Kinetics of the reaction of compound II of horseradish peroxidase with hydrogen peroxide to form compound III

Abstract

The kinetics of the reaction of H 2 0 2 with compound I1 of horseradish peroxidase were studied as a function of pH at 25°C and constant ionic strength of 0.11 M. The reaction of HzOz with compound I1 involves the transient formation of ferric peroxidase and superoxide anion as the first step followed by the reaction of the intermediate species with H z 0 2 to form compound 111. Both reactions are also observed with peracetic acid as substrate, though the amplitude of the first step was too small for the rate to be measured. Observation of the first reaction was not possible below pH 8.5 under the conditions of this investigation. It tends to occur faster at lower pH so an increasing fraction is lost in the dead time of the stopped-flow apparatus. Horseradish peroxidase (HRP; donor : H 2 0 2 oxidoreductase) catalyses the oxidation of a wide variety of compounds by hydroperoxides of the type ROOH. The enzyme derivatives known as compound I and compound 11 have been shown to be involved in the general mechanism (see [l-31 and earlier references contained therein). Details of the events involved in this catalysis have been intensively studied [2]. Observations have also been made on a third enzyme derivative called compound 111 which can be obtained from horseradish peroxidase and excess HzOz [4, 51 and which was not formed when other oxidizing agents were used [6]. This compound is known not to participate in the normal peroxidatic cycle of horseradish peroxidase but it is the peroxidase analog of oxyinyoglobin and oxyhemoglobin 12, 71. The spectra and reactivity of compound 111 correspond to that of the Fe(II)02 adduct [X, 91. Therefore, the mechanism of its formation and its structure and reactivity, of intrinsic interest in their own right, take on a broader significance.Compound 111 has been formed through a number of pathways: by the addition of a large excess of H 2 0 2 to the native enzyme [4, 51, reduction of native enzyme to ferroperoxidase followed by oxygen addition [lo], reaction of H 2 0 z with compound 11 [6, 11, 121 and the addition of superoxide anion to ferriperoxidase [13,14]. The kinetics of the formation of compound TI1 from ferrous peroxidase [Fe(ll)HKP] and

Stabilization of Activity of Oxidoreductases by Their Immobilization onto Special Functionalized Glass and Novel Aminocellulose Film Using Different Coupling Reagents

“…17 To investigate the decrease of HRP activity with operating time, the activity of the enzyme immobilized on NH 2 -glass and NH 2 -cellulose films, respectively, by the coupling reagents shown in Figure 1 was continuously measured in a test solution containing 10 mM H 2 O 2 at pH 5.0 for a time period of 30-40 min. As described in the literature and confirmed by measuring the timedependent activity decrease of HRP dissolved in the abovementioned test solution, the inactivation of HRP in the presence of H 2 O 2 follows a pseudo-first-order kinetic in a good approximation (P < 0.05).…”

“…As described in the literature and confirmed by measuring the timedependent activity decrease of HRP dissolved in the abovementioned test solution, the inactivation of HRP in the presence of H 2 O 2 follows a pseudo-first-order kinetic in a good approximation (P < 0.05). 17 The half-life of immobilized HRP was determined by fitting a pseudo-first-order function to the data of the H 2 O 2 / HRP-enzyme reaction. As seen in Table 2, in all cases, immobilized HRP was stabilized against H 2 O 2 -induced inactivation compared to the native enzyme.…”

“…The half-life of the enzyme was calculated following a procedure suggested by Adediran and Lambeir whereby the half-life time (τ 1/2 ) was calculated by τ 1/2 ) ln(0.5)/k. 17 The pseudo-first-order rate constant k was determined by mathematical exponential curve fitting of the first-order kinetic of HRP activity decrease (V ) V 0 e -(kt) ) by means of Sigma Plot 5.0.…”

Biossensores amperométricos para determinação de compostos fenólicos em amostras de interesse ambiental

“…O mecanismo de transferência de elétrons mediado em eletrodos modificados com peroxidase é mostrado na Figura 6. É conhecido que uma alta concentração de peróxido pode levar a uma forma inativa da peroxidase 95 . Deste modo, os fenóis podem atuar como doadores de elé-trons na reação da peroxidase com peróxidos 94 , sendo este o princípio da utilização de eletrodos modificados com peroxidase para a detecção de espécies fenólicas 32,33,[96][97] .…”

Steady-state kinetics of &lt;i&gt;Roystonea regia&lt;/i&gt; palm tree peroxidase

“…In the absence of substrate, or when they are exposed to high hydrogen peroxide concentrations, peroxidases show the kinetic behavior of suicide inactivation, in which hydrogen peroxide is the suicide substrate that converts Compound II into a highly reactive peroxy-iron(III) porphyrin freeradical termed Compound III [36]. Compound III does not form part of the peroxidase cycle, but is produced under excessive exposure of protonated Compound II to oxidative species in a reaction that is partially mediated by free superoxide radicals [37].…”