Abstract
A new method for determining the intrinsic parameters of reaction in processes involving a high initial rate has been developed. The usefulness of this alternative, which consists of determining several sets of apparent parameters at different times and then extrapolating these to time zero, is demonstrated proved by the linear dependence obtained between the apparent parameters and the reaction time. The method permitted the values of the intrinsic parameters (enzyme specific activity and Michaelis-Menten constants of both substrates) to be obtained for the system under study and was checked with experimental reaction rate data for the soybean peroxidase/phenol/hydrogen peroxide system.
Highlights
The intrinsic parameters of enzymatic systems with soluble enzymes are determined from experimental data of the reaction rate in the first instants of the process
Varying the concentration of hydrogen peroxide: These experiments were carried out in the same conditions of pH, temperature and enzyme concentration of (SBP) as those used in the above assays and a phenol concentration of 2.0 mM
Varying the concentration of phenol: These experiments were carried out in the same conditions of Materials: Soybean peroxidase enzyme (SBP) (EC 1.11.1.7, 25,000 Units), catalase enzyme (EC 1.11.1.6, 2,860 units mg 1 of protein), hydrogen peroxide (35% w/v), phenol pH, temperature and enzyme concentration of (SBP) as those used in the above assays and a hydrogen peroxide concentration of 2.0 mM
Summary
The intrinsic parameters of enzymatic systems with soluble enzymes are determined from experimental data of the reaction rate in the first instants of the process. The object of the present work was to study the kinetic of the SBP/phenol system in a discontinuous reactor, decreasing the buffer load and without adding polyethylene glycol, in an attempt to confirm whether or not the above system follows a bisubstrate ping-pong kinetic In such enzymatic systems, the reaction rate in the first minutes is very high and its variation is not linear with the time, which makes it impossible to obtain reliable data for the values of the kinetic constants. Kinetic parameter determinations: proposed method: Differentiating both parts of the Eq (4) with respect to the initial variable concentration of hydrogen peroxide gives [ ] [ ] d d HO This equation reveals the limitations of this type of representation for the system under study. 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mM, were assessed
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