Inhibition of Enzymatic Reactions. A Rapid Method to Determine the Index pI50

2002 ◽  
Vol 57 (5-6) ◽  
pp. 496-499 ◽  
Author(s):  
David Kulhavý ◽  
Alexander Čegan ◽  
Karel Komers ◽  
Jaromír Mindl
Keyword(s):  
Rapid Method ◽  
Mixed Type ◽  
Enzymatic Reaction ◽  
Reaction Rates ◽  
Fast Method ◽  
Enzymatic Reactions ◽  
Initial Substrate ◽  
The One ◽  
Hydrolysis Of

The activity of every substance I inhibiting an enzymatic reaction can be approximately evaluated by the index pI50. This paper describes a simple and fast method of estimate and/or determination of this index. The method is based on the linearity of the dependence of the ratio of reaction rates of uninhibited and inhibited reaction vs. concentration of the inhibitor at constant initial substrate and enzyme concentrations for fully competitive, noncompetitive, uncompetitive and mixed type of inhibition by the one inhibitor. The validity of the method is demonstrated by four inhibitors of hydrolysis of acetylthiocholine by butyrylcholine esterase.

Enzymatic colorimetry of lecithin and sphingomyelin in aqueous solution.

1983 ◽  
Vol 29 (8) ◽  
pp. 1513-1517 ◽  
Author(s):  
M W McGowan ◽  
J D Artiss ◽  
B Zak
Keyword(s):  
Aqueous Solution ◽  
Hydrogen Peroxide ◽  
Amniotic Fluid ◽  
Enzymatic Reaction ◽  
Detergent Solution ◽  
Enzymatic Determination ◽  
Red Color ◽  
Hydrolysis Of ◽  
Zwitterionic Detergent

Abstract A procedure for the enzymatic determination of lecithin and sphingomyelin in aqueous solution is described. The phospholipids are first dissolved in chloroform:methanol (2:1 by vol), the solvent is evaporated, and the residue is redissolved in an aqueous zwitterionic detergent solution. The enzymatic reaction sequences of both assays involve hydrolysis of the phospholipids to produce choline, which is then oxidized to betaine, thus generating hydrogen peroxide. The hydrogen peroxide is subsequently utilized in the enzymatic coupling of 4-aminoantipyrine and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate, an intensely red color being formed. The presence of a non-reacting phospholipid enhances the hydrolysis of the reacting phospholipid. Thus we added lecithin to the sphingomyelin standards and sphingomyelin to the lecithin standards. This precise procedure may be applicable to determination of lecithin and sphingomyelin in amniotic fluid.

Simple and Rapid Determination of Phytase Activity

1994 ◽  
Vol 77 (3) ◽  
pp. 760-764 ◽  
Author(s):  
Adrianus J Engelen ◽  
Fred C Van Der Heeft ◽  
Peter H G Randsdorp ◽  
Ed L C Smtt
Keyword(s):  
Enzymatic Activity ◽  
Rapid Method ◽  
Rapid Determination ◽  
Phytase Activity ◽  
Sodium Phytate ◽  
Microbial Phytase ◽  
Hydrolysis Of

Abstract A simple and rapid method is described for determining the enzymatic activity of microbial phytase. The method is based on the determination of inorganic orthophosphate released on hydrolysis of sodium phytate at pH 5.5.

Automated Enzymatic Determination of L-Lactate in Serum, with Use of a Miniature Centrifugal Anlyzer

1976 ◽  
Vol 22 (12) ◽  
pp. 2038-2041 ◽  
Author(s):  
T P Hadjiioannou ◽  
S I Hadjiioannou ◽  
S D Brunk ◽  
H V Malmstadt
Keyword(s):  
Lactate Dehydrogenase ◽  
Reaction Rate ◽  
Enzymatic Reaction ◽  
Reaction Rates ◽  
Enzymatic Determination ◽  
Analytical Recovery ◽  
Rate Method ◽  
Relative Errors ◽  
Lithium Lactate

Abstract We describe an automated enzymatic reaction-rate method for spectrophotometric determination of lactate in serum with a miniature centrifugal analyzer. The L(+)-lactate is selectively oxidized in the presence of lactate dehydrogenase (EC 1.1.1.27) and NAD+ to from NADH, whitch is measured from its absorption. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer0generated calibration curve with aqueous lithium lactate standards. Lactte concentrations in the range 0.32-1.6 µg/4 µl (80-400 mg/liter) of sample were determined with relative errors and coefficient of variation of 4.8%. Analytical recovery of lactate added to pooled serum was 89-112% (average, 101%). Comparison with a kit ("Rapid Lactate") method gave a correlation coefficient squared of 0.979 over a concentration range of 39-779 mg/liter.

scholarly journals Application Enzymatic Method for Analysis Vaginal Tabletas “Fluomizin”

2021 ◽  
Vol 62 (3) ◽  
pp. 260-269
Author(s):  
Koval'ska Olena Vasilivna ◽  
Blazheyevskіy Mykola
Keyword(s):  
Hydrogen Peroxide ◽  
Enzymatic Reaction ◽  
Chloride Content ◽  
Solution Concentration ◽  
Photometric Method ◽  
Maximal Velocity ◽  
Buffer Solution ◽  
Dequalinium Chloride ◽  
Hydrolysis Of

Introduction: Development of a new enzymatic kinetic-photometric method for analysis of Dequalinium chloride, based on the use of enzymatic hydrolysis of acetylcholine, for determine the amount of Dequalinium chloride in the vaginal tablets “Fluomizin” No 6. Method: The amount of Dequalinium chloride (DCh) was determined by the degree of inhibition of the enzymatic reaction, which was evaluated by the residual unreacted substrate - acetylcholine. Determination of the residual amount of acetylcholine in the reaction mixture was performed by a kinetic-photometric method using an indicator oxidation reaction of p-phenetidine with peracetic acid, which is formed during the auxiliary reaction of perhydrolysis with addition of excess hydrogen peroxide in the reaction mixture over a period of time. Results: Optimal conditions were determined: pH 8.35 was selected, influence of nature of buffer solution, concentration of acetylcholine (0.05 mg / ml), cholinesterase (0.4 mg / ml), hydrogen peroxide (10,0%) and p-phenethedine (1 %), the incubation time (10 min). Conclusions: The procedure for determination of Dequalinium chloride content in the vaginal tablets “Fluomizin” No 6 was developed. The kinetic parameters Km (Michaelis–Menten constant) and Vmax (maximal velocity) were determined.

scholarly journals Implementation of an Enzyme Membrane Reactor to Intensify the α-O-Glycosylation of Resveratrol Using Cyclodextrins

2021 ◽  
Vol 14 (4) ◽  
pp. 319
Author(s):  
Irina Ioannou ◽  
Eduardo Barboza ◽  
Gaëlle Willig ◽  
Thomas Marié ◽  
Andreïa Texeira ◽  
...  
Keyword(s):  
Membrane Reactor ◽  
Enzymatic Reaction ◽  
Reaction Medium ◽  
Polymeric Membrane ◽  
Partial Hydrolysis ◽  
Enzymatic Reactions ◽  
Solubility In Water ◽  
Recent Developments ◽  
Hydrolysis Of ◽  
Enzymatic Membrane

The O-glycosylation of resveratrol increases both its solubility in water and its bioavailability while preventing its oxidation, allowing a more efficient use of this molecule as a bioactive ingredient in pharmaceutical and cosmetic applications. Resveratrol O-glycosides can be obtained by enzymatic reactions. Recent developments have made it possible to selectively obtain resveratrol α-glycosides from the β-cyclodextrin–resveratrol complex in water with a yield of 35%. However, this yield is limited by the partial hydrolysis of the resveratrol glycosides produced during the reaction. In this study, we propose to intensify this enzymatic reaction by coupling the enzymatic reactor to a membrane process. Firstly, membrane screening was carried out at the laboratory scale and led to the choice of a GE polymeric membrane with a cut-off of 1 kDa. This membrane allowed the retention of 65% of the β-cyclodextrin–resveratrol complex in the reaction medium and the transfer of 70% of the resveratrol α-O-glycosides in the permeate. In a second step, this membrane was used in an enzymatic membrane reactor and improved the yield of the enzymatic glycosylation up to 50%.

scholarly journals Enhanced Performance of Reagent-Less Carbon Nanodots Based Enzyme Electrochemical Biosensors

Sensors ◽  
2019 ◽  
Vol 19 (24) ◽  
pp. 5576 ◽  
Author(s):  
Iria Bravo ◽  
Cristina Gutiérrez-Sánchez ◽  
Tania García-Mendiola ◽  
Mónica Revenga-Parra ◽  
Félix Pariente ◽  
...  
Keyword(s):  
Enzymatic Reaction ◽  
Lactate Concentration ◽  
Standard Addition ◽  
Carbon Nanodots ◽  
Fast Method ◽  
Lactate Oxidase ◽  
The One ◽  
Linear Concentration Range ◽  
Serum Extract

This work reports on the advantages of using carbon nanodots (CNDs) in the development of reagent-less oxidoreductase-based biosensors. Biosensor responses are based on the detection of H2O2, generated in the enzymatic reaction, at 0.4 V. A simple and fast method, consisting of direct adsorption of the bioconjugate, formed by mixing lactate oxidase, glucose oxidase, or uricase with CNDs, is employed to develop the nanostructured biosensors. Peripherical amide groups enriched CNDs are prepared from ethyleneglycol bis-(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid and tris(hydroxymethyl)aminomethane, and used as precursors. The bioconjugate formed between lactate oxidase and CNDs was chosen as a case study to determine the analytical parameters of the resulting L-lactate biosensor. A linear concentration range of 3.0 to 500 µM, a sensitivity of 4.98 × 10−3 µA·µM−1, and a detection limit of 0.9 µM were obtained for the L-lactate biosensing platform. The reproducibility of the biosensor was found to be 8.6%. The biosensor was applied to the L-lactate quantification in a commercial human serum sample. The standard addition method was employed. L-lactate concentration in the serum extract of 0.9 ± 0.3 mM (n = 3) was calculated. The result agrees well with the one obtained in 0.9 ± 0.2 mM, using a commercial spectrophotometric enzymatic kit.

Use of Chromogenic Substrate S-2251 for Determination of Plasminogen Activator in Rat Ovaries

1981 ◽  
Vol 46 (02) ◽  
pp. 507-510 ◽  
Author(s):  
H Shimada ◽  
T Mori ◽  
A Takada ◽  
Y Takada ◽  
Y Noda ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Enzymatic Reactions ◽  
Chromogenic Substrate ◽  
Assay Procedure ◽  
One Step ◽  
Reproducible Method ◽  
Plasmin Inhibitors ◽  
Endogenous Activity ◽  
Hydrolysis Of

SummaryA simple, specific and reproducible method for determination of plasminogen activator activity in rat ovaries has been developed by using the chromogenic substrate S-2251. The two steps of enzymatic reactions, i. e. activation of plasminogen and subsequent hydrolysis of the substrate was performed in one step incubation. A linear relationship was observed between the amount of chromogen produced and activator activity in the range of the optical density from 0.05 to 1.20 for 30 min’s incubation. Endogenous activity of non-specific proteases, plasmin or plasmin inhibitors which might be contained in rat ovaries turned out not to interfere with the specificity of a standardized assay procedure. Reproducibility was firmly established with coefficient of variation not exceeding 10%. Using this method, a marked increase followed by a drastic decrease in the activator activity was shown with rat ovaries around the time of ovulation after the injection of human chorionic gonadotropin.

scholarly journals Real-Time Interferometric Refractive Index Change Measurement for the Direct Detection of Enzymatic Reactions and the Determination of Enzyme Kinetics

Sensors ◽  
2019 ◽  
Vol 19 (3) ◽  
pp. 539
Author(s):  
Søren Jepsen ◽  
Thomas Jørgensen ◽  
Henrik Sørensen ◽  
Søren Kristensen
Keyword(s):  
Refractive Index ◽  
Enzyme Kinetics ◽  
Adenosine Triphosphate ◽  
Direct Detection ◽  
Enzymatic Reaction ◽  
Refractive Index Change ◽  
Direct Determination ◽  
Initial Reaction ◽  
Enzymatic Reactions

Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, KM was found to be 0.33 mM using Michaelis–Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics.

Automated enzymatic determination of L-lactate in serum, with use of a miniature centrifugal analyzer.

1976 ◽  
Vol 22 (12) ◽  
pp. 2042-2045 ◽  
Author(s):  
T P Hadjiioannou ◽  
S I Hadjiioannou ◽  
S D Brunk ◽  
H V Malmstadt
Keyword(s):  
Lactate Dehydrogenase ◽  
Reaction Rate ◽  
Enzymatic Reaction ◽  
Reaction Rates ◽  
Enzymatic Determination ◽  
Analytical Recovery ◽  
Rate Method ◽  
Relative Errors ◽  
Lithium Lactate

Abstract We describe an automated enzymatic reaction-rate method for spectrophotometric determination of lactate in serum with a miniature centrifugal analyzer. The L(+) -lactate is selectively oxidized in the presence of lactate dehydrogenase (EC 1.1.1.27) and NAD+ to form NADH, which is measured from its absorption. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer-generated calibration curve with aqueous lithium lactate standards. Lactate concentrations in the range 0.32-1.6 mug/4 mul (80-400 mg/liter) of sample were determined with relative errors and coefficient of variation of 4.8%. Analytical recovery of lactate added to pooled serum was 89-112% (average, 101%). Comparison with a kit ("Rapid Lactate") method gave a correlation coefficient squared of 0.979 over a concentration range of 39-779 mg/liter.

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