Horseradish peroxidase hydroperoxide determination

Horseradish peroxidase (HRP) is a member of the large class of peroxidases, which are enzymes defined as oxidoreductases using hydroperoxide as electron acceptor. HRP has been widely used for the construction of amperometric biosensor for the determination of H202 and small organic and inorganic substrates. 

S-cyclodextrin condensation, 630 horseradish peroxidase mimic, 628-9, 630 hydrogen peroxide determination, 639 hydroperoxide determination, 681, 708 Hemoglobin see Haemoglobin Henry s law constants... 

Altschul et al. (1, 2) originally discovered that cytochrome c peroxidase reacts with a stoichiometric amount of hydroperoxide to form a red peroxide compound, which will be referred to hereafter as Compound ES. It has a distinct absorption spectrum, as shown in Fig. 2. The formation of Compound ES from the enzyme and hydroperoxides is very rapid (fci > 10 10 sec"M. No intermediate, which precedes Compound ES, has been thus far detected. In the absence of reductants, or S2, Compound ES is highly stable. The rate constant of its spontaneous decay is of the order of 10 sec 22). The primary peroxide compound (Compound I) of horseradish peroxidase decays much faster at a rate of 10 sec (6). This unusual stability of Compound ES allows one to determine various physical and chemical parameters quantitatively and reliably. Titrations of Compound ES with reductants such as ferrocjHio-chrome c Iff, 20) and ferrocyanide 18, 34) have established that Compound ES is two oxidizing equivalents above the original ferric nnzyme. The absorption spectrum of Compound ES is essentially identical to that of Compound II of horseradish peroxidase which contains one oxidizing equivalent per mole in the form of Fe(IV). In addition, EPR examinations have revealed that Compound ES contains a stable free radical, the spin concentration of which is approximately one equivalent per mole (Fig. 3). Therefore, it is reasonable to conclude that two oxidiz-... 

The first step within this project was to improve the HPLC-fluorescence method [1] in order to determine not only hydrophilic but also lipophilic hydroperoxides of up to seven carbon atoms. Among the aspects investigated were colunm length, mixed solvent and gradient elution and the use of microperoxidase instead of horseradish peroxidase in the detection system [2, 3]. The use of microperoxidase let us add secondary, tertiary and other branched hydroperoxides to the list of peroxide species we can determine. For full analysis a number of hydroperoxides were synthesised as standards for retention time and quantitation methyl hydroperoxide (MHP), ethyl hydroperoxide (EHP), 1- and 2-propyl hydroperoxides (PHPs), 1-butyl hydro-peroxide (1-BHP), hydroxymethyl hydroperoxide (HMHP), 1-hydroxyethyl hydroperoxide (1-HEHP), 2-hydroxyethyl hydroperoxide (2-HEHP), 1-hydroxypropyl hydroperoxide (1-HPHP), 1-hydroxybutyl hydroperoxide (1-HBHP), 1- hydroxypentyl hydroperoxide (1-HPentHP),... 

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