Purification of Paracoccidioides brasiliensis catalase P: subsequent kinetic and stability studies.
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.
Chagas
Ronney Fernandes
RF
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Brazil.
Bailão
Alexandre Melo
AM
Fernandes
Kátia Flávia
KF
Winters
Michael S
MS
Pereira
Maristela
M
Soares
Célia Maria de Almeida
CM
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
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D016877:Oxidants; D006861:Hydrogen Peroxide; D002374:Catalase; D000640:Amitrole