Relation between redox potentials and rate constants in reactions coupled with the system oxygen-superoxide.

Univalent oxidation-reduction reactions coupled with the oxygen-superoxide system were investigated in the reactions shown in eq 3 and 8, where Q and Q.- stand for p-benzoquinone and p-benzosemiquinone, respectively. From kinetic experiments the following rate constants were obtained at pH 7.0:k3 = 4.5 x 10(4) M-1 sec-1 and k8 = 3 x 10(-2) M-1 sec-1. With known values of k-3 and k-8, and of E0' for the systems Q-Q.- (0.10 V) and Cyt c3+ - Cyt c2+ (0.255 V), the calculated values of E0(O2-O2.-) were found to lie in the range between -0.27 and -0.33 V.     

Reduction of methemerythrin-anion adducts by dithionite ion

The reduction by dithionite ion (in excess) of methemerythrin-anion adducts, Hr+X-, to deoxyhemerythrin, Hr degree, has been examined at 25 degrees and pH 6.3 and 8.2. The results accord with the scheme: S2O42- in equilibrium 2SO2- rapid Hr+X- in equilibrium Hr++X- k-1, k1 Hr++SO2- leads to PRODUCT k2 with X- = Br-, HCO2-, CNO-, and F-, k2[SO2-] greater than k1[X-], and the pseudo first-order rate constant, kobs (= k-1), is independent of [X-] and [S2O42-]. Only with X- = NCS- is k2[SO2-] approximately k1[X-] and kobs = a[S2O42-]1/2 (b[NCS-] + [S2OR2-]1/2)-1. Values at pH 6.3 of k-1 (sec-1) and k1 (M-1 sec-1), obtained by anation and anion displacement reactions, are 2.3 x 10(-3), 1.6 x 10(-2) (Br-); 1.5 x 10(-3), 1.2 x 10(-2) (HCO2-); 1.3 x 10(-4), 0.52 (CNO-) and approximately 2 x 10(-4), 3.3 x 10(-3) (CN-, pH 7.0). Values of k-1 from reduction and displacement methods are in good agreement with each other. The value of k2 (1.6 x 10(5) M-1 sec-1, pH 6.3) in somewhat smaller than that for reduction of the met form of hemoproteins. There is only a small effect of pH on rates. Direct reduction of Hr+CN- does not occur, in contrast with Mb+CN-.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

Relaxation studies on the interaction of hexamethonium with acetylcholine-receptor channels inAplysia neurons

The mode of action of the cholinergic antagonist hexamethonium on the excitatory responses of voltage-clamped Aplysia neurons to acetylcholine (ACh) has been examined by voltage- and concentration-jump relaxation analysis. At steady-state concentrations of ACh hyperpolarizing command steps induced inward current relaxations to a new steady-state level (Iss). The time constants of these inward relaxations, tau f, which approximate the mean single-channel lifetime, were increased both by increasing the membrane potential and by lowering the bath temperature (Q10 = 3) but were not affected by increasing the ACh concentration over the dose range employed. In the presence of hexamethonium hyperpolarizing command steps produced biphasic relaxations of the agonist-induced current. tau f was reduced in a voltage-dependent manner, the degree of reduction increasing with hyperpolarization. Slow, inverse relaxations were also triggered in the presence of hexamethonium. The time constant of this relaxation was reduced by increasing membrane potential and hexamethonium concentration. Both the estimated association (kf = 5 X 10(4) M-1 . sec-1) and the estimated dissociation (kb = 0.24-0.29 sec-1) rate constants derived from a three-state sequential model for block by hexamethonium were independent of the membrane potential. Similar rate constants were estimated from experiments with the concentration-jump technique, which were also independent of the membrane potential over the range -50 to -110 mV. It is suggested that the voltage-dependent actions of hexamethonium may originate either from an alteration of the channel opening and closing rate constants through an allosteric interaction with the ACh receptor, rather than through an influence of the transmembrane electric field on the rate of drug binding, or through a fast reaction which is rate-limited by voltage-independent diffusion.     

Kinetics of Pseudomonas aeruginosa cytochrome c551 and cytochrome oxidase oxidation by Co(phen)3(3+) and Mn(CyDTA)(H2O)-

The reaction between reduced Pseudomonas cytochrome c551 and cytochrome oxidase with two inorganic metal complexes, Co(phen)3(3+) and Mn(CyDTA)(H2O)-, has been followed by stopped-flow spectrophotometry. The electron transfer to cytochrome c551 by both reactants is a simple process, characterized by the following second-order rate constant: k = 4.8 X 10(4) M-1 sec-1 in the case of Co(phen)3(3+) and k = 2.3 X 10(4) M-1 sec-1 in the case of Mn(CyDTA)(H2O)-. The reaction of the c-heme of the oxidase with both metal complexes is somewhat heterogeneous, the overall process being characterized by the following second-order rate constants: k = 1.7 X 10(3) M-1 sec-1 with Co(phen)3(3+) and k = 4.3 X 10(4) M-1 sec-1 with Mn(CyDTA)(H2O)- as oxidants; under CO (which binds to the d1-heme) the former constant increases by a factor of 2, while the latter does not change significantly. The oxidation of the d1-heme of the oxidase by Co(phen)3(3+) occurs via intramolecular electron transfer to the c-heme, a direct bimolecular transfer from the complex being operative only at high metal complex concentrations; when Mn(CyDTA)(H2O)- is the oxidant, the bimolecular oxidation of the d1-heme competes successfully with the intramolecular electron transfer.     

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.     

Aquo-pentamine Co(III) complexes as models for carbonic anhydrase

The hydrolysis of 4-nitrophenyl acetate by metal complexes Co(en)2(imH)H2O3+, Co(en)2(bzmH)H2O3+, and Co(en)2(imCH3)H2O3+ (imH = imidazole, bzmH = benzimodazole, imCH3 = methyl imidazole) has been investigated in the pH range 5.4-8.9. The small difference in nucleophilic reactivity in the pH range 5.4-6.7 is assumed to be due to hydrogen bonding abilities of the imidazole and substituted imidazole ligands and small pKa differences (k2(imH) = 2.2 X 10(-2) M-1 sec-1, k2(bzmH) = 5.68 X 10(-2) M-1 sec-1, k2(imCH3) = 1.35 X 10(-2) M-1 sec-1, 40 degrees C, 1 = 0.3 NaClO4, pKa(imH) = 6.2, pKa(imCH3) = 6.2 and pKa(bzmH) = 5.9). In the pH range 7.8-8.9, the differences in nucleophilic reactivity (k3(imH) = 85.5 X 10(-2) M-1 sec-1, k3(bzmH) = 33.4 X 10(-2) M-1 sec-1, 40 degrees C, I = 0.3 NaClO4) are reconciled with a significant steric factor outweighing the acidity of the benzimidazole complex. In the pH region 6.7-7.7, the deviation from linearity is presumably due to both hydroxo and imido ligands functioning as nucleophiles, the latter being about 40 times stronger than the former.     

A kinetic study of protein-protein interactions.

Kinetic studies have been carried out of the monomer-dimer interaction of insulin, beta-lactoglobulin, and alpha-chymotrypsin using stopped-flow and temperature-jump techniques. The pH indicators bromothymol blue, bromophenol blue, and phenol red were used to monitor pH changes associated with the monomer-dimer interaction. In all three cases a kinetic process was observed which could be attributed to a simple monomer-dimer equilibrium, and association (k1) and dissociation (k-1) rate constants were determined. The results obtained are as follows: for insulin at 23 degrees C, pH 6.8, 0.125 M KNO3, k1 = 1.14 X 10(8) M-1 s-1, k-1 - 1.48 X 10(4)s(-1); for beta-lactoglobulin AB at 35 degrees C, pH 3.7, 0.025 M KNO3, d1 = 4.7 X 10(4) M-1 s-1, k-1 = 2.1 s-1; for alpha-chymotrypsin at 25 degreesC, pH 4.3, 0.05 M KNO3 k1 - 3.7 X 10(3) M-1 s-1, k-1 - 0.68 s-1. The kinetic behavior of the separated beta-lactoglobulin A and B was similar to that of the mixture. In the case of chymotrypsin, bromophenol blue was found to activate the enzyme catalyzed hydrolysis of p-nitrophenyl acetate, and a rate process was observed with the temperature jump which could be attributed to a conformational change of the indicator-protein complex. The association rate constant for dimer formation of insulin approaches the value expected for a diffusion-controlled process, while the values obtained for the other two proteins are below those expected for a diffusion-controlled reaction unless unusally large steric and electrostatic effects are present.     

Hemes and hemoproteins. 3. The reaction of microperoxidase-8 with cyanide: comparison with aquocobalamin and hemoproteins

The monomeric heme octapeptide from cytochrome c, microperoxidase-8, (MP-8), coordinates CN- with log K = 7.55 +/- 0.04 at 25 degrees C in 20% (v/v) aqueous methanol. Log K values are independent of pH between 6 and 9. A spectrophotometric titration of cyanoMP-8 between pH 5.5 and 13.8 gave a single pKa greater than or equal to 13.5 ascribed to ionization of the proximal His ligand. A study of the kinetics of the reaction of MP-8 with cyanide between pH 5.5 and 12, at 25 degrees C and mu = 0.1, indicates that formation of cyanoMP-8 occurs via three routes: attack of CN- on Fe(III) (k1 = 6.0 +/- 0.3 X 10(5) M-1 sec-1); attack of HCN on Fe(III) (k2 = 4.8 +/- 2.0 X 10(3) M-1 sec-1), followed by deprotonation and isomerization to form the C-bound species; and displacement of OH- by CN- when the proximal His ligand is ionized (k5 = 1.8 +/- 0.1 X 10(5) M-1 sec-1). These results are compared with available data for the reaction of cyanide with aquocobalamin and with various hemoproteins.     

Kinetics and mechanism of Zn(II) complexation with reduced glutathione

The kinetics of formation and dissociation of mono and bis complexes of Zn(II) with reduced glutathione (H4L+ = fully protonated form) were studied in aqueous solution at 25.0 +/- 0.1 degrees C and ionic strength 0.30 M (NaNO3) in the pH range 4.58 to 4.98 by temperature-jump. The reaction was found to proceed via two different mechanisms depending on degree of ligand protonation. In both cases, complex formation is predominantly if not completely through the sulfur. Reaction with the form HL-2 (only the amino nitrogen protonated), the dominant form of this species, proceeds by the expected rat limiting water loss (dissociative or Eigen) mechanism with rate constants of 9.3 X 10(7) M-1 sec-1 (+/- 24%) for mono and 5.1 X 10(7) M-1 sec-1 (+/- 25%) for bis complex formation. Reaction with H2L--(sulfur protonated) yields rate constants of 3.9 X 10(3) M-1 sec-1 (+/- 43%) for mono and 1.95 X 10(3) M-1 sec-1 (+/- 43%) for bis complex formation. The decrease in rate constant is attributed to blockage of the complexing site on reduced glutathione by intramolecular hydrogen bonding, with proton removal being the rate determining step.     

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.     

The kinetics of mebendazole binding to Haemonchus contortus tubulin.

The kinetics of the binding of mebendazole (MBZ) to tubulin from the third-stage (L3) larvae of the parasitic nematode, Haemonchus contortus, have been characterized. In partially purified preparations, the association of [3H]MBZ to nematode tubulin was rapid, k1 = (2.6 +/- 0.3) x 10(5) M-1 min-1, but dissociation was slow, k-1 = (1.58 +/- 0.02) x 10(-3) min-1. The affinity constant (K(a)) for the interaction, determined by the ratio k1/k-1, was (1.6 +/- 0.2) x 10(8) M-1. Similar results were obtained with crude cytosolic fractions. In equilibrium studies, performed with partially purified nematode tubulin under similar conditions, a K(a) of (5.3 +/- 1.6) x 10(6) M-1 was obtained. The best estimate for the K(a) of the MBZ-nematode tubulin interaction is considered to be the 'kinetic' value determined from the ratio of rate constants. The slow dissociation of MBZ from nematode tubulin, which contrasts with the rapid dissociation of MBZ from mammalian tubulin, supports the hypothesis that the selective toxicity of the benzimidazole anthelmintics results from a difference between the affinities of mammalian and nematode tubulins for these drugs.     

Sodium binding sites of gramicidin A: sodium-23 nuclear magnetic resonance study.

In ethanol-water mixtures (90:10), the gramicidin dimer binds Na+ cations at well-defined sites, with a binding constant K = 4 M-1. Partial desolvation of Na+ occurs upon binding, as judged from the magnitude of the quadrupolar coupling constant (1.7 MHz) for bound sodium. The binding sites are identified with the outer sites flanking the channel entrances. The rate constants for binding and release are k+ less than or equal to 2.2 X 10(9) M-1 s-1 and k- less than or equal to 5.5 X 10(8) s-1, respectively.     

Evidence that a cAMP binding protein from Dictyostelium discoideum carries S-adenosyl-L-homocysteine hydrolase activity

A cAMP-adenosine binding protein partially purified from exponentially growing Dictyostelium discoideum cells carries S-adenosyl-L-homocysteine (SAH) hydrolase activity. This protein is present throughout the developmental cycle and has many properties in common with a cAMP binding activity previously reported from this laboratory (Gunzburg and Véron, 1981). Direct binding measurements with radioactive ligands indicate a dissociation constant of 0.2 microM for adenosine and 9 nM for cAMP, a value in good agreement with measurements of the rate constants for cAMP binding (k+1 = 2.4 X 10(4) M-1 sec-1) and dissociation (k-1 = 1.1 X 10(-4) sec-1). The binding of cAMP is completely abolished in the presence of 1 microM adenosine; a maximum 60 per cent inhibition of adenosine binding can be achieved with cAMP concentrations as high as 0.1 microM, suggesting that at least some of the cAMP and adenosine binding sites are not identical. The protein has a sedimentation coefficient of 9.2S and a native molecular weight of 190,000, as judged by gel filtration. Labeling with the photoaffinity ligand 8-azido-[3H]-cAMP followed by SDS polyacrylamide gel electrophoresis results in a single band of 47,000 MW, suggesting that the protein may be a tetramer. The physiological importance of the protein and its association with SAH hydrolase activity is discussed in relation to a possible role in the regulation of protein and phospholipid methylation that occurs during chemotaxis.     

Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Kinetics and mechanism of the reduction of horse heart ferricytochrome c by glutathione.

A detailed investigation of the reduction of cytochrome c by glutathione has shown that the reaction proceeds through several steps. A rapid combination of the reducing agent with the cytochrome leads to the formation of a glutathione-cytochrome intermediate in which the glutathione most likely interacts with the edge of the heme moiety. The electron transfer takes place in a subsequent slower step. Since cytochrome c(III) exists in two conformational forms at neutral pH [Kujundzic, N., & Everse, J. (1978) Biochem. Biophys. Res. Commun. 82, 1211], the reduction of cytochrome c by glutathione may be represented by cyt c(III) + GS- reversible K1 cyt c(III) ... GS- reversible k1 products cyt c*(III) + GS- reversible K2 cyt c*(III) ... GS- reversible k2 products At 25 degrees C, pH 7.5, and an ionic strength of 1.0 (NaCl), k1 = 1.2 X 10(-3) S-1, k2 = 2.0 X 10(-3) S-1, k1 = 2.9 X 10(3) M-1, and K2 = 5.3 X 10(3) M-1. The reaction is catalyzed by trisulfides, and second-order rate constants of 4.55 X 10(3) and 7.14 X 10(3) M-1 S-1 were obtained for methyl trisulfide and cysteine trisulfide, respectively.     

Monoclonal antibody to rat apoC: multiple binding to apoC-I on lipoproteins increases its affinity constant

Using solid phase systems, the kinetics of binding of monoclonal antibody (LRB 45, IgG2b,kappa) to apoC-I and apoC-I on lipoproteins were investigated. At 25 degrees C, the association constant of LRB 45 antibody to apoC-I (3.56 X 10(6) M-1 X sec-1) was almost three times slower than the association constant LRB 45 antibody to lipoproteins (10.4 X 10(6) M-1 X sec-1). However, the dissociation constant of apoC-I from LRB 45 antibody (0.865 X 10(-4) sec-1) was also slower than the dissociation constant of lipoprotein from antibody (1.5 X 10(-4) sec-1). Thus, the calculated affinity constant (association constant/dissociation constant) of LRB 45 antibody for apoC-I was approximately half of that for lipoproteins (4.12 X 10(10) M-1 vs. 6.92 X 10(10) M-1). The intrinsic affinity constants for antibody binding to apoC-I and apoC-I on lipoproteins were determined by Scatchard analysis. The intrinsic affinity constant of antibody bound to apoC-I was estimated to be 5.49 X 10(10) M-1 whereas that of antibody binding to lipoproteins was 30 to 200 times less. Furthermore, ascites fluid from LRB 45 cell lines could immunoprecipitate serum lipoproteins. Thus, it is concluded that there is multiple binding of antibody to apoC-I on lipoproteins. This binding appears to increase the calculated affinity constant (avidity) for antibody-antigen interaction.     

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.     

Kinetics of the cooperative association of actin to actin filaments.

The cooperative formation of actin filaments from monomers was followed by light scattering and electron microscopy. The results are well described by a simple model mechanism in which the growth and destruction of filaments occurs by stepwise addition or dissociation of protomers. All steps except the dimerisation step are assumed to have identical rate constants. These were found to be 5 X 10(3) M-1 - sec-1 and 3 X 10(-2) sec-1 for the association and dissociation, respectively (at pH 7.5 and in the presence of 10(-3) M calcium chloride). The equilibrium constant of elongation as obtained from the critical concentration is 1.7 X 10(5) M-1. The corresponding equilibrium constant of dimerisation is about 10 million times smaller (cooperativity parameter sigma = 2 X 10(-7)). This makes the nucleation extremely difficult and cooperativity very high. A best fit of the model to the experimental data is achieved when the destruction of a dimer is much faster than the addition of a third protomer (fast monomer- dimer pre-equilibrium). The size of the nucleus from which propagation becomes faster than dissociation is 3.     

Strychnine decreases the voltage-dependent Ca2+ current of bothAplysia and frog ganglion neurons

1. The effects of strychnine on the voltage-dependent Ca2+ current (ICa) were studied in physically isolated Aplysia neurons and enzymatically dissociated frog sensory neurons of the dorsal root ganglion. Neurons were studied under the internal perfusion and the voltage clamp condition. 2. Strychnine decreased the ICa with threshold concentrations for effect at 1 to 10 microM. The depression of ICa increased with strychnine dose without effects on the current-voltage relation of ICa. The effects of low concentrations of strychnine were reversible, but recovery was incomplete at higher concentrations. The potency of strychnine was about 10 times less than that of diltiazem, an organic Ca2+ antagonist. At 100 microM the ICa of Aplysia neurons was reduced to about half of the control. This concentration of strychnine also reduced the peak amplitude of ICa of frog sensory neurons. 3. These results indicate that, in addition to its actions on transmitter responses and on Na+ and K+ currents, strychnine has effects on ICa that have not previously been appreciated.