Mahy, J.-P.

FEBS Lett. 1996, 395, 73-76, 10.1016/0014-5793(96)01006-X

In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)α,α,α,β‐mesotetrakis‐orthocarboxyphenyl‐porphyrin (Fe(ToCPP))‐KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG, and 1 IgG2a, were found to bind both Fe(ToCPP) and the free base ToCPPH2 with similar binding constants. None of those antibodies was found to bind tetraphenylporphyrin. Those results suggest that the recognition of Fe(ToCPP) by the antibodies was mainly due to the binding of the carboxylate groups to some amino acid residues of the protein. True K d values of 2.9 × 10−9 M and 5.5 × 10−9 M have been determined for the two IgG1‐Fe(ToCPP) complexes. Those values are the best ones ever reported for iron‐porphyrin‐antibody complexes. UV‐vis. studies have shown that the two IgG1‐Fe(ToCPP) complexes were highspin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2α‐Fe(ToCPP) complex was a low‐spin hexacoordinate iron(III) complex with two strong ligands binding the iron atom. Both IgG1 ‐Fe(ToCPP) complexes were found to catalyze the oxidation of 2,2′‐azinobis (3ethylbenzothiazoline‐6‐sulfonic acid (ABTS) 5‐fold more efficiently than Fe(ToCPP) alone whereas the binding of IgG2a to this iron‐porphyrin had no effect on its catalytic activity. k cat values of 100 min−1 and 63 min−1 and k cat/K m. values of 105 M−1 s−1 and 119 M−1 s−1 have been found respectively for the two IgG1‐Fe(ToCPP) complexes.

Mahy, J.-P.

FEBS Lett. 1999, 443, 229-234, 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 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 H2O2 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 K m(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.

Imanaka, T.

FEBS Lett. 1995, 375, 273-276, 10.1016/0014-5793(95)01224-3

In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin. TCPP (meso‐tetrakis(4‐carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03‐1, 13‐1), that produce monoclonal antibody against TCPP, were obtained. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03‐1 and Mab13‐1 have specific interaction with TCPP. Furthermore, the recombinant antibody L chain from Mab13‐1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS (2,2‐azinobis‐3‐ethylbenzthiazolin‐6‐sulfonic acid) but not with catechol. This new catalytic antibody was extremely thermostable. Optimum temperature of the peroxidase reaction by the complex of 13‐1L chain and TCPP Fe(III) was 90°C, while that the TCPP Fe(III) alone was 60°C.