Analytical aspects of enzyme reversible inhibition (original) (raw)
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Biosensors, 2021
An amperometric biosensor based on tyrosinase, immobilized onto a carbon black paste electrode using glutaraldehyde and BSA was constructed to detect competitive inhibitors. Three inhibitors were used in this study: benzoic acid, sodium azide, and kojic acid, and the obtained values for fifty percent of inhibition (IC50) were 119 µM, 1480 µM, and 30 µM, respectively. The type of inhibition can also be determined from the curve of the degree of inhibition by considering the shift of the inhibition curves. Amperometric experiments were performed with a biosensor polarized at the potential −0.15 V vs. Ag/AgCl and using 0.1 M phosphate buffer (pH 6.8) as an electrolyte. Under optimized conditions, the proposed biosensor showed a linear amperometric response toward catechol detection from 0.5 µM to 38 µM with a detection limit of 0.35 µM (S/N = 3), and its sensitivity was 66.5 mA M−1 cm−2. Moreover, the biosensor exhibited a good storage stability. Conversely, a novel graphical plot for ...
Diagnosis of enzyme inhibition based on the degree of inhibition
Biochimica et Biophysica Acta (BBA) - General Subjects, 2003
In this work, a method for the diagnosis of kinetic inhibition, based on the dependence of the degree of inhibition (e i ) on the inhibitor concentration [I] and on the substrate concentration [S], is presented. Because the degree of inhibition is a ratio between rates, kinetic data are normalized by the introduction of an internal control-the rate of the uninhibited reaction. Therefore, the error associated with the kinetic measurements decreases and less experimental measurements are necessary to achieve the diagnosis. The process described, which uses graphical and/or non-linear fitting procedures, allows distinguishing between 20 different kinds of inhibition, including not only linear and hyperbolic, but also parabolic and rational 2,2 inhibitions. Rational 2,2 indicates a new type of inhibition corresponding to an incomplete parabolic inhibition, i.e. mechanistically it corresponds to an inhibitor that binds to two inhibition sites producing enzymatic complexes that are still active. In spite of its comprehensiveness, the diagnosis process is greatly facilitated by the division of the diagnosis of the inhibition in a step-by-step procedure, where only two rival models are evaluated in each step. In the non-linear fittings, the choice between rival models uses a test based on information statistics theory, the Akaike information criterion test, in order to penalize complex models that tend to be favoured in fittings. Finally, equations that allow the determination of inhibition kinetic constants were also deduced. The formalism presented was tested by examining inhibition of acid phosphatase by phosphate (a linear competitive inhibitor).
Mechanistic and Kinetic Studies of Inhibition of Enzymes
Cell Biochemistry and Biophysics, 2000
A graphical method for analyzing enzyme data to obtain kinetic parameters, and to identify the types of inhibition and the enzyme mechanisms, is described. The method consists of plotting experimental data as v/(V o -v) vs 1/(I) at different substrate concentrations. I is the inhibitor concentration; V o and v are the rates of enzyme reaction attained by the system in the presence of a fixed amount of substrate, and in the absence and presence of inhibitor, respectively. Complete inhibition gives straight lines that go through the origin; partial inhibition gives straight lines that converge on the 1-I axis, at a point away from the origin. For competitive inhibition, the slopes of the lines increase with increasing substrate concentration; with noncompetitive inhibition, the slopes are independent of substrate concentration; with uncompetitive inhibition, the slopes of the lines decrease with increasing substrate concentrations. The kinetic parameters, K m , K i , K i ′, and β (degree of partiality) can best be determined from respective secondary plots of slope and intercept vs substrate concentration, for competitive and noncompetitive inhibition mechanism or slope and intercept vs reciprocal substrate concentration for uncompetitive inhibition mechanism. Functional consequencs of these analyses are represented in terms of specific enzyme-inhibitor systems.
Current Enzyme Inhibition, 2015
Using wheat germ acid phosphatase and sodium orthovanadate as a competitive inhibitor, a novel method for analyzing reversible inhibition was carried out. Our alternative approach involves plotting the initial velocity at which product is formed as a function of the ratio of substrate concentration to inhibitor concentration at a constant enzyme concentration and constant assay conditions. The concept of initial concentrations driving equilibrium leads to the chosen axes. Three apparent constants can be derived from this plot: K max , K min , and K inflect. K max and K min represent the substrate to inhibitor concentration ratio for complete inhibition and minimal inhibition, respectively. K inflect represents the substrate to inhibitor concentration ratio at which the enzyme-substrate complex is equal to the inhibitory complex. These constants can be interpolated from the graph or calculated using the first and second derivative of the plot. We conclude that a steeper slope and a shift of the line to the right (increased x-axis values) would indicate a better inhibitor. Since initial velocity is not a linear function of the substrate/inhibitor ratio, this means that inhibition changes more quickly with the change in the [S]/ [I] ratio. When preincubating the enzyme with substrate before the addition of inhibitor, preincubating the enzyme with inhibitor before the addition of substrate or with concurrent addition of both substrate and inhibitor, modest changes in the slopes and y-intercepts were obtained. This plot appears useful for known competitive and non-competitive inhibitors and may have general applicability.
Molecules
Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition (iD), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of iD with respect to the initial inhibitor concentration [I]0 allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC50), thus avoidi...
Biosensing of tyrosinase inhibitors in nonaqueous solvents
Electroanalysis, 1995
An amperometric biosensor based on a Clark oxygen electrode coupled with a biocatalytic layer containing tyrosinase has been developed for measurement in nonaqueous solvents. Glass filter paper was soaked with the enzyme and fixed on top of the electrode by a dialysis membrane. Thiourea derivatives were detected in hexane by evaluating the decrease in the response towards phenol. This method was fast and highly sensitive with detection limits in the range of 13-181 nM depending on the structure of the inhibitor compounds. The response time was up to 10min and depended on the inhibitor's structure. The mechanism of inhibition is discussed using kinetic parameters.
Simulated Analysis of Linear Reversible Enzyme Inhibition with SCILAB
SCILAB is a lesser-known program (than MATLAB) for numeric simulations and has the advantage of being free software. A challenging software-based activity to analyze the most common linear reversible inhibition types with SCILAB is described. Students establish typical values for the concentration of enzyme, substrate, and inhibitor to simulate realistic kinetic data. To extract kinetic parameters, linear and nonlinear (hyperbolic) data fitting are used. The actual distinction between competitive and mixed-type inhibitions and between noncompetitive and mixed-type inhibitions is discussed on the basis of nonlinear data fitting. The activity was performed as a part of an advanced undergraduate course, "Chemical Kinetics", and added to students' diverse preexisting knowledge.
Enzyme Inhibition and Activation: A General Theory
The rate of an enzymatic reaction may be changed by a moderator. Usually, the effect is to reduce the rate, and this is called inhibition. Sometimes the rate of enzyme reaction is raised, and this is called activation. Not only enzyme activation is subject of a less detailed presentation, but also enzyme inhibition and activation are very often discussed independently in enzymology. I attempt to introduce a general model of enzyme inhibition and activation to allow one to interpret inhibition and activation from a mechanistic or physical perspective using the significance of cooperativity as a new approach. The magnitude of interaction between substrate and inhibitor binding sites is given by the D parameter and the magnitude of increasing catalytic reaction constant is given by the E parameter, which both parameter values characterize the type of inhibition and activation. The moderation of mushroom tyrosinse is described by application of the model as a typical.
Bahauddin Zakariya University, Multan (Pakistan)., 2016
A simple, accurate and precise spectroscopic method was developed for simultaneous estimation of Irbesartan and atorvastatin in synthetic mixture using simultaneous equation Method. In this spectroscopic method, 226.00 nm and 246.00 nm wavelengths were selected for measurement of absorptivity. Both the drugs show linearity in a concentration range of 05-30 μg/ml at their respective λmax. Accuracy, precision and recovery studies were done by QC samples covering lower, medium and high concentrations of the linearity range. The relative standard deviation for accuracy, precision studies were found to be within the acceptance range (<2%). The limit of determination was 0.033μg/ml and 0.125 μg/ml for Irbesartan and atorvastatin, respectively. The limit of quantification was 0.1008 μg/ml and 0.3792 μg/ml for Irbesartan and atorvastatin, respectively. Recovery of Irbesartan and atorvastatin were found to be 99.75 % and 99.52% respectively confirming the accuracy of the proposed method. The proposed method is recommended for routine analysis since they are rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent extraction.
Inhibited enzyme electrodes. Part 1: Theoretical model
Biosensors and Bioelectronics, 1990
ABSTMCT A theoretical model is developed for an electrochemical sensor for toxic substances which works by measuring the inhibition of the enzyme activiry. The enzyme is assumed to followMichaelis44enten kinetics and the dtjusion kinetic equation describing the concentration prorle of the enzyme's substrate in the electrolyte layer between the electrode and the membrane covering the electrode is solved. A complete set of analytical solutions is found which correspond to a number of dtrerent rate limitingprocesses. Theset of solutions is described in a case diagram. The use of cytochrvme oxidase in particular is discussed.