Estudo voltamétrico da interação dos íons Zn2+, Cu2+, Cd2+ e Pb2+ com a enzima catalase

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Catalase (CAT), which is an important enzyme that catalyzes the decomposition of hydrogen peroxide (H O ) to water (H O) and oxygen (O ), contains 2 2 2 2 some metal binding sites (Cys, His). The binding of metal ions to CAT can change or stabilize its secondary structure, resulting in its inhibition or activation, respectively. The inhibition of CAT, which occurs when metal ions are in excess, contributes to the increase of the generation of reactive oxygen species, resulting in oxidative stress. In 2+ 2+ 2+ 2+ this work, the interaction of Zn , Cu , Cd and Pb ions with catalase was investigated, near physiological conditions, by using differential pulse anodic stripping voltammetry (DPASV), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The developed methodology allows the direct determination of quantitative data (stoichiometry, dissociation constant (K ) and standard reduction d 0 2+ potential (E )) of metal ion-CAT systems and the theoretical identification of Zn , 2+ 2+ 2+ Cu , Cd e Pb binding sites. The acquired data will be useful to understand the 2+ 2+ 2+ role of metal ions in the oxidative stress mediated by catalase. Zn , Cu , Cd and 2+ Pb binding sites were identified in CAT, considering only the sites containing hystidine and cysteine residues without steric impediment, through comparison of physical-chemical parameters (MM, pI e MH) of standard peptides that bind zinc, copper, cadmium and lead ions and those from the peptides generated by the cleavage of CAT with GluC2 and V8-proteinase enzymes. The formation of a stable 2+ complex between zinc and catalase, with a stoichiometry of 20 Zn ions per one -11 -1 CAT, was attested by using DPASV (K = 1,62 x 10 mol L ) and CV (K = 2,98 x d d -11 -1 10 mol L ). The formation of a stable complex between copper and catalase, with a 2+ stoichiometry of 16 Cu ions per one CAT molecule, was attested by the decrease of oxidation/reduction currents of the metal with incremental additions of catalase and -10 -1 -10 by the dissociation constant (K = 1,73 x 10 mol L - DPASV, K = 1,25 x 10 mol d d -1 -10 -1 L - CV, K = 1,81 x 10 mol L DPV). The small difference between theoretical d 2+ 2+ : 1 CAT; 24 Cu : 1CAT) and experimental stoichiometry values found for (28 Zn these complexes can be assigned to the steric impediment of some sites identified in the theoretical studies, caused by the size of the enzyme and its conformation on the 2+ electrode surface and by the pH of the medium. The calculated K values for Zn d 2+ CAT and Cu CAT complexes suggest the involvement of these two metal ions in the inhibition of catalase by the formation of a stable complex with the enzyme, 2+ 2+ CAT and Pb CAT contributing in this way to the oxidative stress. For Cd systems, it was not possible to determine the stoichiometry and dissociation constant, because it was observed a small decrease (the oxidation current was not completely eliminated) of the oxidation currents of cadmium and lead in the presence of CAT in the voltammograms, taking to the following hypotheses: the binding sites for these metals ions have already been occupied, there is a steric impediment for the bindings, the interactions are not stable, or the concentration of metal ions in solution does not allow the interactions with catalase.

ASSUNTO(S)

voltametria catalase íons metálicos quimica estresse oxidativo

ACESSO AO ARTIGO

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