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Peroxidase activity of the iron-porphyrin microperoxidase-8.

Bachelor, John L. (1994) Peroxidase activity of the iron-porphyrin microperoxidase-8. Doctoral thesis, University of Surrey (United Kingdom)..

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Microperoxidase-8 (MP-8), a haem octapeptide derived from cyctochrome c and retaining His as one axial ligand, has been further developed as a model for the "protein-free" co-factor for comparison with the peroxidase enzymes (eg. horseradish peroxidase, HRP) and myoglobin (Mb). pH profiles have been obtained allowing a general comparison of MP-8, HRP and Mb. The reaction of MP-8 is pH-independent at low pHs, indicating reaction of neutral H2O2 molecules, whilst it is pH-dependent in the range pH 5-10, indicating reaction of HO2-. The protein shifts the diagonal of a plot of log k2 versus pH of the CO-factor 4 units to higher pH in Mb and ?7 pH units to the left in peroxidase. This shows that the protein acts to bind H2O2 as HO2" with the simultaneous uptake of a proton. The initial reaction of H2O2 with Fe(III) of MP-8 at pH 7.0, which is the rate-limiting step, follows the rate equation, rate = k2[HO2-]1[MP-8] 1. This can be significantly increased by the addition of (protonated) quinine (Q), through the formation of a QH+HO2- ion pair. The reaction forms an active intermediate, (a MP-8 analogue of peroxidase enzymes Compound I) containing a FeO3+ species, two oxidising equivalents above Fe(III). Comparisons of the peroxidase reactions of acetylated MP-8 (AcMP-8) and MP-8 show comparable rates (log k2 = 3.81 +/- 0.10) for the two species at pH 7; it is deduced that formation of MP-8 Compound I proceeds via a species equivalent to Compound 0 identified for HRP and AcMP-8. Over limited concentration ranges rates of reaction of MP-8 are independent of substrate concentration for a wide range of sub-strates, with second-order rate constants, IC2. In such regions log k2 = 3.81 +/- 0.10 for all substrates ie. the initial activation of MP-8 is independent of the nature of the substrate. Rate versus substrate concentration profiles obtained and compared for the reactions of MP-8 and HRP with a range of substrates have been characterised into two distinct types, termed Types A and B. A dimer of guaiacol, "diguaiacol", has been prepared and shown to be a competent peroxidase substrate, being oxidised to a similar product to "tetraguaiacol", the unstable oxidation product of guaiacol. In this work high concentrations of guaiacol have been shown to accelerate decomposition of this product, thereby explaining the observed drop in the rate of the peroxidase reaction at guaiacol concentrations above 0.01 M. A means of quantifying the extent to which different substrates protect MP-8 from decomposition or "bleaching" has been found by calculating a ratio termed the Relative Catalytic Efficiency (RCE) of MP-8. The RCE of MP-8 depends on the nature of the substrate but is independent of the concentration of a particular substrate. The latter indicates the presence of a hitherto unsuspected substrate-dependent decomposition pathway for MP-8 Compound I. The enzymic oxidation products of guaiacol, 4-methoxyphenol and 4-methoxy-l-naphthol with H2O2 and MP-8/HRP have been studied in detail to learn more about the oxidation mechanism.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors :
Date : 1994
Depositing User : EPrints Services
Date Deposited : 09 Nov 2017 12:11
Last Modified : 15 Mar 2018 14:49

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