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Characterization of the Myoglobin peroxidase activity in the absence and in the presence of the proteolytic enzyme TIMET-oligopeptidase wild type with different gluthatiolation degrees and mutants in cystein residues

Grant number: 07/06230-7
Support Opportunities:Scholarships in Brazil - Master
Effective date (Start): March 01, 2008
Effective date (End): February 28, 2010
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Principal Investigator:Iseli Lourenço Nantes Cardoso
Grantee:Juliana Conrado Ferreira
Host Institution: Departamento de Bioquímica. Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil


A probable influence of THIMET-oligopeptidase in the reduced glutathione/oxidized glutathione ratio(GSH/GSSG) in the cell and thus, in the cell oxidative stress, suggest that this mechanism can be related to a peroxidase catalytic cycle. Besides catalase and glutathione peroxidase, the peroxidase activity in cells can be exhibited by other hemeproteins of the globin family, neuroglobin (Ngb) e cytoglobin (Cygb), that also exhibit multiple activities.Considering the structural similarity of Cygb with myoglobin (Mb), in this project, it will be used the ferric muscle hemeprotein to study the capacity of THIMET-oligopeptidase to participate in a peroxidase cycle as a reducing agent in a mechanism controlled by the degree of enzyme glutathiolation. In this study it will be characterized the peroxidase catalytic cycle of myoglobin (Mb) in the presence of the the enzyme THIMET-oligopeptidase wild type, treated with different GSH concentrations and in the totally reduced form. It will be also used mutants of THIMET-oligopeptidase in which cys246 and cys248 were be replaced by serine. The kinetic of Mb-Compound II reduction generated by hydrogen peroxide, in the absence and in the presence of the enzyme THIMET-oligopeptidase native, glutatiolated and reduced forms, as well as, the mutants equimolar with Mb, will be investigated by electronic absorption spectrometry. The spin state of the Mb high valence intermediates generated by hydrogen peroxide (Composto I e II) in the absence and in the presence of THIMET-oligopeptidase as well as free radicals will be characterized by EPR technique. The activity of THIMET-oligopeptidase glutathiolated, reduced e mutants before and after the participation in the peroxidase catalytic cycle, will be also determined by the technique of fluorescent substrates. In the future, it will be obtained recombinant Cyb recombinante and the characterization of its catalytic cycle in the presence of THIMET-oligopeptidase will be done.

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