scholarly journals Kinetics of the interaction of chymotrypsin with eglin c

1991 ◽  
Vol 280 (1) ◽  
pp. 27-32 ◽  
Author(s):  
B Faller ◽  
J G Bieth
Keyword(s):  
Rate Constant ◽  
Proteinase Inhibitor ◽  
Enzyme Inhibitor ◽  
Order Rate Constant ◽  
Stopped Flow ◽  
First Order ◽  
Wide Range ◽  
Eglin C ◽  
Pseudo First Order ◽  
Kinetics Of

The kinetics of binding of recombinant eglin c to bovine pancreatic chymotrypsin was studied by conventional and stopped-flow techniques. With nanomolar enzyme and inhibitor concentrations, the inhibition was fast and pseudo-irreversible (k(assoc.) = 4 x 10(6) m-1.s-1 at 7.4 and 25 degrees C). Reaction of the enzyme-inhibitor complex with alpha 1-proteinase inhibitor, an irreversible chymotrypsin ligand, resulted in a slow release of free eglin c, which was monitored by electrophoresis (k(dissoc.) approximately 1.6 x 10(-6) s-1, t1/2 approximately 5 days). The proflavin displacement method and a stopped-flow apparatus were used to monitor the association of chymotrypsin with eglin c under a wide range of inhibitor concentration and under pseudo-first-order conditions. At pH 7.4 and 25 degrees C or 5 degrees C, or at pH 5.0 and 25 degrees C, the pseudo-first-order rate constant of proflavin displacement increased linearly with eglin c up to the highest concentration tested, suggesting a one-step bimolecular association reaction: E + I in equilibrium with EI. However, kassoc. is much lower than the rate constant for a bimolecular reaction and its activation energy (66 kJ.mol-1 at pH 7.4 and 78 kJ.mol-1 at pH 5.0) is far too high for a diffusion-controlled step. The enzyme-inhibitor association may therefore occur via a loose pre-equilibrium complex EI* (Ki* much greater than 5 x 10(-4) M) that rapidly isomerizes (k2 much greater than 2 x 10(3) s-1) into an extremely stable final complex (Ki approximately 4 x 10(-13) M). Unlike other proteinase-inhibitor systems, the chymotrypsin-eglin association is virtually pH-independent.

Kinetics of reconstitution of apotyrosinase by copper

1990 ◽  
Vol 68 (2) ◽  
pp. 476-479
Author(s):  
Donald C. Wigfield ◽  
Douglas M. Goltz
Keyword(s):  
Rate Constant ◽  
Copper Concentration ◽  
Copper Ions ◽  
Copper Ion ◽  
Order Rate Constant ◽  
First Order ◽  
Order Rate ◽  
Pseudo First Order ◽  
Rate Limiting ◽  
Kinetics Of

The kinetics of the reconstitution reaction of apotyrosinase with copper (II) ions are reported. The reaction is pseudo first order with respect to apoenzyme and the values of these pseudo first order rate constants are reported as a function of copper (II) concentration. Two copper ions bind to apoenzyme, and if the second one is rate limiting, the kinetically relevant copper concentration is the copper originally added minus the amount used in binding the first copper ion to enzyme. This modified copper concentration is linearly related to the magnitude of the pseudo first order rate constant, up to a copper concentration of 1.25 × 10−4 M (10-fold excess), giving a second order rate constant of 7.67 × 102 ± 0.93 × 102 M−1∙s−1.Key words: apotyrosinase, copper, tyrosinase.

scholarly journals Kinetic evidence for a two-step mechanism for the binding of chymotrysin to α1-proteinase inhibitor

1989 ◽  
Vol 259 (3) ◽  
pp. 929-930 ◽  
Author(s):  
M Bruch ◽  
J G Bieth
Keyword(s):  
Reaction Mechanism ◽  
Rate Constant ◽  
Proteinase Inhibitor ◽  
Stopped Flow ◽  
Inhibitor Concentration ◽  
Displacement Method ◽  
First Order ◽  
Flow Apparatus ◽  
Pseudo First Order ◽  
Step Mechanism

We have used the proflavin displacement method and a stopped-flow apparatus to measure the rate constant for the binding of 2 microM-chymotrypsin to 20-125 microM-alpha 1-proteinase inhibitor. The observed pseudo-first-order constant showed a hyperbolic dependence on alpha 1-proteinase inhibitor concentration, suggesting a reaction mechanism in which a fast pre-equilibrium (K = 0.19 mM) is followed by a first-order formation of the final complex (k = 252 s-1).

Kinetics of Reactions of 8-Quinolinol and Acetate with Hydroxyaluminum Species in Aqueous Solutions. 1. Polynuclear Hydroxyaluminum Cations

1971 ◽  
Vol 49 (10) ◽  
pp. 1683-1687 ◽  
Author(s):  
R. C. Turner ◽  
Wan Sulaiman
Keyword(s):  
Acetic Acid ◽  
Rate Constant ◽  
Empirical Equation ◽  
Decomposition Reaction ◽  
Order Rate Constant ◽  
Acetate Concentration ◽  
First Order ◽  
Order Rate ◽  
Pseudo First Order ◽  
Kinetics Of

The effect of varying 8-quinolinol and acetate concentration on the rate of decomposition of poly-nuclear hydroxyaluminum cations was studied. It was found that the concentration of the undissociated 8-quinolinol and acetic acid molecules determined the magnitude of the first order rate constant for the decomposition of the polynuclear hydroxyaluminum cations, except when the acetate concentrations were relatively high. With high acetate concentrations, it appeared that polynuclear acetate species were involved in the reactions. An empirical equation was developed showing the effect of 8-quinolinol and acetic acid molecule concentrations on the pseudo first order rate constant for the decomposition reaction.

Oxidation of Nickel(II) and Manganese(II) by Peroxodisulfate in Aqueous Solution in the Presence of Molybdate. Crystal Structure of the (NH4)6[NiMo9O32].6H2O Product

1992 ◽  
Vol 45 (12) ◽  
pp. 1943 ◽  
Author(s):  
SJ Dunne ◽  
RC Burns ◽  
GA Lawrance
Keyword(s):  
Rate Constant ◽  
Linear Dependence ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
X Ray Diffraction ◽  
X Ray ◽  
First Order ◽  
Oxidant Concentration ◽  
Ph Range ◽  
Kinetics Of

Oxidation of Ni2+,aq, by S2O82- to nickel(IV) in the presence of molybdate ion, as in the analogous manganese system, involves the formation of the soluble heteropolymolybdate anion [MMogO32]2- (M = Ni, Mn ). The nickel(IV) product crystallized as (NH4)6 [NiMogO32].6H2O from the reaction mixture in the rhombohedra1 space group R3, a 15.922(1), c 12.406(1) � ; the structure was determined by X-ray diffraction methods, and refined to a residual of 0.025 for 1741 independent 'observed' reflections. The kinetics of the oxidation were examined at 80 C over the pH range 3.0-5.2; a linear dependence on [S2O82-] and a non-linear dependence on l/[H+] were observed. The influence of variation of the Ni/Mo ratio between 1:10 and 1:25 on the observed rate constant was very small at pH 4.5, a result supporting the view that the precursor exists as the known [NiMo6O24H6]4- or a close analogue in solution. The pH dependence of the observed rate constant at a fixed oxidant concentration (0.025 mol dm-3) fits dequately to the expression kobs = kH [H+]/(Ka+[H+]) where kH = 0.0013 dm3 mol-1 s-1 and Ka = 4-0x10-5. The first-order dependence on peroxodisulfate subsequently yields a second-order rate constant of 0.042 dm3 mol-1 s-1. Under analogous conditions, oxidation of manganese(II) occurs eightfold more slowly than oxidation of nickel(II), whereas oxidation of manganese(II) by peroxomonosulfuric acid is 16-fold faster than oxidation by peroxodisulfate under similar conditions.

scholarly journals Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?

1976 ◽  
Vol 153 (2) ◽  
pp. 495-497 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Schiff Base ◽  
Rate Constant ◽  
Active Site ◽  
Order Rate Constant ◽  
Lysine Residue ◽  
Prolonged Incubation ◽  
Enzyme Active Site ◽  
First Order ◽  
Substrate Analogue ◽  
Pseudo First Order

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 × 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.

An automated approach to characterize, in simple kinetic terms, the generation and inactivation of thrombin and the interaction of fibrinogen with thrombin.

1988 ◽  
Vol 34 (10) ◽  
pp. 1971-1975 ◽  
Author(s):  
D R Hoak ◽  
S K Banerjee ◽  
G Kaldor
Keyword(s):  
Prothrombin Time ◽  
Rate Constant ◽  
Thrombin Generation ◽  
Order Rate Constant ◽  
Clinical Use ◽  
First Order ◽  
Pathological Conditions ◽  
Pseudo First Order ◽  
Instantaneous Concentration

Abstract Here, we used a fully automated, computer-directed centrifugal analyzer (which permitted simultaneous turbidimetry and calculation of results) and purified thrombin, fibrinogen, and various inhibitors to study clot formation. The Km and Vm for these reactions were useful in detecting and partly characterizing anticoagulants. We also explored the generation and inactivation of thrombin, using the two-stage prothrombin time and antithrombin activity tests. The amount of thrombin instantaneously generated and inactivated was monitored under artificially created pathological conditions. The pseudo-first-order rate constant for thrombin generation and inactivation and the instantaneous concentration of enzymatically active and inactive thrombin were used in the characterization of these conditions. We believe this approach is suitable for routine clinical use.

Kinetics and mechanisms of oxidations by metal ions. Part IV. Oxidation of phenylphosphinic acid by aquavanadium(V) ions

1985 ◽  
Vol 63 (3) ◽  
pp. 663-666 ◽  
Author(s):  
Raj Narain Mehrotra
Keyword(s):  
Acidic Solution ◽  
Active Form ◽  
Equilibrium Mixture ◽  
Order Rate Constant ◽  
First Order ◽  
Inactive Form ◽  
Pseudo First Order ◽  
Independent Path ◽  
Kinetics Of ◽  
Vanadium Ion

The kinetics of the oxidation of phenylphosphinic acid by quinquevalent vanadium ion have been investigated in aqueous perchlorate media under pseudo-first order conditions (phenylphosphinic acid in excess). The reaction has a first order dependence in [V(V)] and [phenylphosphinic acid] and the observed pseudo-first order rate constant kobs is given by kobs = a + b[H+].The acid-independent path is considered to be due to the reaction between VO2+ (aq.) and C6H5P:(OH)2, the active form of phenylphosphinic acid, while the reaction between V(OH)32+ (aq.) and C6H5P(O)(OH)H, the inactive form of phenylphosphinic acid, is considered to explain the acid-dependent path. Phenylphosphinic acid in aqueous acidic solution is known to exist as an equilibrium mixture of the active and inactive forms. The composite activation and thermodynamic parameters associated with the constants a and b are reported.

scholarly journals Inactivation of the endogenous argininosuccinate lyase activity of duck δ-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate

1993 ◽  
Vol 293 (2) ◽  
pp. 537-544 ◽  
Author(s):  
H J Lee ◽  
S H Chiou ◽  
G G Chang
Keyword(s):  
Enzyme Activity ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Histidine Residue ◽  
Argininosuccinate Lyase ◽  
First Order ◽  
Diethyl Pyrocarbonate ◽  
Order Rate ◽  
Lyase Activity ◽  
Pseudo First Order

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

Decolorization of Methyl Orange Simulated Wastewater by Chlorine Dioxide with Ultrasound

2011 ◽  
Vol 255-260 ◽  
pp. 2904-2908
Author(s):  
Li Jie Huang ◽  
Ting Xu ◽  
Shuang Fei Wang
Keyword(s):  
Methyl Orange ◽  
Rate Constant ◽  
Chlorine Dioxide ◽  
Oxidation Process ◽  
Order Rate Constant ◽  
Pseudo First Order Reaction ◽  
First Order ◽  
Effective Technology ◽  
Simulated Wastewater ◽  
Pseudo First Order

Experiments were conducted to investigate the decolorization of methyl orange simulated wastewater in order to assess the effectiveness and feasibility of ultrasound(US) enhanced high-purity chlorine dioxide(ClO2) oxidation process. The results showed that in ClO2/US system the decolorization rate of methyl orange was up to 96%, which was increased by 8% as compared to ClO2treatment alone. The decolorization of methyl orange with/without ultrasonic irradiation follows apparent pseudo-first-order reaction kinetics. The apparent pseudo-first-order rate constant kappwas 0.19min-1in the ClO2/US system, which was a little higher than 0.13min-1of rate constant achieved in ClO2treatment alone. It shows that ClO2/US system can be an effective technology for the decolorization of azo dyes in wastewater.

scholarly journals The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide

1975 ◽  
Vol 151 (1) ◽  
pp. 51-59 ◽  
Author(s):  
S R Parr ◽  
M T Wilson ◽  
C Greenwood
Keyword(s):  
Cytochrome Oxidase ◽  
Flash Photolysis ◽  
Order Rate Constant ◽  
Second Order ◽  
Stopped Flow ◽  
First Order ◽  
Order Rate ◽  
Co Concentration ◽  
Pseudo First Order ◽  
Two Phases

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 × 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 × 10(4)M-1-s-1 and 1.6 × 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.

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