Temperature dependence of the nucleation constant rate in beta amyloid fibrillogenesis

Beta-amyloid peptide (A beta), in fibrillar form, is the primary constituent of senile plaques, a defining feature of Alzheimer's disease (AD). In solution assays, fibrils form with a lag time, interpreted as a nucleation/condensation-dependent process. The kinetics of fibrillogenesis is controlled by two key parameters: nucleation and elongation rate constants. We report here the study of the temperature dependence of the nucleation rate constant on an A beta monomer concentration of 18.4 microM at pH 7.4 and at temperatures ranging from 302 to 318 K. We found that the nucleation constant varied as in the Arrhenius law, giving an activation energy of 311.2 kJ mol(-1). The corresponding values of enthalpy of activation (deltaH*), entropy of activation (deltaS*) and Gibbs energy of activation (deltaG*) were evaluated by Eyring's equation of absolute reaction rate. A Gibbs energy of activation of approximately 110 kJ mol(-1) was obtained.     

Linear relationships between the ligand binding energy and the activation energy of time-dependent inhibition of steroid 5alpha-reductase by delta 1-4-azasteroids

The inhibition of steroid 5alpha-reductase (5AR) by Delta(1)-4-azasteroids is characterized by a two-step time-dependent kinetic mechanism where inhibitor combines with enzyme in a fast equilibrium, defined by the inhibition constant K(i), to form an initial reversible enzyme-inhibitor complex, which subsequently undergoes a time-dependent chemical rearrangement, defined by the rate constant k(3), leading to the formation of an apparently irreversible, tight-binding enzyme-inhibitor complex (Tian, G., Mook, R. A., Jr., Moss, M. L., and Frye, S. V. (1995) Biochemistry 34, 13453-13459). A detailed kinetic analysis of this process with a series of Delta(1)-4-azasteroids having different C-17 substituents was performed to understand the relationships between the rate of time-dependent inhibition and the affinity of the time-dependent inhibitors for the enzyme. A linear correlation was observed between ln(1/K(i)), which is proportional to the ligand binding energy for the formation of the enzyme-inhibitor complex, and ln(1/(k(3)/K(i))), which is proportional to the activation energy for the inhibition reaction under the second order reaction condition, which leads to the formation of the irreversible, tight-binding enzyme-inhibitor complex. The coefficient of the correlation was -0.88 +/- 0.07 for type 1 5AR and -1.0 +/- 0.2 for type 2 5AR. In comparison, there was no obvious correlation between ln(1/K(i)) and ln(1/k(3)), which is proportional to the activation energy of the second, time-dependent step of the inhibition reaction. These data are consistent with a model where ligand binding energies provided at C-17 of Delta(1)-4-azasteroids is fully expressed to lower the activation energy of k(3)/K(i) with little perturbation of the energy barrier of the second, time-dependent step.     

Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.     

Absolute reaction rate and kinetics of protein adsorption at solid-liquid interfaces.

Extents of adsorption of bovine serum albumin from aqueous solution to the surface of alumina, silica, carbon and chromium powder have been studied as function of time for various values of bulk protein concentration, pH, ionic strength and temperature. The rates of adsorption in all cases have been observed to fit in the first order rate equation with two different rate constants Ka1 and Ka2. Effects of addition of SDS, CTAB and neutral salts on values of Ka1 and Ka2 have also been studied. Using Arrhenius equation the activation energy values Ea1 and Ea2 have been evaluated from the values of Ka1 and Ka2 at three different temperatures, respectively. The corresponding values of enthalpy of activation (delta H*), entropy of activation (delta S*), and free energy of activation (delta G*) have been evaluated using Eyring's equation of absolute reaction rate. The mechanism of protein adsorption has been discussed in the light of basic principles of absolute reaction rate. It has been found that for Ka1 the delta H*1 greater than T delta S*1 and for Ka2 T delta S*2 greater than H*2, i.e. the anchorage and binding of protein to the surface are enthalpy controlled processes whereas the surface denaturation as well as rearrangement and folding is an entropy controlled process. The role of diffusion on rate of adsorption has also been discussed.     

Influence of sodium dodecyl sulfate/TritonX-100 micelles on the oxidation of D-fructose by chromic acid in presence of HClO(4)

The kinetics of oxidation of D-fructose by chromic acid in aqueous and aqueous surfactant (sodium dodecyl sulfate, SDS, and alkylphenyl polyethylenglykol, TX-100) media have been investigated in the presence of HClO(4). The reaction is acid catalyzed and is associated with an induction period which is dependent on [H(+)], [surfactant] and temperature. The order of oxidation during induction under [D-fructose]>[chromic acid] conditions is fractional in each reagent in both media. The rate constant was found to increase with [Mn(II)]. A mechanism has been proposed for the reaction. The micelles produce a catalytic effect in the range of SDS and TX-100 concentrations used, and the effect is explained by means of the pseudo-phase mass-action model. In the presence of SDS, the reaction is inhibited by electrolytes (NH(4)Br, NaBr, LiBr), and the inhibition order Na(+)>Li(+)>NH(4)(+) is explained on the basis of electrostatic considerations. The rate constant (k(m)), binding constants (K(S) and K(F)), and corresponding activation parameters (E(a), delta H( not equal ) and delta S( not equal )) have been evaluated and discussed. The order of reactivities of different sugars is found as: D-fructose>D-arabinose>D-xylose approximately D-glucose.     

Simultaneous modelling of the thermal degradation kinetics of pectin methylesterase in lettuce (Lactuca sativa L.) and carrot (Daucus carota L.) extracts: analysis of seasonal variation and tissue type

The thermal degradation kinetics of pectin methylesterase (PME) from carrot and lettuce were studied. Fresh extracts were exposed to temperatures from 55 to 70 degrees C until the enzyme was inactivated. A model based on the presence of two forms of the enzyme, one active and one non-active, is proposed. The natural variability of the PME activity was taken into the model in the form of normally distributed random effects. The common model parameters obtained (cleavage constant (0.0395+/-0.0062 s(-1)), degradation constant (0.556+/-0.112 s(-1)), cleavage energy of activation (469+/-23 kJ mol(-1)) and degradation energy of activation (488+/-18 kJ mol(-1))) show that the PME degradation kinetics of the two vegetables can be explained with a single set of parameters.     

Purification and characterization of an extracellular alpha-D-glucuronidase from Phlebia radiata

Alpha-D-glucuronidase was isolated from the culture filtrate of Phlebia radiata grown on wheat bran and purified to homogeneity by chromatographic methods. The final enzymic preparation was purified 65-fold with an activity yield of 58%; it showed a high level of specific activity (over 23,000 nkat/mg protein). The molecular and hydrolytic properties of the purified enzyme were studied. The secreted alpha-glucuronidase had a molecular weight of 110 kDa, as established by gel permeation chromatography (GP HPLC), had a determined pI just below 4.4, and was stable at pH 5.5 for prolonged times. The carbohydrate content in protein molecules was found to be 15%. The activity of alpha-D-glucuronidase peaked at pH 3,8 and 60 degrees C with aldouronic acids preparation as the substrate. The Michaelis-Menten constant (K(m)), the maximum reaction velocity (V(max)), and the activation energy (E(a)) were 0.18 mM, 0.13 microM/min and 5.91 kJ/mol, respectively. The alpha-glucuronidase was active mainly on small substituted xylooligomers. When this enzyme was used with endoxylanase for the degradation of oat xylan, synergistic effects were observed.     

Kinetics of complexing activation by the magnesium ion on green crab (Scylla serrata) alkaline phosphatase.

As with mammalian enzymes, green crab (Scylla serrata) alkaline phosphatase can be activated by Mg2+ through a time-dependent course. The activation is mainly a Vmax effect. Tsou's method was used to study the kinetic course of activation. The results show that the enzyme was activated by a complexing scheme that had not been previously identified: the enzyme first reversibly and quickly binds Mg2+ and then undergoes a slow reversible course to activation, with a relatively high activation energy (78 +/- 4 kJ/mol) and a slow conformational change. The activation reaction is a single molecule reaction, and the apparent activation rate constant is independent of Mg2+ concentration if the concentration is sufficiently high. The microscopic rate constants of activation and the association constant were determined from the measurements. The proposed scheme may also be applied to the Mg2+ activation mechanism for mammalian enzyme, to explain why the activation rate is time-dependent and not diffusion controlled. Substrate binding was also shown to affect the activation rate constant.     

Reaction kinetics for laccase-catalyzed polymerization of 1-naphthol

Laccase-catalyzed oxidative polymerization of 1-naphthol was carried out in a closed system containing acetone and sodium acetate buffer. The effects of initial 1-naphthol and dissolved oxygen concentrations on the initial reaction rate were investigated. A multiplicative mathematical model, using a function of 1-naphthol and dissolved oxygen concentrations, was developed for enzymatic polymerization and the corresponding biokinetic parameters have been evaluated for the first time. The activation energy and reaction rate constant of the laccase-catalyzed 1-naphthol polymerization were calculated as 57 kJ/mol and 311 l/s, respectively. The activation energy calculated was in the typical range of 30-60 kJ/mol and rate constant was of the order of magnitude of previously reported values for laccase-catalyzed reactions with different monomers.     

Fluorescence polarization and intensity kinetic studies of antifluorescein antibody obtained at different stages of the immune response.

Kinetic studies of reactions between fluorescein and antifluorescein antibody produced during early, intermediate, and late stages of the immune response have been carried out utilizing both fluorescence intensity and polarization measurements in the static (time constant similar to 5 sec) and in the stopped-flow modes (time constant similar to 5 msec). During maturation of the immune response, it was found that the "on" second-order association rate constant increased its value only by a factor of three, whereas the "off" dissociation first-order rate constant decreased by a factor of over 1000. Hence, it is the rate of dissociation which largely determines the stability of the hapten-antihapten complex. Furthermore, since second-order rate behavior was found for even heterogeneous antibody, most of the heterogeneity with respect to binding affinity occurs as a result of the heterogeneity in the rate of dissociation of the hapten-antihapten complex and not from the primary combination of hapten and antibody. Antifluorescein antibody which exhibits both high binding affinity (K similar to 5 x 10(11) M-1) and homogeneity with respect to equilibrium binding has been shown to obey second-order association kinetics over wide ranges in concentration. Despite the fact that the value of the second-order rate constant for this fluorescein-antifluorescein reaction is as large as that for most other hapten-antihapten reactions (1.4 x 10(8) M-1 sec-1), the binding reaction has an appreciable activation energy (7 kcal/mol). This is true for both divalent and univalent antibody. Furthermore, the reaction rate parameters are markedly affected by specific anions. The value of the second-order rate constant (18.5 degrees) increases according to the following scheme: salicylate less than trichloroacetate less than SCN- less than ClO4- less than Cl- less than F- less than phosphate. The activation energy increases as follows: trichloroacetate less than phosphate less than F- less than Cl- less than ClO4- less than SCN- less than salicylate, whereas estimates of the entropy of activation indicate that deltaS++ increases as follows: tricholroacetate less than phosphate similar to F- less than Cl- less than ClO4- less than SCN- less than salicylate. The same mechanism which was previously proposed by us for the antigen-antibody reaction is also consistent with the kinetics of the fluorescein-antifluorescein reaction. This mechanism postulates a bimolecular process with structural rearrangements (conformational changes and/or the loss of water) in the formation of the transition state complex. The reaction between the fluorescein hapten and its antibody hence is not diffusion limited.     

Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: role of local internal viscosity in protein dynamics

Upon activation of trypsinogen four peptide segments flanked by hinge glycine residues undergo conformational changes. To test whether the degree of conformational freedom of hinge regions affects the rate of activation, we introduced amino acid side chains of different characters at one of the hinges (position 193) and studied their effects on the rate constant of the conformational change. This structural rearrangement leading to activation was triggered by a pH-jump and monitored by intrinsic fluorescence change in the stopped-flow apparatus. We found that an increase in the size of the side chain at position 193 is associated with the decrease of the reaction rate constant. To analyze the thermodynamics of the reaction, temperature dependence of the reaction rate constants was examined in a wide temperature range (5-60 degrees C) using a novel temperature-jump/stopped-flow apparatus developed in our laboratory. Our data show that the mutations do not affect the activation energy (the exponential term) of the reaction, but they significantly alter the preexponential term of the Arrhenius equation. The effect of solvent viscosity on the rate constants of the conformational change during activation of the wild type enzyme and its R193G and R193A mutants was determined and evaluated on the basis of Kramers' theory. Based on this we propose that the reaction rate of this conformational transition is regulated by the internal molecular friction, which can be specifically modulated by mutagenesis in the hinge region.     

Scanning calorimetry measurements of the energy parameters of a plasmochemical polymer oxidation reaction

The contributions of heat fluxes of different nature to the total heat flux from a weakly ionized oxygen plasma of a low-pressure (20–120 Pa) RF discharge onto the calorimeter surface, on which a chemical reaction between atomic oxygen and a polymer proceeds, are distinguished. The activation energy (ΔE≈0.37 eV), the reaction heat (H≈27 kJ/g), and the rate constant for heat release in a surface plasmochemical reaction are determined.     

Shear breakage of DNA.

Determinations were made of the mean length of fragments produced after shearing long (greater than 100 kb) native Hela DNA in a VirTis homogenizer. (VirTis Co., Inc., Gardiner, N.Y.). The mean length (L) is a function of the speed of rotation of the homogenizer blades (omega), time of shearing (t), water concentration ([H2O]), solvent viscosity (eta), temperature (T), and energy of activation (E*), but not a function of the initial length so long as the starting molecules sustain an average of three or more breaks. The relationship of the parameters is expressed by the equation L = (b/omegat1/2eta1/2[H2O])eE*/2kBT, where kB is the Boltzmann constant and b is a constant of proportionality. The breakage rate constant k was determined to have the relationship k = (omega2L2eta[H2O]2/2b2)e-E*/kBT. These equations are valid throughout large ranges of the parameters, and a simple method is described which chooses a final mean length between at least 0.15 and 36 kb by choosing the appropriate shearing conditions and initial fragment length. The heterogeneity of shearing conditions within the shearing vessel permits use of the equations at all breakage rates tested. Based on the work of others using more homogeneous shearing conditions and initial fragment lengths, more complicated forms of the equations are necessary at low breakage rates but not at high ones. A proposed model of the breakage mechanism suggests that molecules with stress-induced localized denaturations break at a rate different from that for native DNA.     

Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange

Nitric oxide (NO), a highly reactive redox molecule, can react with protein thiols and protein metal centers to regulate a multitude of physiological processes. NO has been shown to promote guanine nucleotide exchange on the critical cellular signaling protein p21Ras (Ras) by S-nitrosylation of a redox-active thiol group (Cys(118)). This increases cellular Ras-GTP levels in vivo, leading to activation of downstream signaling pathways. Yet the process by which this occurs is not clear. Although several feasible mechanisms for protein S-nitrosylation with NO and NO donating have been proposed, results obtained from our studies suggest that Ras can be S-nitrosylated by direct reaction of Cys(118) with nitrogen dioxide (*NO(2)), a reaction product of NO with O(2), via a Ras thiyl-radical intermediate (Ras-S*). Results from our studies also indicate that Ras Cys(118) can be S-nitrosylated by direct reaction of Cys(118) with a glutathionyl radical (GS*), a reaction product derived from homolytic cleavage of S-nitrosoglutathione (GSNO). Moreover, we present evidence that reaction of GS* with Ras generates a Ras-S* intermediate during GSNO-mediated Ras S-nitrosylation. The Ras-S(*) radical intermediate formed from reaction of the Ras thiol with either *NO(2) or GS*, in turn, reacts with NO to complete Ras S-nitrosylation. NO and GSNO modulate Ras activity by promoting guanine nucleotide dissociation from Ras. Our results suggest that formation of the Ras radical intermediate, Ras-S*, may perturb interactions between Ras and its guanine nucleotide substrate, resulting in enhancement of guanine nucleotide dissociation from Ras.     

Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split.

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.     

Chemical kinetics and interactions involved in horseradish peroxidase-mediated oxidative polymerization of phenolic compounds

The primary objective of this research was to evaluate various factors that affect the reaction rate of oxidative coupling (OXC) reaction of phenolic estrogens catalyzed by horseradish peroxidase (HRP). Kinetic parameters were obtained for the conversion of phenol as well as natural and synthetic estrogens estrone (E(1)), 17β-estradiol (E(2)), estriol (E(3)), and 17α-ethinylestradiol (EE(2)). Molecular orbital theory and Autodock software were employed to analyze chemical properties and substrate binding characteristics. Reactions were first order with respect to phenolic concentration and reaction rate constants (k(r)) were determined for phenol, E(3), E(1), E(2) and EE(2) (in increasing order). Oxidative coupling was controlled by enzyme-substrate interactions, not collision frequency. Docking simulations show that higher binding energy and a shorter binding distance both promote more favorable kinetics. This research is the first to show that the OXC of phenolics is an entropy-driven and enthalpy-retarded process.     

An amperometric glucose biosensor based on poly(o-aminophenol) and Prussian blue films at platinum electrode

Prussian blue (PB), as a good catalyst for the reduction of hydrogen peroxide, has been combined with nonconducting poly(o-aminophenol) (POAP) film to assemble glucose biosensor. Compared with PB-modified enzymatic biosensor, the biosensor based on glucose oxidase immobilized in POAP film at PB-modified electrode shows much improved stability (78% remains after 30 days) in neutral medium. Additionally, the biosensor, at an applied potential of 0.0 V, exhibits other good characteristics, such as relative low detection limit (0.01 mM), short response time (within 5s), large current density (0.28 mA/cm2), high sensitivity (24 mAM(-1)cm(-2)), and good antiinterferent ability. The apparent activation energy of enzyme-catalyzed reaction and apparent Michaelis-Menten constant are 34.2 KJmol(-1) and 10.5 mM, respectively. In addition, effects of temperature, applied potential used in the determination, pH value of the detection solution, and electroactive interferents on the amperometric response of the sensor were investigated and are discussed.     

Successful removal of p-quinone with chitosan in an aqueous phase in relation to degree of deacetylation

Phenol oxidant is successfully removed by using chitosan particles in the aqueous phase. Removal of p-quinone by chitosan from crab shells was investigated kinetically from molecular weight (MW) of chitosan, deacetylation degree (DD) and reaction temperature. The rate constant assuming first-ordered reaction on removal of p-quinone in aqueous phase primarily depended on the MW of chitosan, not on the DD. Quantities of chitosan exceeding 5 x 10(5) MW are able to obtain a sufficiently high rate constant (10(-3) s(-1)). At higher temperatures, higher rate constants were obtained in the entire experimental MW and DD. The activation energy obtained was 43.8 kJ x mol(-1).     

Autoxidation studies of extracellular hemoglobin of Glossoscolex paulistus at pH 9: cyanide and hydroxyl effect

The complex oligomeric assembly of the hemoglobin subunits may influence the autoxidation rate. To understand this relation, the rate of autoxidation was studied at pH 9.0, where the Glossoscolex paulistus Hemoglobin (GpHb) dissociates. At alkaline pH, this hemoglobin is dissociated into monomers, trimers and tetramers, allowing the study of the integral protein and monomer subunit autoxidation on independent experiments. The autoxidation rate was evaluated in the presence and absence of cyanide (CN(-)), a strong field ligand to the ferric ion. The oxidation kinetic was monitored using the UV-vis absorption at 415 nm, and resulted in: i) bi-exponential kinetics for the whole hemoglobin (indicating a fast and a slow oxidative process) and ii) mono-exponential for the monomer (indicating a single process). To understand the specific characteristics of each autoxidation process, Arrhenius plots allowed the determination of the activation energy. The experimental results indicate for the whole hemoglobin in the absence of CN(-) an activation energy of 150 +/- 10 kJ mol(-1) for the fast and the slow processes. Under the same conditions the monomer displayed an activation energy of 160 +/- 10 kJ mol(-1), very close to the value obtained for the integral protein. The pseudo-second order rate constant for the whole protein autoxidation by CN(-) showed two different behaviors characterized by a rate constant k(CN1)' = 0.11 +/- 0.02 s(-1) mol(-1) L for CN(-) concentrations lower than 0.012 mol L(-1); and k(CN1)" = 0.76 +/- 0.04 s(-1) mol(-1) L at higher concentrations for the fast process, while the slow process remain constant with k(CN2) = 0.033 +/- 0.002 s(-1) mol(-1) L. The monomer has a characteristic rate constant of 0.041 +/- 0.002 s(-1) mol(-1) L for all cyanide concentrations. Comparing the results for the slow process of the whole hemoglobin and the oxidation of the monomer, it is possible to infer that the slow process has a strong contribution of the monomer in the whole hemoglobin kinetic. Moreover, as disulfide linkers sustain the trimer assembly, cooperativity may explain the higher kinetic constant for this subunit.     

Effect of trypsin microenvironment on the rate constants of elementary stages of Nalpha-benzoyl-L-arginine ethyl ester hydrolysis

The hydrolysis reaction of Nalpha-benzoyl-L-arginine ethyl ester catalyzed by trypsin from pig pancreas was comparatively studied in an aqueous buffer solution and in the system of reversed micelles of Aerosol OT in octane (pH 8.5) to determine the mechanisms of influence of the enzyme microenvironment on the rate constants of the elementary stages of the enzymatic reaction. The temperature dependences of the catalytic constant kcat and the rate constant of the second order kcat/Km (s, catalysis efficiency) allowed the determination of the rate constants and the activation energy of elementary stages of the enzymatic reaction. It was revealed that a decrease in the efficiency of catalytic action of trypsin in inverted mycelles in comparison with an aqueous solution is first of all determined by a decrease in the rate constant of formation of the enzyme-substrate complex k1. Possible mechanisms of the effect of the microenvironment on the elementary stages of catalytic action of the enzyme are discussed.