Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.     

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.     

Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells

Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C.     

beta-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

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).     

The kinetics of antibody binding to membrane antigens in solution and at the cell surface.

The reaction kinetics of 125I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+1), dissociation rate constants (k-1) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-1 antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-1 for the univalent interaction, in which case all binding was bivalent. (ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 10(5)--1.1 x 10(6) M-1.s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently. However, the k-1 values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.     

Mechanism of association of a specific aldehyde inhibitor, leupeptin, with bovine trypsin

Leupeptin (acyl peptidyl-L-argininal) is a potent inhibitor of trypsin and related proteases. We analyzed the association of leupeptim with bovine trypsin kinetically, assuming that it proceeds by a pathway which involves two steps: E + I in equilibrium K1 Complex I k-2 in equilibrium k+2 Complex II. The observed dissociation constant (K1) for the first step was 1.24 X 10(-3) M (at pH 8.2 15 degrees C) and the two first-order rate constants (k+2 and k-2) were 166 s-1 and 1.75 X 10(-3.s-1, respectively (at pH 8.2, 15 degrees C). The dissociation constant (Kd) for the whole process was calculated from these parameters to be 1.34 X 10(-8) M. This value is compatible with that determined directly by an independent static method (2.36 X 10(-8) M). We also measured Kd for the leupeptine complex of anhydrotrypsin, a trypsin derivative in which the active-site hydroxyl group is missing. The observed value was about 5 orders of magnitude larger than Kd and was rather similar to K1 in native trypsin. A elupeptin isomer which contains a D-argininal residue did not show strong affinity towards trypsin. These findings suggest that complex II consists of a covalent hemiacetal adduct formed between the serine hydroxyl group in the enzyme active site and the aldehyde group in the inhibitor. The pH dependencies of the dissociation constant and other parameters show that deprotonation of the charge-relay sustem in the active site is important for the formation and stabilization of complex II.     

Reversible DIDS binding to band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([3H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS--Band 3' complex at 0 degree C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k+1 was determined to be (3.72 +/- 0.42) x 10(5) M-1 s-1, while the dissociation rate constant K-1 was determined to be (9.40 +/- 0.68) x 10(-3) s-1. The dissociation constant KD, calculated from the measured values of k-1 and k+1, was found to be 2.53 x 10(-8) M. The standard thermodynamics parameters characterizing reversible DIDS binding to Band 3 protein at 0 degree C were calculated. The mean values of the activation energies for the association and dissociation steps in the DIDS binding mechanism were determined to be (34 +/- 9) kJ mole-1 and (152 +/- 21) kJ mole-1, respectively. The results provide, for the first time, evidence for the reversibility of DIDS binding to Band 3 protein at 0 degree C. The existence of a stimulatory site is suggested, nearby the transport site on the Band 3 protein. The binding of an anion to this site can facilitate (through electrostatic repulsion interaction between two anions) the transmembrane movement of another anion from the transport site.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Benzodiazepine receptors along the nephron: [3H]PK 11195 binding in rat tubules

Binding of [3H]PK 11195, an isoquinoline carboxamide derivative, was measured in microdissected tubule segments of rat nephron. High specific binding capacities (1.1-1.8 fmol X mm-1) were found in the thick ascending limb of the Henle's loop and in the collecting tubule, whereas specific binding could not be detected in the proximal tubule. In the medullary collecting tubule, the association and dissociation rate constants at 4 degrees C were k1 = 3.0 X 10(6) M-1 X min-1 and k-1 = 0.021 min -1; the ratio k-1/k1 = 7.0 nM was in agreement with the estimated equilibrium dissociation constant (Kd = 2.4 nM). [3H]PK 11195 binding sites from medullary ascending limb and medullary collecting tubule revealed the following sequence of specificity: PK 11195 = Ro 5-4864 much greater than clonazepam, indicating that tubule binding sites might be the peripheral benzodiazepine receptors of the rat kidney.     

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.     

A kinetic study of cyclic adenosine 3':5'-monophosphate binding and mode of activation of protein kinase from Drosophila melanogaster embryos.

Cyclic AMP-dependent protein kinase and its regulatory subunit were isolated from Drosophila melanogaster embryos. The profiles of cyclic AMP binding by these proteins were significantly different. In order to explain such a difference and to find the mode of enzyme activation by cyclic AMP, a kinetic study of cyclic AMP binding was carried out. First, the association rate constant k1 and dissociation rate constant k-1 in the cyclic AMP-regulatory subunit interaction at 0 degrees C were estimated to be 2.3 X 10(6)M-1s-1 and 1.1 X 10(-3)s-1, respectively. Secondly, the three possible modes of enzyme activation by cyclic AMP were mathematically considered and could be described by a unique formula: r=APt + BQt (A + B=1) in which the parameters A, B, P, and Q are equivalent to rate constants in the sense that the rate constants are simply expressed by these parameters. Thirdly, the values of the parameters and subsequently the values of rate constants involved in the possible mechanisms were evaluated using a curve-fitting technique and compared with experimental observation. It was then found that the following mechanism was the only one which fitted the experimental observations. Namely, RC + L k3 equilibrium k-3 LRC k4 equilibrium k-4 RL + C where R, C, and L represent the regulatory and catalytic subunits and cyclic AMP as a ligand. Thus, our results indicate that in the presence of cyclic AMP the active enzyme (C) is released from a ternary intermediate which is the primary product of the cyclic AMP-holoenzyme interaction. The estimated values of the rate constants are: k3=3.5 X 10(6)M-1s-1;k-3=7.3 X 10(-1)s-1;and k4=3.8 X 10(-2)s. These estimates indicate that the reaction LRC leads to RL + C is relatively slow and limits the rate of the overall reaction. By comparing k-3 and k4, it is apparent that a large part of newly formed ternary intermediate reverts to the holoenzyme.     

Photophysics of the fluorescent K+ indicator PBFI.

The fluorescent indicator PBFI is widely used for the determination of intracellular concentrations of K+. To investigate the binding reaction of K+ to PBFI in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surface was analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution at pH 7.2: k01 = 1.1 x 10(9) s-1, k21 = 2.7 x 10(8) M-1s-1, k02 = 1.8 x 10(9) s-1, and k12 = 1.4 x 10(9) s-1. k01 and k02 denote the respective deactivation rate constants of the K+ free and bound forms of PBFI in the excited state. k21 represents the second-order rate constant of binding of K+ to the indicator in the excited state whereas k12 is the first-order rate constant of dissociation of the excited K(+)-PBFI complex. From the estimated values of k12 and k21, the dissociation constant Kd* in the excited state was calculated. It was found that pKd* (-0.7) is smaller than pKd (2.2). The effect of the excited-state reaction can be neglected in the determination of Kd and/or the K+ concentration. Therefore, intracellular K+ concentrations can be accurately determined from fluorimetric measurements by using PBFI as K+ indicator.     

Guanine nucleotide regulation of B2 kinin receptors. Time-dependent formation of a guanine nucleotide-sensitive receptor state from which [3H]bradykinin dissociates slowly

We have examined the binding of [3H]bradykinin to bovine myometrial membranes and assessed its sensitivity to guanine nucleotides. Total binding displayed a typical B2 kinin receptor specificity. However, saturation binding isotherms were resolved into at least two components with KD values of 8 pM (45%) and 378 pM (55%). Low affinity binding exhibited relatively rapid rates of association (kobs = 1.40 x 10(-2) s-1) and dissociation (k-1 = 3.82 x 10(-3) s-1), while high affinity binding exhibited considerably slower rates (kobs = 9.52 x 10(-4) s-1 and k-1 = 4.43 x 10(-5) s-1). Pre-equilibrium dissociation kinetics revealed that formation of high affinity binding was characterized as a time-dependent accumulation of the slow dissociation rate at the expense of at least one other more rapid dissociation rate. In the presence of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), at least two binding components were resolved with KD values of 37 pM (12%) and 444 pM (88%). Gpp(NH)p apparently specifically perturbed high affinity binding by completely preventing the accumulation of the slow dissociation phase. Instead, two more rapid dissociation rates (k-1 = 8.53 x 10(-3) s-1 and 4.43 x 10(-4) s-1) were observed. These results suggest that [3H]bradykinin interacts with at least two B2 kinin receptor-like binding sites in bovine myometrial membranes. A three-state model for the guanine nucleotide-sensitive agonist interaction with the high affinity binding sites is proposed.     

Stopped-flow fluorescence kinetic studies of Glu-plasminogen. Conformational changes triggered by AH-site ligand binding

Binding of 6-aminohexanoic acid to the AH-site, a weak lysine binding site in Glu-plasminogen, alters the conformation of the molecule. The kinetics of the binding and the accompanying conformational change are investigated at pH 7.8, 25 degrees C. Changes of intrinsic protein fluorescence were measured as a function of time after rapid mixing in a stopped-flow apparatus. The results reflect a two-step reaction mechanism: Rapid association of Glu-plasminogen and 6-aminohexanoic acid (K1 = 44 mM) followed by the conformational change (k2 = 69 s-1 and k-2 = 3 s-1) with an overall dissociation constant Kd = 2.0 mM. Thus the conformational change is rather fast, t12 = 0.01 s. Its importance for the rates of Glu-plasminogen activation reactions is discussed.     

Evaluation of 5-enolpyruvoylshikimate-3-phosphate synthase substrate and inhibitor binding by stopped-flow and equilibrium fluorescence measurements

The binding of substrates and the herbicide N-(phosphonomethyl)glycine (glyphosate) to enolpyruvoylshikimate-3-phosphate (EPSP) synthase was evaluated by stopped-flow and equilibrium fluorescence measurements. Changes in protein fluorescence were observed upon the binding of EPSP and upon the formation of the enzyme-shikimate 3-phosphate-glyphosate ternary complex; no change was seen with either shikimate 3-phosphate (S3P) or glyphosate alone. By fluorescence titrations, the dissociation constants were determined for the formation of the enzyme binary complexes with S3P (Kd,S = 7 +/- 1.2 microM) and EPSP (Kd,EPSP = 1 +/- 0.01 microM). The dissociation constant for S3P was determined by competition with EPSP or by measurements in the presence of a low glyphosate concentration. At saturating concentrations of S3P, glyphosate bound to the enzyme--S3P binary complex with a dissociation constant of 0.16 +/- 0.02 microM. Glyphosate did not bind significantly to free enzyme, so the binding is ordered with S3P binding first: (formula; see text) where S refers to S3P, G refers to glyphosate, and E.S.G. represents the complex with altered fluorescence. The kinetics of binding were measured by stopped-flow fluorescence methods. The rate of glyphosate binding to the enzyme--S3P complex was k2 = (7.8 +/- 0.2) X 10(5) M-1 s-1, from which we calculated the dissociation rate k-2 = 0.12 +/- 0.02 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photophysics of the fluorescent Ca2+ indicator Fura-2.

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.     

Equilibrium and kinetic studies on the binding of gluconolactone to almond beta-glucosidase in the absence and presence of glucose

The binding of glucono-1,5-lactone (gluconolactone) with almond beta-glucosidase was studied at pH 5.0 and 25 degrees C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, delta Fmax, were determined to be 12.7 microM and 14.7%, respectively. From the study of the temperature dependence of Kd, delta G degrees, delta H degrees and delta S degrees for the binding were evaluated to be -6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25 degrees C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism: (formula; see text) which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k-1/k+1 = 4.5 mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2 = 94 s-1, k-2 = 0.36 s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.     

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)