Studies of the reactivity of artificial peroxidase‐like hemoproteins based on antibodies elicited against a specifically designed <i>ortho</i>‐carboxy substituted tetraarylporphyrin

Abstract

The temperature and pH dependence as well as the selectivity of the peroxidase activity of a complex associating a monoclonal antibody 13G10 with its iron(III)-alpha,alpha,alpha,beta-mesotetrakis(ortho-carboxyphenyl) porphyrin (Fe(ToCPP)) hapten have been studied and compared to those of Fe(ToCPP) alone. It first appears that the peroxidase activity of the 13G10-Fe(ToCPP) complex is remarkably thermostable and remains about 5 times higher than that of Fe(ToCPP) alone until at least 80 degrees C. Secondly, this complex is able to use not only H2O2 as oxidant but also a wide range of hydroperoxides such as alkyl, aralkyl and fatty acid hydroperoxides and catalyze their reduction 2-6-fold faster than Fe(ToCPP) alone. It is also able to catalyze the oxidation by H202 of a variety of reducing cosubstrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB) and 3,3'-dimethoxybenzidine 3-8-fold faster than Fe(ToCPP) alone, the bicyclic aromatic ABTS and TMB being the best reducing cosubstrates. Finally, a pH dependence study, between pH 4.6 and 7.5, of the oxidation of ABTS by H2O2 in the presence of either 13G10-Fe(ToCPP) or Fe(ToCPP) shows that Km(H2O2) values vary very similarly for both catalysts, whereas very different variations are found for the k(cat) values. With Fe(ToCPP) as catalyst the k(cat) value remains constant around 100 min(-1) whereas with the 13G10-Fe(ToCPP) complex, it increases sharply below pH 5 to reach 540 min -1 at pH 4.6. This could be due to the participation of a carboxylic acid side chain of the antibody protein, as a general acid-base catalyst, to the heterolytic cleavage of the O-O bond of H2O2 leading to the highly reactive iron(V)-oxo intermediate in the peroxidase mechanism. Accordingly, the modification of the carboxylic acid residues of antibody 13G10 by glycinamide leads to a 50% decrease of the peroxidase activity of the 13G10-Fe(ToCPP) complex.