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.

Inactivation Of Purified Human Platelet Cyclic Amp Phosphodiesterase By The Affinity Label 2’-O-Iodohydrin-P-Cyclic Amp

1981 ◽  
Author(s):  
P G Grant ◽  
R F Colman ◽  
A K Sinha ◽  
R W Colman
Keyword(s):  
Cyclic Amp ◽  
Rate Constant ◽  
Active Site ◽  
Human Platelet ◽  
Order Rate Constant ◽  
Supernatant Fraction ◽  
First Order ◽  
Cyclic Amp Phosphodiesterase ◽  
Order Rate ◽  
Pseudo First Order

Cyclic AMP phosphodiesterase (PDE) is a regulatory enzyme in human platelets. Inhibitors of this enzyme raise intracellular cAMP which prevents platelet activation. Little is known about the biochemistry of this enzyme. PDE was isolated from human platelet concentrates by nitrogen bomb cavitation. The specific activity of PDE in cell lysate was 0.064 nmoles cAMP hydrolysed/min/mg protein at 22° , 1 µM cAMP. Eighty percent of the activity appeared in the 100,000 × g supernatant fraction. Chromatography was performed on DEAE cellulose equibrated with 50 mM Tris- acetate pH 6.0, 3.75 mM 2-mercaptoethanol. A linear gradient with a limiting salt concentration of 1.0 M Na acetate separated two peaks of PDE activity. The first had a for cAMP of >100 µM; the second had a Km for cAMP of 5 µM. The lower enzyme was further purified by adsorption on blue dextran Sepharose in 50 mM Tris pH 7.5, 2 mM MgCl2 followed by affinity elution with 1 mM cAMP in the same buffer. These steps resulted in a 1700 purification of the enzyme (113 nmoles/min/mg). The compound 2’-0-iodohydrin- p-cAMP (IH-cAMP) is a cAMP derivative with an alkylating side chain. Incubation of PDE with 5 mM IH-cAMP at 37° resulted in 88% inactivation of the enzyme at 15 hours, compared to a control, with a corrected pseudo-first-order rate constant of 0.144 h-1. When cAMP (100 µM) was included in the inactivation mixture the corrected pseudo-first-order rate constant decreased to 0.064 h-1l. Thus, a 20-fold excess of cAMP protected 56% against inactivation by IH- cAMP. The inhibition was not reversed by gel filtration of the inactivated enzyme which removed IH-cAMP. These results suggest that IH-cAMP reacts with the active site of PDE to irreversibly inactivate the enzyme. IH-cAMP should prove to be a useful tool in understanding the chemistry of the active site of this important enzyme.

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.

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.

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.

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.

Optical kinetic method for calibration of spectrophotometer temperature: demonstration with the Cobas-Bio analyzer.

1987 ◽  
Vol 33 (10) ◽  
pp. 1891-1895 ◽  
Author(s):  
E K Armitage ◽  
W G Miller
Keyword(s):  
Analysis Of Variance ◽  
Rate Constant ◽  
Kinetic Method ◽  
Order Rate Constant ◽  
Time Intervals ◽  
First Order ◽  
Order Rate ◽  
Analytical Imprecision ◽  
Pseudo First Order ◽  
Temperature Errors

Abstract The pseudo-first-order rate constant for the Jaffé reaction with creatinine varies logarithmically with temperature and was calibrated in the range 25 to 37 degrees C to measure the temperature of the liquid in the lightpath of spectrophotometric instrumentation. The reagent concentrations can be adjusted to permit rate-constant measurements in time intervals from a few seconds to several minutes. The temperature increment that can be resolved is limited only by the analytical imprecision of the instrumentation used to measure the rate constant and the calibration temperature. In this investigation, a temperature SD of 0.03 degrees C could be measured. Two Cobas-Bio centrifugal analyzers, used to demonstrate the utility of this technique, were found to have temperature errors from -1.0 to -1.7 degrees C in the 30 to 37 degrees C range and overall temperature SD of 0.19 and 0.36 degree C, respectively, at 37 degrees C. Analysis of variance gave between-rotor SD of 0.14 and 0.34 degrees C and within-rotor SD of 0.13 and 0.11 degree C, respectively. We found temperature differences of 0.3 degree C between cuvets in a rotor, and gradients of 0.3 and 0.4 degree C, respectively, from the top to bottom of an individual cuvet in the two instruments.

scholarly journals Kinetic study on the removal of mercury by fly ash

2014 ◽  
Vol 16 (2) ◽  
pp. 385-392 ◽  
Keyword(s):  
Experimental Data ◽  
Fly Ash ◽  
Rate Constant ◽  
Contact Time ◽  
Order Rate Constant ◽  
First Order ◽  
Pseudo Second Order ◽  
Pseudo First Order ◽  
Percent Removal ◽  
Adsorbent Dose

<div> <p>The removal of mercury by adsorption process using fly ash was investigated in this study. Mercury removal capacity of fly ash was performed by batch mode adsorption experiment with the effect of various parameters i.e., contact time (0.5-3.5) h, pH of 2-10, concentration of adsorbate (1, 5 and 10)<br /> mg l<sup>-1</sup>, adsorbent dose (100-1000) mg per 100 ml solution and temperature (303, 313 and 323) K. Mercury concentration (10 mg l<sup>-1</sup>) was chosen for all parameters except adsorbent dose. The experimental data were showed that the adsorbent dose of 200, 400 and 600 mg per 100 ml were sufficient to maximum removal of mercury (98 percent) from aqueous solution of mercury (1, 5 and 10) mg.L<sup>-1</sup> at equilibrium and 89 percent mercury was removed when concentration was 10 mg l<sup>-1</sup> at 303K temperature. Adsorbent dose of 100 mg per 100 ml solution showed 74 percent removal of mercury for 2 hours contact time and 90 percent removal at pH 10. The experimental data were fitted with pseudo first order and pseudo second order kinetics which was proposed by Lagergreen. The value of pseudo first order rate constant, k<sub>1</sub> is 0.697 h<sup>-1</sup> and pseudo second order rate constant k<sub>2</sub> is 0.135 l mg<sup>-1</sup> h<sup>-1</sup>.</p> </div> <p>&nbsp;</p>

scholarly journals Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5′-isothiocyanate

1992 ◽  
Vol 285 (3) ◽  
pp. 737-743 ◽  
Author(s):  
C M Tzeng ◽  
L H Hsu ◽  
R L Pan
Keyword(s):  
Rate Constant ◽  
Mung Bean ◽  
Large Subunit ◽  
Blue Shift ◽  
Order Rate Constant ◽  
Lysine Residue ◽  
Double Logarithmic Plot ◽  
Hydrophobic Domain ◽  
First Order ◽  
A Subunit

Fluorescein 5′-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.

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