method for measuring binding constants. Chloranil (tetrachlorobenzoquinone), a well known electron acceptor, was combined with Aldrich humic materials (2.7 mg C/l) giving rise to a shift in the chloranil 290 nm band. Spectra of several such solutions with differing chloranil concentrations when analyzed versus a chloranil reference contained a negative peak at this wavelength due to the binding interaction. The concentration of bound chloranil could be directly determined by measurement of the negative peak. The binding constant (K doc) for the chloranil-humic system was determined to be 1.2 × 10 5. Binding continued to occur over a period of several weeks, indicating that studies which measured binding constants for relatively short periods of time may have generated binding constants which were lower than those applicable to the environment. A charge transfer mechanism can be postulated for the chloranil-humic system as well as for similar systems.

Interaction of monoclonal anti-peptide antibodies with lysozyme.

The interaction of monoclonal anti-peptide antibodies with the free peptide and its protein counterpart has been evaluated for hen egg white lysozyme and the peptide constituting residues 38 to 45. Fluorescence methodology has been developed for the measurement of association constants based on resonance energy transfer between the excited tryptophan of antibody and bound peptide ligand conjugated to a fluorescent probe. Five antibodies, four IgM and one IgG, have been assayed by ELISA, and have demonstrated binding to the adsorbed peptide alone, to the adsorbed lysozyme alone, or to both. Multivalent interaction with the adsorbed ligand is a key factor in the efficacy of binding. Measurement of binding constants in homogeneous solution, by equilibrium dialysis and energy transfer, demonstrated that lysozyme was bound to an IgG antipeptide antibody with an association constant (4 X 10(2) M-1) 200-fold less than that for the free peptide (8 X 10(4) M-1). It was also inferred for IgM that an association constant of the order of 10(2) M-1 was sufficient to effect selective interaction in a system providing multivalent interaction. The shared conformations between protein and peptide, implied by the specific reactivity of the anti-peptide antibody with the protein, points to structural fluctuations of the surface regions and residues of globular proteins.

Selective binding of amino acid residues to tRNAPhe.

The interaction of amino acid amides with tRNAPhe was studied by measurements of the Wye base fluorescence. Binding of phenylalanine-, tyrosine- and tryptophan-amides leads to considerable quenching, whereas the amides of e.g. glycine and leucine do not induce quenching under the same conditions. Binding constants at 0.13 M salt - 100 M-1 for Phe-, 110 M-1 for Tyr- and 300 M-1 for Trp-amide - are about a factor of 6 higher than those evaluated from independent measurements for binding to simple single-stranded polynucleotides; the corresponding factor is 10 for double-stranded polynucleotides. Since the apparent enthalpy changes derived from measurements at different temperatures remains relatively low (-9 to -20 kJ/mol), the increased affinity appears to be mainly due to an increase of the entropy changes. Titration experiments performed in the presence of Mg2+ indicate cooperative interactions of the aromatic residues with the anticodon loop that are consistent with preferential binding to one of two loop conformations. Measurements of binding constants at different pH-values indicate the protonation of a tRNA residue in the tryptophanamide-tRNAPhe complex characterised by a pK value of about 7.0.

Measurement of binding constants for divalent antibody-immobilized antigen interaction by antigen-sepharose 4B chromatography.

Binding constants for the human IgG-anti human IgG heavy chain were determined from elution profiles obtained by loading human IgG-Sepharose 4B columns with the antibody and eluting it at different NaI concentrations. The agreement between the value obtained at zero NaI concentration and the value determined in solution by equilibrium molecular sieving was good. An application of this column method should be useful for comparison of avidity values of antibody populations of the same specificity.

Measurement of apparent binding constant between copper ion and apo-bovine superoxide dismutase

The binding constants of copper ions to apo-bovine superoxide dismutase were measured by the method of equilibrium dialysis. The binding constant (10 8.9 M −1) of copper ion to the native copper site was much larger (10 6 times) than that to the native zinc site at pH 4.0. The two native binding sites of copper ions are identical and show no interaction in the measurement of the binding constants. The binding of copper ions to the native zinc sites involved release of two protons and competition between these two protons and copper ion was governed by the pH dependence of copper binding constant to the native zinc sites.

Lanthanide Shift Reagents. Paper 19. Equilibrium Binding Constants for Alicyclic Ketones, Ethers and Epoxides

Abstract Previous measurements of the binding constants of the lanthanide reagent, Eu(thd)3, with (mainly) open chain ketones showed that moderately strong complexes were formed1,2. We have now extended these studies using nine alicyclic ketones to examine the effects of ring size and carbonyl conjugation. The influence of Europium and Ytterbium fluroinated shift reagents on five ethers and epoxides is also discussed.

Use of difference boundary sedimentation velocity to investigate nonspecific protein-nucleic acid interactions.

The difference boundary sedimentation velocity technique of Schachman and co-workers is demonstrated to be applicalbe to the measurement of binding constants (Kobsd) in the range 10(2)-10(5) M(-1) for the nonspecific interactions of proteins with DNA. The difference technique can reproducibly detect a 2% change in the sedimentation coefficient of the DNA upon binding ligands, corresponding to average extents of association as low as 10 molecules of protein (in the cases of Escherichia coli lac repressor and E. coli RNA polymerase) per molecule of bacteriophage T7 DNA. At these low binding densities, it is plausible to assume that the primary effect of ligand binding is on the buoyant mass of the complex and not on the frictional coefficient of the flexible DNA coil. Binding constants calculated by using this assumption agree well with literature values for the nonspecific interactions of RNase and lac repressor proteins with double-stranded DNA. Advantages of the method are that it is relatively rapid, requires the optical detection of the DNA only, and can be performed on small amounts of sample. The method appears useful for surveying (to an accuracy of +/-50% in Kobsd or +/-10% in log Kobsd) the effects of solution variables on Kobsd of protein-DNA interactions. Applications of the method to the nonspecific interactions of RNA polymerase core and holoenzymes with T7 DNA are discussed.

Coordination chemical studies on metalloenzymes: Measurement of binding constant between apo-tyrosinase and copper ion

The behavior of complexing agents for the copper removal reaction was studied by the equilibrium dialysis method. In the copper removal reaction, complexing agents are divided into two types: those that are reducing agents and those that are not. Sodium cyanide and sodium thiosulfate are of the first type, and 8-hydroxyquinoline-5-sulfonic acid, 2,2′-bipyridyl, and picolinic acid are of the second type. From equilibrium dialysis with the first type of complexing agent, the apparent binding constant (pH 6.0) between cuprous ions and apotyrosinase was calculated to be 10 15 m −1. Similarly, the apparent binding constant (pH 6.0) between cupric ions and apo-tyrosinase was about 10 13 m −1, which was calculated from equilibrium dialysis with the second type of complexing agent. The apparent binding constant between cuprous ions and apo-tyrosinase was larger than that between cupric ions and apo-tyrosinase.

Lanthanide Shift Reagents. Paper 16. Binding Constants for Europium Shift Reagent with Sterically Hindered Amine Donors

Abstract In continuation of our work on the measurement of binding constants of organic substrates with Europium shift reagents, we have studied the steric effects of one or more alkyl groups on the binding of a series of eleven amines (anilines, pyridines and piperidines). Both steric and inductive parameters have proved to be important in determining binding constants for carbinols1, aromatic aldehydes and ketones2,3 and esters3,4. The balance of these parameters is discussed for the amines and the results for the three anilines compared with figures derived by Infrared spectroscopy in an earlier study.

Corrections - Measurement of Macromolecular Equilibrium Binding Constants by a Sucrose Gradient Band Sedimentation Method. Application to Protein-Nucleic Acid Interactions

Corrections - Measurement of Macromolecular Equilibrium Binding Constants by a Sucrose Gradient Band Sedimentation Method. Application to Protein-Nucleic Acid Interactions

Measurement of macromolecular equilibrium binding constants by a sucrose gradient band sedimentation method. Application to protein-nucleic acid interactions

Measurement of macromolecular equilibrium binding constants by a sucrose gradient band sedimentation method. Application to protein-nucleic acid interactions

Measurement of binding constants for protein-DNA interactions by DNA-cellulose chromatography.

We describe the application of DNA-cellulose chromatography to the determination of binding constants for the nonspecific interaction between proteins and DNA. The method involves loading a small DNA-cellulose column to a low binding density and subsequently eluting the protein with buffer at a constant salt concentration. The elution is conveniently followed by monitoring the protein fluorescence of the eluate. From the shape of the elution profile, the binding constant for the interaction can be calculated. Employing columns containing double-stranded calf thymus DNA-cellulose, we have measured binding constants in the range of 10(4) to 10(6) M-1. Extension of this range is possible by varying the amount of DNA on the column. For lac repressor, agreement between our measurements and those of Revzin, A., and von Hippel, P.H. [(1977) Biochemistry 16 (second of five papers in a series in this issue)], who employ an absolute boundary sedimentation method, is good. The column method should be useful for the rapid screening of the effect of a large number of variables on protein-DNA binding constants.

A boundary sedimentation velocity method for determining nonspecific nucleic acid-protein interaction binding parameters

An absolute method for measuring affinity constants and binding site sizes for the non-sequence-specific binding of protein to single- or double-stranded polynucleotide chains is described, based on the difference in the rate of sedimentation of free protein and the protein-nucleic acid complexes. In the form outlined, the technique can measure binding constants ranging from 10 3 to 10 6 m −1; methods for extending the range of measurable binding constants are also described. Data obtained for the noncooperative binding of ribonuclease to DNA are presented as fully worked-out examples of the method, and the extension of this approach to other noncooperative and cooperative protein-nucleic acid binding systems is discussed.

The use of protamine to study [6,7-3H] oestradiol-17-beta binding in rat uterus.

1. The binding of [6,7-(3)H]oestradiol-17beta to uteri has been studied by using sucrose-gradient analysis and also the property of oestradiol receptors to form insoluble complexes with protamine. 2. Protamine precipitates the 8S and part of the 4S oestradiol-binding proteins in uterine cytoplasm from mature rats. It does not precipitate the oestradiol-17beta-binding proteins present in cytoplasm from non-target tissues or serum. No tritium-labelled material was precipitated by protamine after equilibration of [6,7-(3)H]oestradiol-17beta with either serum albumin or phosvitin. 3. Protamine precipitated a small amount of progesterone but not testosterone or cortisol that had been equilibrated with uterine cytoplasm. It did not precipitate any tritium radioactivity from muscle cytoplasm that had been equilibrated with either [1,2-(3)H]testosterone sulphate or [1,2-(3)H]dehydroepiandrosterone. 4. A simple method has been devised for measuring binding constants of tissue extracts for [6,7-(3)H]oestradiol-17beta, based on precipitation with protamine. Reasonable agreement was obtained between the values obtained by this method and those obtained by sucrose-gradient analysis. 5. This method has been used to study the effect of maturity, ovariectomy, adrenalectomy and hypophysectomy on the cytoplasmic binding of [6,7-(3)H]oestradiol-17beta. None of these procedures affected the dissociation constant K(d) or the number of binding sites/mg of cytoplasmic protein. When measured per uterus or per mg of DNA, ovariectomy and hypophysectomy decreased the number of binding sites. Adrenalectomy had no effect. 6. The properties of the 4S oestradiol-binding protein present in cytoplasm from mature uteri have been studied. It is not present in uteri from immature, ovariectomized, or hypophysectomized rats and it does not bind testosterone or cortisol. Unlabelled oestradiol-17beta, U-11,100A, N-ethylmaleimide and N-bromosuccinimide all decrease the binding of [6,7-(3)H]oestradiol-17beta to both 8S and 4S receptors. Binding to both 8S and 4S receptors decreases when oestradiol is transported to the nucleus. The 4S receptor is not the same as the 4S binding component formed by salt dissociation of the 8S receptor.