title: Purification of Paracoccidioides brasiliensis catalase P: subsequent kinetic and stability studies.
abstract: Catalases are essential components of the cellular equipment to cope with oxidative stress. Here we have purified a highly abundant catalase P of Paracoccidioides brasiliensis (PbCatP) that is preferentially expressed in the parasitic yeast phase. This oxidative stress-induced protein was isolated from yeast cells grown in the presence of 15 mM of hydrogen peroxide (H(2)O(2)). We have used consecutive steps of protein precipitation and gel filtration chromatography to achieve the purified protein. Protein purification was validated using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and bioinformatics analysis. The purified enzyme showed strong similarity to small-subunit catalases. Like most monofunctional catalases, PbCatP is a homotetramer, resistant to inactivation by acidic conditions, temperature and denaturants. Furthermore, the kinetic behaviour of catalase P was observed to be different at low compared to high H(2)O(2) concentrations. The results demonstrated that a purified PbCatP is a homotetrameric enzyme, classified as a small subunit catalase.
authors.affiliation: Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Brazil.
authors.forename: Ronney Fernandes
authors.initials: RF
authors.lastname: Chagas
authors.forename: Alexandre Melo
authors.initials: AM
authors.lastname: Bailão
authors.forename: Kátia Flávia
authors.initials: KF
authors.lastname: Fernandes
authors.forename: Michael S
authors.initials: MS
authors.lastname: Winters
authors.forename: Maristela
authors.initials: M
authors.lastname: Pereira
authors.forename: Célia Maria de Almeida
authors.initials: CM
authors.lastname: Soares
mesh_terms: D000595:Amino Acid Sequence; D000640:Amitrole / Q000494:pharmacology; D002374:Catalase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000008:administration & dosage; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D016877:Oxidants / Q000008:administration & dosage; D018384:Oxidative Stress; D010228:Paracoccidioides / Q000187:drug effects / Q000201:enzymology*; D010229:Paracoccidioidomycosis / Q000201:enzymology / Q000382:microbiology; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D016877:Oxidants; D006861:Hydrogen Peroxide; D002374:Catalase; D000640:Amitrole