title: Purification and structural stability of white Spanish broom (Cytisus multiflorus) peroxidase.
abstract: New plant peroxidase has been isolated to homogeneity from the white Spanish broom Cytisus multiflorus. The enzyme purification steps included homogenization, NH(4)SO(4) precipitation, extraction of broom colored compounds and consecutive chromatography on Phenyl-Sepharose, HiTrap™ SP HP and Superdex-75 and 200. The novel peroxidase was characterized as having a molecular weight of 50 ± 3 kDa. Steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the structural stability of C. multiflorus peroxidase (CMP) at pH 7.0. Thus changes in far-UV CD corresponded to changes in the overall secondary structure of enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. It is shown that the process of CMP denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N ⟶ kD, where k is a first-order kinetic constant that changes with temperature following the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.
authors.affiliation: Water Research and Development Centre (CIDTA), Universidad de Salamanca, 37007 Salamanca, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Perez Galende
authors.affiliation: Instituto de Estudios Biofuncionales, Departamento de Química-Física II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Nazaret
authors.initials: N
authors.lastname: Hidalgo Cuadrado
authors.affiliation: Instituto de Recursos Naturales y Agrobiologia (IRNASA-CSIC), 37008 Salamanca, Spain.
authors.forename: Juan B
authors.initials: JB
authors.lastname: Arellano
authors.affiliation: Departamento de Bioquimica y Biologia Molecular, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Gavilanes
authors.affiliation: Department of Biochemistry, Microbiology and Biotechnology, Far Eastern Federal University, 690600 Vladivostok, Russia.
authors.forename: Eduard Ya
authors.initials: EY
authors.lastname: Kostetsky
authors.affiliation: Water Research and Development Centre (CIDTA), Universidad de Salamanca, 37007 Salamanca, Spain.
authors.forename: Galina G
authors.initials: GG
authors.lastname: Zhadan
authors.affiliation: Departamento de Bioquimica y Biologia Molecular, Universidad de Salamanca, 37007 Salamanca, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Villar
authors.affiliation: Water Research and Development Centre (CIDTA), Universidad de Salamanca, 37007 Salamanca, Spain; Departamento de Quimica Física, Universidad de Salamanca, 37008 Salamanca, Spain. Electronic address: mgr@usal.es.
authors.forename: Manuel G
authors.initials: MG
authors.lastname: Roig
authors.affiliation: Chembiotech Laboratorios, Kyrewood House, Tenbury Wells, Worcestershire WR15 8SG, UK.
authors.forename: John F
authors.initials: JF
authors.lastname: Kennedy
authors.affiliation: Departamento de Bioquimica y Biologia Molecular, Universidad de Salamanca, 37007 Salamanca, Spain.
authors.forename: Valery L
authors.initials: VL
authors.lastname: Shnyrov
mesh_terms: D002942:Circular Dichroism; D029865:Cytisus / Q000201:enzymology*; D004795:Enzyme Stability; D009195:Peroxidase / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D009195:Peroxidase
keywords: Circular dichroism; Cytisus multiflorus; Intrinsic fluorescence; Peroxidase; Protein stability