Requirements for reliable determination of binding affinity constants by saturation analysis approach

Accurate calculation of the equilibrium association constant (K) and binding site concentration (N) related to a receptor (R)/ligand (L) interaction, via R saturation analysis, requires exact determination of the specifically bound L concentration (B(S)) and the unbound L concentration (U) at equilibrium. However, most binding determinations involve a procedure for separation of bound and unbound L. In such situations, it was previously shown that correct calculation of B(S) and U from binding data requires prior determination of alpha, i.e. the procedure parameter representing the proportion of equilibrium B(S) recovered after running the separation process, and of kn, i.e. the equilibrium nonspecific binding coefficient. For the simplest model of R/L interaction, the consequences of alpha neglect and/or kn neglect on determination of K and N, via R saturation analysis, are investigated. When alpha but not kn has been determined, B(S) can be accurately calculated, whereas U is overestimated by factor (kn + 1). Consequently the type (linear or hyperbolic) of theoretic curves obtained by usual representations (such as the Scatchard, the Lineweaver-Burk or the Michaelis-Menten plot) of the R/L binding is unchanged; these curves afford correct N and underestimation of K by factor (kn + 1). When alpha (alpha < 1) has not been determined B(S) and U are underestimated and overestimated, respectively. Then erroneous representations of the R/L binding result (e.g. instead of regular straight line segments, Scatchard plot and Lineweaver-Burk plot involve convex-upward and convex-downward hyperbola portions, respectively, suggestive of positive cooperativity of L binding), which leads to incorrect N and K. Errors in N and K would depend on (i) the binding (K, N and kn) and method (alpha) parameters and (ii) the expressions used to calculate approximate B(S) and U values. Simulations involving variable alpha, KN and kn values indicate that: (1) the magnitude of error in N determination (mainly involving moderate underestimation) directly depends on the alpha value; (2) the magnitude of K underestimation mainly depends on the KN value; it is moderate (usually < two-fold) with KN values < 1, but could become very high (e.g. > 100-fold), when KN > 10(2). In this case, the K underestimation is modulated by the alpha and kn values. Practical situations which afford high KN and thus might result in very marked underestimation of K are discussed. A single R dilution method is proposed to assess the validity of K determinations using the R saturation analysis approach.     

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.     

1,4萘醌和谷胱甘肽对Ryanodine受体初始结合率的影响及其意义

采用ｒｙａｎｏｄｉｎｅ 动态结合方法,考察了１,４ 萘醌（１,４ＮＱ） 和谷胱甘肽（ＧＳＳＧ） 作用于兔骨骼肌肌质网（ＳＲ）ｒｙａｎｏｄｉｎｅ 敏感钙通道（ＲｙＲ） 所产生的对ｒｙａｎｏｄｉｎｅ 初结合率（Ｒ０） 的影响。类似于１,４ＮＱ 对ｒｙａｎｏｄｉｎｅ 平衡结合的实验结果［７ ］ ,１ ,４ＮＱ 对Ｒ０ 也诱导出浓度依赖性的多相性变化规律,还原态１ , ４ＮＱ 的Ｒ０ 曲线则没有这种多相性。而ＧＳＳＧ 在３７°Ｃ 时所有浓度或时间区间都没有对ｒｙａｎｏｄｉｎｅ 结合对或Ｒ０ 诱导出明显的多相性行为;还原态的谷胱甘肽（ＧＳＨ） 亦只能产生出单相的抑制作用。这些氧化还原试剂也对ｒｙａｎｏｄｉｎｅ 结合率系数ｋ ＋ １ ,但不是ｋ － １ ,产生了相应的影响。     

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.     

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.     

Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions

Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.     

Affinity parameters of amino acid derivative binding to molecularly imprinted nanospheres consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)

The binding of L-Boc-phenylalanine anilide (BFA) and L-Boc-phenylalanine (phe) to molecularly imprinted and non-imprinted polymer nanoparticles consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)] has been investigated by adsorption experiments and mathematical modeling. The experimental isotherms have been mathematically adapted following the models of Freundlich, Langmuir, Langmuir-Freundlich, Bi-Langmuir, and extended Langmuir. The extended Langmuir model differentiated between specific and nonspecific binding of the ligand to the receptor nanoparticles and rendered excellent fitting of the experimental data. It delivered a thermodynamic and kinetic parameter set on the experimental association curves of L-BFA by L-BFA-imprinted nanospheres in suspension experiments with the equilibrium constant KD= 4.09 +/- 0.69 micromol L(-1) and the kinetic association rate constant Ka= 5.60 mL micromol(-1) min(-1).     

Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

Reactivation of tetrameric porcine skeletal muscle lactic dehydrogenase after dissociation and extensive unfolding of the monomers by 6 M guanidine hydrochloride (Gdn . HCl) is characterized by sigmoidal kinetics, indicating a complex mechanism involving rate-limiting folding and association steps. For analysis of the association reactions, chemical cross-linking with glutaraldehyde may be used [Hermann, R., Jaenicke, R., & Rudolph, R. (1981) Biochemistry 20, 2195-2201]. The data clearly show that the formation of a dimeric intermediate is determined by a first-order folding reaction of the monomers with k1 = (8.0 +/- 0.1) x 10(-4) s-1. The rate constant of the association of dimers to tetramers which represents the second rate-limiting step on the pathway of reconstitution after guanidine denaturation, was then determined by reactivation and cross-linking experiments after dissociation in 0.1 M H3PO4 containing 1 M Na2SO4. The rate constant for the dimer association (which is the only rate-limiting step after acid dissociation) was k2 = (3.0 +/- 0.5) x 10(4) M-1 s-1. On the basis of the given two rate constants, the complete reassociation pattern of porcine lactic dehydrogenase after dissociation and denaturation in 6 M Gdn . HCl can be described by the kinetic model (formula: see text).     

Characterization of human somatotropin binding to detergent-solubilized lactogenic receptors from rat liver.

Lactogenic receptors from rat liver microsomal fraction ('microsomes') were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 x 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 x 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.     

CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).     

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.     

Characterization of [3H]Ro 5-4864 Binding Sites in Rat Vas Deferens

The presence of benzodiazepine binding sites in rat vas deferens was detected using [3H]Ro 5-4864 as a radioligand. The binding of [3H]Ro 5-4864 to the mitochondrial sites is saturable, reversible, and temperature and time dependent. The association rate constant (k1) was 8.7 +/- 0.7 x 10(7) M-1 min-1, and the dissociation rate constant (k-1) was 0.031 +/- 0.003 min-1. The dissociation constant (KD) determined by saturation binding was 5.22 +/- 0.56 nM. The density of binding was 4,926 +/- 565 fmol/mg of protein. The Hill coefficient of binding was 0.99 +/- 0.01, an indication that [3H]Ro 5-4864 binds to a single site. The [3H]Ro 5-4864 binding was inhibited competitively by Ro 5-4864 and 2-hydroxy-5-nitrobenzyl-6-thioguanosine and noncompetitively by PK 11195, nitrendipine, alpha,beta-methylene-ATP, and carboxyatractyloside and was not affected by clonazepam, dicyclohexylcarbodiimide, or protoporphyrin IX. Our data indicate that [3H]Ro 5-4864 binding sites are not identical to those labeled by PK 11195. These binding sites are modulated by the ADP/ATP mitochondrial carrier, and an interaction of dihydropyridines and [3H]Ro 5-4864 binding sites in rat vas deferens is suggested.     

Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance

The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K(D)) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The K(D) measured for the binding of wild type and His(6)-Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased K(D) by 200-300-fold, whereas the A173T substitution increased K(D) by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein.     

Kinetics and thermodynamics of ouabain binding by intact turkey erythrocytes: effects of external sodium ion, potassium ion, and temperature

The kinetics of association and dissociation for the ouabain-Na+,K+- dependent ATPase complex have been studied in intact turkey erythrocytes as a function of external Na+ concentration, K+ concentration, and temperature. At free ligand concentrations substantially exceeding the concentration of available binding sites, the association reaction exhibits pseudo-first-order kinetics with an association rate constant (k1) that is conveniently determined over a wide range of temperatures (5-37 degrees C). The dissociation reaction exhibits strict first-order kinetics with a dissociation rate constant (k-1) that has the unusual property, in the turkey cell, of being sufficiently great to permit its direct determination even at temperatures as low as 5 degrees C. Values for the equilibrium binding constant for the ouabain-ATPase complex (KA) predicted from the ratio of the association and dissociation rate constants agree closely with independently measured values of KA determined directly under conditions of equilibrium binding. KA is a sensitive function of the composition of the external ionic environment, rising with increasing Na+ concentration and falling with increasing K+ concentration. These changes in KA are shown to be quantitatively attributable to changes in the rate constant k1, k-1 in contrast being unaffected at any given temperature by even very large changes in Na+ or K+ concentration. Arrhenius plots of k1 and k-1 both yield straight lines over the entire temperature range corresponding to activation energies for association and dissociation of 29.5 and 24.2 kcal/mol, respectively. These observations have made it possible to calculate the following standard values for the ouabain binding reaction in the presence of 150 mM Na+: delta G degree = -9.8 kcal/mol; delta H degree = +5.3 kcal/mol; delta S degree = +48.7 cal/degree/mol. The large positive value of delta S degree presumably reflects a highly ordered configuration of the ouabain-free ATPase molecule that is lost upon ouabain binding and that "drives" the reaction despite the positive value of delta H degree.     

Binding of colchicine to purified microtubule protein.

The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.     

Kinetics of interaction of trypsin with an anionic inhibitor of trypsin BWI-1a from buckwheat seeds

The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k(ass)) was 2.2 x 10(6) M-1 x sec-1 and the dissociation rate constant (k(off)) of the enzyme--inhibitor complex was 3.5 x 10(-3) sec-1; the inhibition constant Ki was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.     

Binding of [3H] Cocaine in Mouse Brain: Kinetics and Saturability

Abstract

Kinetic experiments indicate that association of [³H]- cocaine to its binding site in brain occurs rapidly (seconds). Dissociation of membrane-bound cocaine is also rapid, with a dissociation half-life in seconds; this raises the question of whether membrane-bound cocaine is released during the time required for rapid filtration of tissue-containing filters. Results from experiments with increasing numbers of filterwashes indicate that there is no significant loss of [³H]-cocaine saturably bound to brain membranes within the timescale of the rapid filtration procedure. In addition, saturation analysis of binding data obtained with the filtration procedure and with the centrifugation method give similar estimates of the affinity (dissociation constant: 0.8 μM) and the maximal binding (5 pmol/mg of protein) of cocaine. However, the nonspecific binding and the experimental error in the saturable binding are considerably greater in centrifugation assays than in filtration assays.     

Sequence specific recognition of ssDNA by a lupus autoantibody: kinetics and mechanism of binding.

11F8 is a pathogenic anti-ssDNA monoclonal autoantibody isolated from a lupus-prone mouse. Previous studies have established that 11F8 is sequence specific. To determine the basis for the observed binding specificity, stopped-flow fluorescence spectroscopy was used to measure the kinetic parameters and establish the mechanisms for the association of 11F8 with its target sequence, noncognate, and nonspecific ssDNA ligands. The data revealed that sequence-specific binding follows a two-step mechanism where the initial association step is second order. Values of k(1) are fast and above the modified Smoluchowski limit for a diffusion limited interaction (10(5)-10(6)M(-1)s(-1)). The dependency of k(1) on [salt] and solvent polarity indicates that electrostatic steering is responsible for this rapid association rate. The second association step is rate limiting and is characteristic of an isomerization process during which binding interfaces are optimized. This step apparently is driven by the desolvation of hydrophobic surfaces within the binding interface. The differences in the rate of dissociation for the various DNA ligands suggest that specificity is governed primarily through the dissociation of the final complexes.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Kinetic characterization of the pH-dependent oligomerization of R67 dihydrofolate reductase.

Protein-protein recognition results from the assembly of complementary surfaces on two molecules that form a stable, noncovalent, specific complex. Our interest was to describe kinetic aspects of the recognition in order to understand the subtle molecular mechanism of association. R67 dihydrofolate reductase (DHFR) provides an ideal model to investigate kinetic parameters of protein-protein association since it is a homotetramer resulting from the pH-dependent dimerization of homodimers. We took advantage of the presence of a tryptophan residue at the dimer-dimer interface to monitor pH-dependent oligomerization of R67 DHFR using stopped-flow fluorescence techniques. Except for pH near neutrality where dissociation exhibited biphasic kinetics, association and dissociation followed monophasic kinetics fitted on a two-state model. Apparent rate constants of association k(on) and dissociation k(off) were determined at various pHs and pointed to the key role of a histidine located at the dimer-dimer interface in the pH control of tetramerization. The values of the tetramer-dimer equilibrium dissociation constant were calculated from the ratio k(off) /k(on) and correlated well with those previously measured at equilibrium. The thermodynamic parameters and the activation energies of both the association and dissociation were determined and indicated that the association is enthalpy driven and suggested that the formation of four hydrogen bonds (one per monomer) is responsible for the thermodynamic stability of the tetramer. Detailed analysis of the biphasic kinetics led to an original model, in which protonation of the tetramer is the triggering event for the dissociation process while the association involves primarily the unprotonated dimers.