Purification and structural stability of white Spanish broom (Cytisus multiflorus) peroxidase.

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.