The binding of Ni2+ to adenylyl-3',5'-adenosine and to poly(adenylic acid)

Studies of the binding of Ni²⁺ to adenylyl-3',5'-adenosine (ApA) at pH 6-0 by ultraviolet spectrophotometry indicate the formation of a 1:1 complex in the presence of a large excess of metal ion. At 25 °C. and ionic strength μ = 0.5 M, the stability constant of Ni(ApA) is evaluated to be K = 2.6 (±0.6) M^?1. The low stability is taken as evidence that the predominant complex species is one in which the ApA acts as a monodentate ligand, mainly through the adenine group. The rate constants for complex formation and dissociation, k_f = 1430 M^?1 s^?1 and k_b = 665 s^?1 (25°C. μ = 0.5M). determined by the temperature-jump relaxation technique, are consistent with this interpretation. The binding strength of Ni²⁺ to poly(adenylic acid) [poly(A)] has been studied at pH 7.0 using murexide as an indicator of the concentration of free Ni²⁺. Within the concentration range [Ni²⁺ = 1 × 10^?5 × 10^?3 M the data can be represented in the form of a linear Scatchard plot. i.e., the process can be described as the binding of Ni²⁺ to one class of independent binding sites. The number of binding sites per monomer is 0.26, and the stability constant K = 8.2×10³ M^?1 (25°C μ = 0.1 M). In kinetic studies of the reaction of Ni²⁺ with poly(A), two relaxation effects due to complex formation were detected, one with a concentration-independent time constant of about 0.4 ms, the other with a concentration-dependent time constant in the millisecond range. The concentration dependence of the longer relaxation time can be accounted for by a three-step mechanism which consists of a fast second-order association reaction followed by two first-order steps. There is evidence, however, that the overall process is more complicated than expressed by the three-step mechanism.     

Lanthanide(III) complexes of amide derivatives of DOTA exhibit an unusual variation in stability across the lanthanide series

Luminescence excitation spectroscopy of the ⁷F₀→⁵D₀ transition of the Eu(III) complex of 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (TCMC, an amide derivative of DOTA) is used to measure the stability constant of the complex (K). A log K value of 10.6 is obtained for [Eu(TCMC)]³⁺ at 25 °C and an ionic strength of 0.1 M. Competition experiments with eleven other members of the lanthanide(III) series give stability constants for their complexes with TCMC. An unusual variation in stability is observed for complexes of [Ln(TCMC)]³⁺ across the lanthanide series with a pronounced optimum for the Sm(III) complex. This variation is quite different from that observed for other Ln(III) macrocyclic complexes, suggesting that the TCMC ligand is uniquely sensitive to Ln(III) ion radius.     

Interaction of anthelmintic benzimidazoles with AscarisSuum embryonic tubulin

The ability of mebendazole and fenbendazole to bind to tubulin in cytosolic fractions from 8-day Ascaris suum embryos was determined by inhibition studies with [³H]colchicine. Colchicine binding in the presence of 1·10^?6 M mebendazole was completely inhibited during a 6 h incubation period at 37°C. Inhibition of colchicine binding to A. suum embryonic tubulin by mebendazole and fenbendazole appeared to be noncompetative. The inhibition constants of mebendazole and fenbendazole for A. suum embryonic tubulin were 1.9·10^?8 M and 6.5·10^?8 M, respectively. Mebendazole and fenbendazole appeared to be competitive inhibitors of colchicine binding to bovine brain tubulin. The inhibition constants of mebendazole and fenbendazole for bovine brain tubulin were 7.3·10^?6 M and 1.7·10^?5 M, respectively. These values are 250–400 times greater than the inhibition constants of fenbendazole and mebendazole for A. suum embryonic tubulin. Differential binding affinities between nematode tubulin and mammalian tubulin for benzimidazoles may explain the selective toxicity. The importance of tubulin as a receptor for anthelmintic benzimidazoles in animal parasitic nematodes is discussed.     

Interaction of elongation factor EF-Tu with γ-amides of GTP and β-amides of GDP bearing the azidoaryl group or the chloroethylaminoaryl group placed at the terminal phosphate

New types of azidoaryl analogs of GTP: γ-(4-azido)anilide of GTP (I), γ-(N-(4-azidobenzyl)-N-methyl)amide of GTP (II) and of GDP: β-(4-azido)anilide of GDP (III), β-(N-(4-azidobenzyl)-N-methyl)amide of GDP (IV) have been synthesized by treatment of the nucleotide in aqueous solution with N-cyclohexyl-N′-β-(4-methylmorpholinium)- ethylcarbodiimidep-toluene sulfonate and the respective amine. The analog of GTP bearing at the γ-phosphate an alkylating 2-chloroethylamino group: γ-(4-N-(2-chloroethyl)-N-methylaminobenzyl)amide of GTP (V) was prepared by the method described previously for the preparation of the analog of ATP (Knorre, D.G., Kurbatov, V.A. and Samukov, V.V. (1976) FEBS Lett. 70, 105–108). Azidoaryl analogs of GTP and GDP as well as the chloroethylaminoaryl analog of GTP compete with GDP in the formation of the binary complex EF-Tu·GDP with the respective K_i values 3.9·10^?7 M (I), 2.9·10^?8 M (II), 6.9·10^?7 M (III), 5.0·10^?7 M (IV) and 3.8·10^?8 M (V) relative to GDP. constants of the complexes of the radioactively-labeled GTP analogs I, II and V with elongation factor Tu were calculated to be 8.5·10^?6 M, 3.4·10^?7 M and 4.6·10^?8 M, respectively, or approx. 1740-, 70- and 9-times greater than that of GDP. GTP analogs I, II and V were found to substitute GTP in the stimulation of EF-Tu-dependent binding of aminoacyl-tRNA to the ribosome-mRNA complex.     

Metal ion complexes of cytidinediphosphocholine.

Cytidine-5′-diphosphocholine (CDPcholine) forms a complex with Mg²⁺ and Mn²⁺ ions as indicated by the broadened ³¹P NMR peaks of CDPcholine in the presence of these ions. Additional evidence for the complex is the decrease in absorption at 360 nm when CDPcholine is added to solutions of 8-hydroxyquinoline and Mg²⁺. The stability constant of the Mg-CDPcholine complex was found to be 20 M^?1.     

Superoxide dismutase: a comparison of rate constants

O₂^?was introduced, at a constant rate, into buffered aqueous solutions, either by mechanical infusion of KO₂, dissolved in tetrahydrofuran, or by the in situ action of xanthine oxidase on xanthine plus oxygen. This O₂^? was allowed to react with ferricytochrome c or with tetranitromethane and the formation of the reaction products, ferrocytochrome c or nitroform, respectively, was monitored spectrophotometrically. That concentration of Superoxide dismutase, which competed equally with given levels of cytochrome c or tetranitromethane and which thus caused 50% inhibition of the rates of accumulation of ferrocytochrome c or of nitroform, was determined. The rate constant for the enzymatic dismutation of O₂^? by the copper and zinc containing enzyme from bovine erythrocytes was then calculated from the known rate constants for the reaction of O₂^? with ferricytochrome c and with tetranitromethane and was found to be 2 × 10⁹m^?1 sec^?1 at pH 7.8 and 8.5. This rate constant was obtained at steady-state concentrations of O₂^? in the 10^?8m → 10^?13m range and is in full agreement with the results of pulse radiolytic investigations which were performed at O₂^? concentrations in the 10^?5m range. The second order rate constant for the enzymatic dismutation of O₂^? is thus independent of the concentration of O₂^? in the range 10^?5 → 10^?13m.Several distinct types of Superoxide dismutase have been described. These include the mangano-enzymes from Escherichia coli and from chicken liver mitochondria and the iron-enzyme from E. coli. The rate constants for the dismutations catalyzed by these enzymes have also been investigated as a function of pH.     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

Cytosol androgen receptor from kidney of normal and testicular feminized (Tfm) mice

Steroid binding has been studied in cytoplasmic extracts of normal mouse kidney, an androgen sensitive organ, and of kidney from mice affected with testicular feminization (Tfm mutant) that have inherited androgen resistance. Macromolecules that bind ³H-5α-dihydrotestosterone (DHT, the presumed active androgen in most testosterone target organs) and sediment in glycerol gradient at 8–9S can be observed in cytosol from kidney of mice of different sex, age, and hormonal history. The 8–9S component from normal females is heat labile, pronase sensitive, and dissociated by high salt to a lower molecular weight entity. The apparent equilibrium dissociation constant (K_d) for the DHT-receptor complex is 1.4 × 10^?9M, and there are about 1500 binding sites per testosterone-sensitive kidney proximal tubule cell. Cytosol from Tfm/Y ^ animals also shows a sharp peak of ³H-DHT-binding activity at 8–9S. The Tfm protein, however, has reduced affinity for DHT and binds only 10–25% as much ³H-DHT as wild-type receptor at ³H-DHT concentrations from 5 × 10^?11M to 1.2 × 10^?8M. Scatchard analysis, and studies involving competition with unlabeled steroids, relative binding of various androgens, and dissociation of the ³H-DHT-binding protein complex after extensive dialysis have led to the conclusion that Tfm kidney contains very little, if any, androgen receptor with properties similar to that found in normal kidney.     

Lanthanide complexation with 1,3,5-triamino 2,4,6-trihydroxycyclohexane

The protonation constants of 1,3,5-triamino-2,4,6-trihydroxycyclohexane (taci), at 25 °C in I = 1.00 M (NaClO₄) were determined to be: pKa₁, 5.57 (0.08); pKa₂, 7.45 (0.02); pKa₃, 9.05 (0.04). The log of the stability constants, log β₃₀₂, at 25°C in I = 1.00 M (NaClO₄) for formation of were measured by potentiometry to be: Nd(III), 25.33 (0.09); Eu(III), 26.42 (0.06); Tm(III), 30.07 (0.10); Lu(III), 33.68 (0.07) ; Y(III), 28.59 (0.07). ¹H NMR spectra were consistent with formation of a single complex from pcH 6 to 10. Laser fluorescence measurements of the ⁷F_o-⁵D_o transition of Eu(III) complexed by taci indicated a single complexed species. The shift in this peak relative to that of Eu³⁺(aq) was significantly greater than the values reported for the complexes of other organic ligands with Eu(III). Luminescence lifetime measurements indicated two water molecules bound to each of the Eu(III) cations in the taci complex.     

Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of ¹²⁵I-labeled α-bungarotoxin to a 34 800 × g pellet of a whole rat brain homogenate has been obtained at levels 2 pmol toxin per g of whole brain with a K_d of 8·10^?9 M. Binding is reduced 90% by 10^?5 M (+)- tubocurarine chloride and 10^?4 M nicotine, whereas concentrations of 10^?4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with ¹²⁵I-labeled α-bungarotoxin and soluble acetylcholine receptor protein preparations form Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character.Extraction of the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds ¹²⁵I-labeled α-bungarotoxin with a K_d of 5·10^?9 M. Nicotine and α-bungarotoxin at concentrations of 10^?5 M abolish toxin- receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35–40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10^?5 M.     

Characterization of a panel of mouse single-chain antibodies against human recombinant interferon β1b

A panel of single-chain Fv-antibodies (ScFv’s) against recombinant human interferon beta 1b (rhIFN-β1b) has been obtained from immune and naïve combinatorial cDNA libraries of the mouse variable immunoglobulin genes. ScFv’s were expressed in Escherichia coli cells. For producers isolated from the immune library a difference in production yield of ScFv’s in periplasm and incubation medium as well as their expression and storage stability have been demonstrated. After sequencing of target DNA the multiple alignment and structural analysis of ScFv’s sequences with different primary structures were carried out and significant difference in both complementarity-determining (CDR) and framework (FR) regions of theirs variable domains has been shown. For the ScFv’s isolated from the immune library, specificity of their binding with native and denatured rhIFN-β1b in ELISA and Western-blotting as well as their high storage stability have been shown. The affinity constants for each representatives of the ScFv’s panel were in the range from 1.96 × 10^?8 to 1.69 × 10^?9 M.     

Probing the fatty acid binding site of β-lactoglobulins

The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10^?7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10^?7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpH's lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.     

Interaction of the regulatory gene product with the operator site in the l-arabinose operon of Escherichia coli

The DNA binding properties of the araC protein in the absence of l-arabinose have been studied in Escherichia coli using the nitrocellulose membrane filter technique. Equilibrium competition experiments demonstrate that the araC protein binds specifically to the ara operator. The apparent K_m of the interaction is 1 × 10^?12m at 20 °C. The rates of association and dissociation of the complex have also been determined. A k_a of 2 × 10⁹m^?1 s^?1at 20 °C is calculated assuming binding to a single site. The half-life of the complex is three minutes. The equilibrium constant calculated from the ratio of k_a to k_d is 2.8 × 10^?12m at 20 °C. The good agreement between the equilibrium and kinetic determinations of the equilibrium constant suggest that the kinetic studies are providing true rate constants. It is calculated that about 1% of the purified araC protein is active with respect to operator binding activity.     

Kinetic control of co-operativity in the oxygen binding of Panulirus interruptus hemocyanin.

The kinetics of the oxygen reaction of Panulirus interruptus hemocyanin have been studied at pH 9.6 under conditions where the protein exists in the undissociated, co-operative state and in the dissociated, non-co-operative state.Temperature-jump relaxation measurements of the undissociated protein at high oxygen saturation levels show one relaxation process which has been assigned to the high oxygen affinity (R) state, the on and off kinetic constants being 3.1 × 10⁷m^?1s^?1 and 60 s^?1, respectively. Stopped-flow measurements of the oxygen dissociation reaction show (1) an autocatalytic time-course of the reaction at pH 9.6 and (2) an increase in the overall oxygen dissociation rate constant, as the pH is decreased from 9.6 to 7.0.Temperature-jump relaxation measurements of the dissociated protein show one relaxation process characterized by a very high oxygen dissociation rate constant (1500 s^?1) and a combination constant which is of the same order of magnitude as reported for undissociated protein (k_on = 4.6 × 10⁷m^?1s${}^{\mathrm{?1}}$). The behaviour of dissociated protein can be considered as characteristic of the low oxygen affinity (T) state.The results presented in this paper, together with data available for other hemocyanins as well as hemoglobins, lead to the conclusion that respiratory proteins show a common feature in the kinetic control of co-operative oxygen binding: the stability of the oxygen-protein complex is largely determined by the value of the dissociation rate constant, the oxygen combination process very often appearing to be diffusion controlled.     

The stabilizing effect of the acyl group on the co-pigmentation of acylated anthocyanins with C-glucosylflavones

The stability of the complex formed between acylated anthocyanins and flavocommelin was compared with that between unacylated anthocyanins and the flavone. At low anthocyanin concentrations (5 × 10^?4 M), the complex involving acylated anthocyanins was much stabler than that involving ordinary anthocyanins. This extra stability is due to the acyl moiety in the acylated anthocyanins. The co-pigmentation constant (Kc) is defined as the affinity between an anthocyanin and its co-pigment.     

Borate buffer inhibition of peroxidatic intermediate formation in the deutero-ferriheme-hydrogen peroxide system

The rate of formation of peroxidatically active reaction intermediate(s) via oxidation of the iron(III)-porphyrin complex, deuteroferriheme, with hydrogen peroxide decreases with increasing borate content of mixed borate-carbonate buffer solutions. Studies at pH = 9.25 in 0.035 M borate buffer and 0.035 M carbonate buffer suggest borate to function as an uncompetitive inhibitor. A comparison of slopes and intercepts of double reciprocal plots for inhibited and uninhbited reactions allows calculation of selected parameters for the deuteroferriheme-H₂O₂ reaction at pH = 9.25 in terms of a typical enzymatic stoichiometric mechanism for heme activity. This includes the Michaelis constant (K_m = 8.1 × 10^?5 M) and the first-order rate constant for conversion of heme-substrate complex to intermediate(s) (k₃ = 7.4 sec^?1). A tentative mechanistic model involving reversible interaction of borate inhibitor with heme-substrate complex is considered, and pseudo-first-order rate constants calculated on the basis of this scheme are in reasonable agreement with those obtained experimentally. It is suggested that comparable inhibitory action may be responsible for some previously reported cases of decreased catalase enzyme activity in borate buffer solutions     

Preparation, crystal structure and luminescent properties of lanthanide picrate complexes with N-benzyl-2-{2′-[(benzyl-methyl-carbamoyl)-methoxy]-biphenyl-2-yloxy}-N-methyl-acetamide (L)

A new amide-based ligand derived from biphenyl, N-benzyl-2-{2′-[(benzyl-methyl-carbamoyl)-methoxy]-biphenyl-2-yloxy}-N-methyl-aceamide (L) was synthesized. Solid complexes of lanthanide picrates with this new ligand were prepared and characterized by elemental analysis, conductivity measurements, IR and electronic spectroscopies. The molecular structure of [Eu(pic)₃L] shows that the Eu(III) ion is nine-coordinated by four oxygen atoms from the L and five from two bidentate and one unidentate picrates. All the coordinate picrates and their adjacent equivalent picrates form intermolecular π-π stacking. Furthermore, the [Eu(pic)₃L] complex units are linked by the π-π stacking to form a two-dimensional (2-D) netlike supramolecule. Under excitation, the europium complex exhibited characteristic emissions. The lifetime of the ⁵D₀ level of the Eu(III) ion in the complex is 0.22 ms. The quantum yield Φ of the europium complex was found to be 1.01 × 10⁻³ with quinine sulfate as reference. The lowest triplet state energy level of the ligand indicates that the triplet state energy level of the ligand matches better to the resonance level of Eu(III) than Tb(III) ion.     

The reaction between cytochrome C1 and cytochrome C

The kinetics of electron transfer between the isolated enzymes of cytochrome c₁ and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c₁ and ferricytochrome c is fast; the second-order rate constant (k₁) is 3.0 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ M^?1 · s^?1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c₁ and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c₁ and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c₁ and ferricytochrome c and the reverse reaction are k₁ = 2.4 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°C). The ‘equilibrium’ constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c²⁺₁ + cytochrome c³⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.