Studies of the reactivity of artificial peroxidase-like hemoproteins based on antibodies elicited against a specifically designed ortho-carboxy substituted tetraarylporphyrin

• Published in: FEBS letters Vol. 443; no. 2; pp. 229 - 234
• Main Authors: de Lauzon, Solange, Desfosses, Bernard, Mansuy, Daniel, Mahy, Jean-Pierre
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 29.01.1999
• Subjects: Antibodies - metabolism; Artificial hemoprotein; Catalysis; Catalytic antibody; Enzyme Stability; Hemeproteins - metabolism; Hydrogen-Ion Concentration; Kinetics; Oxidation-Reduction; Peroxidase; Peroxidases - metabolism; Porphyrin; Porphyrins - immunology; Substrate Specificity; Catalytic antibody; Artificial hemoprotein; Peroxidase; Porphyrin
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)01703-7

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)-α,α,α,β- meso- tetrakis( 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°C. Secondly, this complex is able to use not only H 2O 2 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 H 2O 2 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 H 2O 2 in the presence of either 13G10-Fe(ToCPP) or Fe(ToCPP) shows that K m(H 2O 2) 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 H 2O 2 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.