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International Journal for Parasitology 29 (1999) 655±662

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro
 Elida G. Campos a, b, *, Marcelo Hermes-Lima b, James M. Smith a, Roger K. Prichard a
a

Institute of Parasitology, McGill University, MacDonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, CanadaH9X 3V9 b Departmento de Biologia Celular, Universidade de BrasõÂlia, BrasõÂlia, DF, CEP 70910-900, Brazil Received 14 December 1998; received in revised form 9 March 1999; accepted 9 March 1999

Abstract Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciolahepatica and Schistosoma mansoni. Using partially puri®ed cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII±EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxylradical. Fasciola hepatica cytochrome c peroxidase e€ectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low speci®c activity of the ®nal partially puri®ed sample the e€ects of ascorbate and calcium chloride on cytochrome cperoxidase were investigated. The activity of the partially puri®ed enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially puri®ed enzyme with 1 mM calcium chloride did not have any e€ect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of thehighly toxic hydroxyl radical (ÁOH). We suggest that the capacity of CcP to inhibit ÁOH-formation, by eciently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica. # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved.
Keywords: Cytochrome c peroxidase;Schistosoma mansoni; Fasciola hepatica; Reactive oxygen species; Antioxidant enzyme; Oxidative stress

* Corresponding author. Tel.: 55-61-307-2423; fax: 55-61-349-8411; e-mail: elida@unb.br 0020-7519/$20.00 # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 0 3 0 - 2

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E.G. Campos et al./International Journalfor Parasitology 29 (1999) 655±662

1. Introduction Cytochrome c peroxidase (CcP; EC 1.11.1.5) is a hemeprotein found in yeast (Saccharomyces cerevisiae) and bacteria, in some lower eukaryotes, such as the trematodes Fasciola hepatica, and Schistosoma mansoni [1±3]. Cytochrome c peroxidase oxidises H2O2 using cytochrome c as the electron donor. This enzyme is present in the intermembrane space ofS. cerevisiae mitochondria [4]. Yeast CcP has a characteristic property of undergoing the so-called `induced conversion' which is the formation of the holoenzyme (by attachment of the heme moiety) when anaerobic yeast adapts to oxygen [5]. It has been shown that, under physiological conditions, yeast CcP utilises 53±55% of the H2O2 generated at the level of the mitochondrial respiratory chain,thus preventing H2O2 leakage to the cytosol [6]. Studies of the mechanism of reaction and protein structure of CcP, both from baker's yeast and bacteria, have generated an abundance of information about this enzyme [4, 7]. However, CcP appears to be absent from vertebrates and little is known about its physiological role in the lower multicellular organisms in which it has been described....