Analysis of the binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase from tern and whale using the BIAcore biosensor: effect of immobilization level and flow rate on kinetic analysis.

The binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase, isolated from tern and whale, was measured using an optical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immobilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir binding model. A contribution of mass transport to the kinetic constants was demonstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high flow rates (up to 100 microl/min). Application of a global fitting algorithm to a 1:1 binding model incorporating a correction term for mass transport indicated that mass transport was minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic constants similar to those obtained with the 1:1 Langmuir binding model applied to binding data where mass transport had been minimized experimentally. The binding constant for binding of NC10 Fab to N9 neuraminidase from tern influenza virus (K(A) = 6.3 +/- 1.3 x 10(7) M(-1)) was about 15-fold higher than that for the NC10 Fab binding to N9 neuraminidase from whale influenza virus (K(A) = 4.3 +/- 0.7 x 10(6) M(-1)). This difference in binding affinity was mainly attributable to a 12-fold faster dissociation rate constant of the whale neuraminidase-NC10 Fab complex and may be due to either (i) the long-range structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to Asn(200), which form part of the binding epitope on both neuraminidases to which NC10 Fab binds.     

Kinetics of gonadotropin binding by receptors of the rat testis. Analysis by a nonlinear curve-fitting method.

The kinetics of the reaction between human chorionic gonadotropin (hCG) and specific gonadotropin receptors in the rat testis were determined at 24 and 37 degrees, over a wide range of hormone concentrations. Hormone concentrations were corrected for the binding activity of the (-125I)hCG tracer preparations. Analysis of the experimental data was performed with an interactive nonlinear curve fitting program, based upon the second-order chemical kinetic differential equation. The mean values for the association rate constant (k1) were 4.7 x 10-7 M-1 min-1 at 24 degrees, and 11.0 x 10-7 M-1 min-1 at 37 degrees. At both temperatures, the values of kl were independent of hormone concentration. Initial dissociation rates were consistent with first order kinetics, with dissociation rate constant (k2) of 1.7 x 10 minus -3 and 4.6 x 10 minus -3 min minus -1 at 24 and 37 degrees, respectively. When studied over longer periods at 24 degrees, the dissociation process appeared to be multiexponential. The kinetics of degradation of (-125I)hCG and receptors were determined at both temperatures, and a mathematical model was developed by modification of the second-order chemical kinetic differential equation to take these factors into account. The application of such a model to hCG kinetic binding data demonstrated that reactant degradation had little significant effect on the derivation of the association rate constant (k1), but caused significant overestimation of the dissociation rate constant (k2) values derived from association experiments. The model was also applied by computer simulation to a theoretical analysis of the effects of degradation of free hormone and receptor sites upon kinetic and steadystate binding data. By this method, the initial velocities of hormone binding were shown to be less affected by degradation than the steady-state levels of hormone-receptor complex. Also, reactant degradation in simulated steady-state experiments caused an underestimate of the apparent equilibrium association constant, but had relatively less effect on the determination of binding site concentration.     

Kinetic analysis of the mass transport limited interaction between the tyrosine kinase lck SH2 domain and a phosphorylated peptide studied by a new cuvette-based surface plasmon resonance instrument

We explored the use of a newly developed cuvette-based surface plasmon resonance (SPR) instrument (IBIS) to study peptide-protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant (k(d)) of 0.6+/-0.1 s(-1) was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant (K(D)) of 5.9+/-0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k(a) of 1.1(+/-0.2)x10(8) M(-1) x s(-1) was obtained from k(d)/K(D). The values for k(a) and k(d) obtained in this way were 2-3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.     

Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.     

The efficiency of the red alga Mastocarpus stellatus for remediation of cadmium pollution

This work reports the results of the study for cadmium binding by the dead red macroalga Mastocarpus stellatus. Kinetics sorption experiments demonstrated the high rate of metal biosorption: the system attained over 50% of the total biomass cadmium uptake within 2 min of contact and over 90% in the first 9 min. The kinetic data were successfully described by a pseudo-second order model with rate constants ranging from 1.06 to 10 gmmol(-1)min(-1), as a function of initial metal concentration and temperature. The equilibrium binding was accurately represented in terms of Langmuir and Langmuir-Freundlich models. The sorption isotherms at constant pH showed uptake values as 0.49 mmol g(-1) (at pH 2.4), 0.56 mmol g(-1) (at pH 4) and 0.59 mmol g(-1) (at pH 6), while the affinity constant values were between 0.6 and 5 mmol(-1) L (Langmuir fit). The acid-base properties of the alga were also studied, obtaining the total number of acid groups, 2.5 mmol g(-1), and their apparent pK value, 1.56, using the Katchalsky model. Desorption studies were conducted employing different HNO(3) concentrations and desorption times.     

Inhibition of rat liver steroid 5 alpha-reductase by 3-androstene-3-carboxylic acids: mechanism of enzyme-inhibitor interaction

The interactions of a series of newly discovered inhibitors of delta 4-3-oxo-steroid 5 alpha-reductase (SR; EC 1.3.1.30), the 3-androstene-3-carboxylic acids (steroidal acrylates), have been studied by using a solubilized rat liver enzyme preparation. As exemplified by one member of this series, 17 beta-[N,N-diisopropyl-carbamoyl)androst-3,5-diene-3-carboxylic acid (1a), the dead-end inhibition patterns of selected compounds in this class are best evaluated by a linear uncompetitive kinetic model versus either substrate, testosterone (T) or NADPH. These results were interpreted within the context of the preferentially ordered kinetic mechanism for rat liver SR to arise from the association of inhibitor to the binary complex of enzyme and NADP+. This proposed inhibition mechanism was supported by data from double-inhibition experiments implicating the synergistic binding of steroidal acrylate and NADP+ to SR. Further evidence for the preferential formation of this ternary complex was obtained from filtration binding assays with [3H]-1a, where radioligand association to protein was greatly enhanced in the presence of NADP+. The amount of [3H]-1a binding to protein was proportional to the specific activity of SR in the enzyme preparations, and the estimated dissociation constant from binding data by Scatchard analysis (Kd = 25 nM) was comparable to the inhibition constants estimated for SR activity (Ki = 12-26 nM). From the pH profile for inhibition of the solubilized liver SR with 1a, it is proposed that the anion of the steroidal acrylate (pK1 = 4.7 +/- 0.2) is the active inhibitory species, coordinating to a protonated active site functionality (pK2 = 7.5 +/- 0.1). On the basis of data from similar experiments with structural analogues of 1a, the determinants for binding recognition and inhibitory potency are compared to structural features of the putative enzyme-bound intermediate states. These compounds represent a potential therapeutic alternative in the treatment of 5 alpha-dihydrotestosterone specific androgen dependent disease states.     

Cyanide binding to cytochrome c peroxidase (H52L)

Cyanide binding to a cytochrome c peroxidase (CcP) variant in which the distal histidine has been replaced by a leucine residue, CcP(H52L), has been investigated as a function of pH using spectroscopic, equilibrium, and kinetic methods. Between pH 4 and 8, the apparent equilibrium dissociation constant for the CcP(H52L)/cyanide complex varies by a factor of 60, from 135 microM at pH 4.7 to 2.2 microM at pH 8.0. The binding kinetics are biphasic, involving bimolecular association of the two reactants, followed by an isomerization of the enzyme/cyanide complex. The association rate constant could be determined up to pH 8.9 using pH-jump techniques. The association rate constant increases by almost 4 orders of magnitude over the pH range investigated, from 1.8 x 10(2) M(-1) s(-1) at pH 4 to 9.2 x 10(5) M(-1) s(-1) at pH 8.6. In contrast to wild-type CcP, where the binding of HCN is the dominant binding pathway, CcP(H52L) preferentially binds the cyanide anion. Above pH 8, cyanide binding to CcP(H52L) is faster than cyanide binding to wild-type CcP. Cyanide dissociates 4 times slower from the mutant protein although the pH dependence of the dissociation rate constant is essentially identical for CcP(H52L) and CcP. Isomerization of the CcP(H52L)/cyanide complex is observed between pH 4 and 8 and stabilizes the complex. The isomerization rate constant has a similar magnitude and pH dependence as the cyanide dissociation rate constant, and the two reactions are coupled at low cyanide concentrations. This isomerization has no counterpart in the wild-type CcP/cyanide complex.     

Kinetics parameter estimation for the binding process of salicylic acid to human serum albumin (HSA) with capacitive sensing technique

A novel method for real-time investigating the binding interaction between human serum albumin (HSA) and salicylic acid with capacitive sensing technique was successfully proposed. HSA was immobilized on the surface of a gold electrode modified with an insulating poly (o-phenylenediamine) (o-PD) film and colloid Au nanoparticles layers. The bioactivity of HSA was remained and major binding sites were available because of the excellent biocompatibility of gold nanoparticles. The capacitance and interfacial electron resistance of the sensor were altered, owing to the binding of HSA to salicylic acid. The time courses of the capacitance change were acquired with capacitive sensing technique during the binding process. Based on the capacitance response curves with time, the response model for the binding was derived in theory and the corresponding regression parameters were determined by fitting the real-time experimental data to the model. The binding and the dissociation rate constants (k(1) and k(-1)) were estimated to be 54.8 (mol l(-1))(-1) s(-1) and 2.9 x 10(-3) s(-1), respectively. And the binding equilibrium constant (K(a)) was calculated to be 1.89 x 10(4) (mol l(-1))(-1).     

The binding of guanine-guanine mismatched DNA to naphthyridine dimer immobilized sensor surfaces: kinetic aspects

Naphthyridine dimer composed of two naphthyridine chromophores and a linker connecting them strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. The kinetics for the binding of the G-G mismatch to the naphthyridine dimer was investigated by surface plasmon resonance assay. The sensor surface was prepared by immobilizing naphthyridine dimer through a long poly(ethylene oxide) linker with the ligand density of 9.1 x 10(-12) fmolnm(-2). The kinetic analyses revealed that the binding of the G-G mismatch was sequence dependent on the flanking base pairs, and the G-G mismatches flanking at least one G-C base pair bound to the surface via a two-step process with a 1:1 DNA-ligand stoichiometry. The first association rate constant for the binding of the G-G mismatch in the 5'-CGG-3'/3'-GGC-5' sequence to the naphthyridine dimer-immobilized sensor surface was 3.2 x 10(3)M(-1)s(-1) and the first dissociation rate constant was 1.4 x 10(-2)s(-1). The association and dissociation rate constants for the second step were insensitive to the flanking sequences, and were almost of the same order of magnitude as the first dissociation rate constant. This indicates that the second step had only a small energetic contribution to the binding. The association constant calculated from kinetic parameters was 2.7 x 10(5)M(-1), which is significantly smaller than the apparent association constants obtained from experiments in solution. Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry on the complex produced from the G-G mismatch and naphthyridine dimer showed the formation of the 1:1 complex and a 1:2 DNA-ligand complex in solution. The latter complex became the dominant complex when a six-fold excess of naphthyridine dimer was added to DNA.     

Fast kinetics of Taxol binding to microtubules. Effects of solution variables and microtubule-associated proteins

The kinetics of Taxol association to and dissociation from stabilized microtubules has been measured by competition with the reference fluorescent derivative Flutax-1 (Diaz, J. F., Strobe, R., Engelborghs, Y., Souto, A. A., and Andreu, J. M. (2000) J. Biol. Chem. 275, 26265-26276). The association rate constant at 37 degrees C is k(+) = (3.6 +/- 0.1) x 10(6) m(-1) s(-1). The reaction profile is similar to that of the first step of Flutax-1 binding, which probably corresponds to the binding of the Taxol moiety. The rate constant of the initial binding of Flutax-1 is inversely proportional to the viscosity of the solution, which is compatible with a diffusion-controlled reaction. Microtubule-associated proteins bound to the microtubule outer surface slow down the binding of Flutax-1 and Flutax-2 10-fold. The binding site is fully accessible to Flutax-2 in native cytoskeletons of PtK2 cells; the observed kinetic rates of Flutax-2 microtubule staining and de-staining are similar to the reaction rates with microtubule associated proteins-containing microtubules. The kinetic data prove that taxoids bind directly from the bulk solution to an exposed microtubule site. Several hypotheses have been analyzed to potentially reconcile these data with the location of a Taxol-binding site at the model microtubule lumen, including dynamic opening of the microtubule wall and transport from an initial Taxol-binding site at the microtubule pores.     

Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of 125I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) of 3.5 x 10(-4) s-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.     

Kinetic analysis of the interaction between amphotericin B and human serum albumin using surface plasmon resonance and fluorescence spectroscopy.

The binding interaction between amphotericin B and human serum albumin (HSA) has been studied using surface plasmon resonance (SPR) spectroscopy combined with a fluorescence quenching method to confirm the binding kinetic results. In this paper, the SPR method used to study the drug-protein interaction has been described in detail. The association rate constant, dissociation rate constant and the equilibrium association constant of amphotericin B binding to HSA were obtained using this method. To confirm the feasibility of the SPR method, a fluorescence quenching method was performed to obtain the equilibrium constant. In order to obtain more accurate results, experiment design was used to optimize the fluorescence quenching process. The two equilibrium association constants obtained using the two methods were 4.017 x 10(4) M(-1) (SPR) and 3.656 x 10(4) M(-1) (fluorescence quenching method) respectively.     

An experimental and theoretical study of the morphine binding capacity and kinetics of an engineered opioid receptor

Electrochemical real-time monitoring of ligand binding to an engineered opioid receptor specific for morphine is reported. In the particular systems studied, 90% of the binding was found to be completed after only 85-120 s. Thus, the binding kinetics has proven to be more rapid than previously believed. The observed association rate constant for the morphine binding reaction was calculated to be 215 M(-1)s(-1). A theoretical analysis of the experimental binding data suggested that the binding sites of the engineered opioid receptor could best be described by a model having two populations of binding sites: K(D)=40 microM (13 micromol/g) and K(D)=205 microM (29 micromol/g). Furthermore, a theoretical model was developed in order to explain the observed binding of the engineered opioid receptor. This model suggested that the binding sites on the polymer surface are up to 5.1A deep and they allow 100% of the ligand (morphine) to anchor itself into the site. The predicted theoretical maximum binding capacity for the reported receptor is calculated to be approximately 2 mmol/g polymer (based on an increase of cavity density).     

Covalent binding of a carcinogen as a probe for the dynamics of deoxyribonucleic acid

In order to determine the kinetic parameters of the binding to DNA of two closely related ultimate carcinogens, 2-(N-acetoxy-N-acetylamino)fluorene (N-Aco-AAF) and 2-(N-hydroxyamino)fluorene (N-OH-AF), three kinds of experiments were performed: measurement of the final amount of adduct (N-Aco-AAF and N-OH-AF), determination of the initial rate, and study of the reaction with deoxyguanosine (N-Aco-AAF only) at temperatures between 4 and 50 degrees C. The kinetic treatment of the chemical equations relies on two main assumptions: (i) binding of carcinogen to the C8 of guanine (G) could occur either with the classical B conformation or with a transient conformational state of the sugar--phosphate chain at the level of the guanine and denoted by G*; (ii) the equilibrium between G and G* is fast as compared to the chemical rate of carcinogen binding. These two assumptions have been verified by comparing experimental and calculated values of some of the data. From experimental data it is possible then to determine the characteristic independent parameters of the reaction: the constant K of the G in equilibrium G* and the enthalpy change delta H of the process, the rate constant k3 of the binding to the C8 of G, and the rate constant k1 of hydrolysis of the carcinogen with their corresponding activation enthalpies E3 and E1. Some essential results obtained are as follows: (a) The amount of G* that represents about 10% of the G at room temperature increases with temperature and is higher in denatured than in native DNA. (b) The values of delta H (approximately 9 kcal mol-1) and delta S (approximately 27 cal K-1 mol-1) of the G in equilibrium G* equilibrium are close to those associated with single base pair opening [Wartell, R.M., & Benight, A.S. (1982) Biopolymers 21, 2069]. (c) N-Aco-AAF reacts only with the G* conformation while N-OH-AF binds preferentially to the "classical" G (B conformation). Therefore, the electrophilic carcinogens behave as probes of the dynamic state of the DNA, but the rate of the G in equilibrium G* exchange is fast as compared to the binding rate of the carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p: reinterpretation of recent single molecule experiments

Brewer et al. (Biophys. J. 85 (2003) 2519-2524) have studied the compaction of dsDNA in a double flow cell by observing the extension of stained DNA tethered in buffer solutions with or without Abf2p. They use a Langmuir adsorption model in which one Abf2p molecule adsorbs on one site on the DNA, and the binding constant, K, is given as the ratio of the experimental rates of adsorption and desorption. This paper presents an improved interpretation. Instead of Langmuir adsorption we use the more appropriate McGhee-von Hippel (J. Mol. Biol. 86 (1974) 469-489) theory for the adsorption of large ligands to a one-dimensional lattice. We assume that each adsorbed molecule shortens the effective contour length of DNA by the foot print of Abf2p of 27 base pairs. When Abf2p adsorbs to DNA stretched in the flowing buffer solution, we account for a tension effect that decreases the adsorption rate and the binding constant by a factor of 2 to 4. The data suggest that the accessibility to Abf2p decreases significantly with increasing compaction of DNA, resulting in a lower adsorption rate and a lower binding constant. The kinetics reported by Brewer et al. (Biophys. J. 85 (2003) 2519-2524) lead to a binding constant K=3.6 x 10(6) M(-1) at the beginning, and to K=5 x 10(5) M(-1) near the end of a compaction run, more than an order of magnitude lower than the value K=2.57 x 10(7) M(-1) calculated by Brewer et al. (Biophys. J. 85 (2003) 2519-2524).     

Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.     

Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction

A filter retention assay is used to examine the kinetic and equilibrium properties of the interaction between phage R17 coat protein and its 21-nucleotide RNA binding site. The kinetics of the reaction are consistent with the equilibrium association constant and indicate a diffusion-controlled reaction. The temperature dependence of Ka gives delta H = -19 kcal/mol. This large favorable delta H is partially offset by a delta S = -30 cal mol-1 deg-1 to give a delta G = -11 kcal/mol at 2 degrees C in 0.19 M salt. The binding reaction has a pH optimum centered around pH 8.5, but pH has no effect on delta H. While the interaction is insensitive to the type of monovalent cation, the affinity decreases with the lyotropic series among monovalent anions. The ionic strength dependence of Ka reveals that ionic contacts contribute to the interaction. Most of the binding free energy, however, is a result of nonelectrostatic interactions.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arc repressor is tetrameric when bound to operator DNA

The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition. Here, Arc is shown to bind to its operator site as a tetramer. When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed. This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins. Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer. The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria. In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a). Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization [K1 = 5 (+/- 3) x 10(-9) M]. The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1. The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1. The ratio of these kinetic constants [K2 = 5.7 (+/- 2.3) x 10(-23) M2] provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator. An independent determination of this complex equilibrium constant [K2 = 7.8 (+/- 4.8) x 10(-23) M2] was obtained from equilibrium binding experiments.     

Requirements for reliable determination of binding affinity constants by kinetic approach

The nonspecific binding (equilibrium coefficient kn) of ligand (L) and/or the incomplete recovery (alpha < 1) of the receptor-ligand (RL) complex in binding measurements, could hamper accurate determination of the association and dissociation rate constants of the R/L system. For the simplest model of R/L interaction, characterized by a bimolecular association process (rate constant k1) and a monomolecular dissociation process (rate constant k2), the consequences of kn and/or alpha neglect on k1 and k2 determination were investigated. Various situations that are especially relevant for k1 determination, were examined in which nonspecific binding was: (i) negligible relative to specific binding, or (ii) developed progressively or very rapidly in association kinetics. When only the initial kinetic phase was used, according to the situation (i.e. the nonspecific binding characteristics, and the fact that kn and/or alpha were or were not taken into account to correct the binding measurements), k1 could be accurately determined or generally slightly overestimated or slightly underestimated (in the two latter cases by factors involving mainly kn and/or alpha but not the R concentration or the R/L equilibrium association constant, K), whereas k2 should always be fairly well estimated. Consequently, for the simplest R/L systems, the k1/k2 ratio derived from such kinetic experiments should be much less susceptible to substantial underestimation than K derived from R saturation experiments [Borgna, J. Steroid Biochem. Mol. Biol. (2004)]. Kinetic experiments could also be more appropriate than R saturation experiments to detect cooperative--positive or negative--binding of L to R.