An electron spin resonance (ESR) study on the mechanism of ascorbyl radical production by metal-binding proteins |
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Authors: | A. Mouithys-Mickalad C. Deby G. Deby-Dupont M. Lamy |
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Affiliation: | (1) Centre for the Biochemistry of Oxygen, Institut de Chimie, Universit de Lige Domaine Universitaire duSart, Tilman, Belgium;(2) Department of Anaesthesia and Intensive Care Medicine, University Hospital of Lige Domaine Universitaire du Sart, Tilman, Belgium |
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Abstract: | The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable pro-duction of AFR was detected, which was increased by a 10 M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive inter-actions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo. © Rapid Science 1998 |
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Keywords: | ascorbate albumin ceruloplasmin copper electron spin resonance iron transferrin |
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