here that antibodies elicited against a more sophisticated hapAbstract In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(Ill)-ten, the ironllll) -meso-c~,c~,c¢,~-tetrakis-orthocarboxyphenylct,ct,ct,[~-mesotetrakis-orthocarboxyphenyl-porphyrin (Feporphyrin (Fe(ToCPP), 1) bind 1 with the highest affinity (ToCPP))-KLH conjugates. Monocional antibodies have been reported so far for iron-porphyrins. A detailed spectroscopic produced by the hybridoma technology. Three antibodies, 2 IgG1 and kinetic study of the resulting IgG-1 complexes shows that and 1 IgG2a, were found to bind both Fe(ToCPP) and the free they are either high-spin or low-spin Fe(III) complexes and base ToCPPH2 with similar binding constants. None of those only the high spin complexes exhibit a peroxidase activity 5-antibodies was found to bind tetraphenylporphyrin. Those results fold higher than 1 alone and stable until complete conversion suggest that the recognition of Fe(ToCPP) by the antibodies was of the reducing cosubstrate. mainly due to the binding of the carboxylate groups to some amino acid residues of the protein. True Kd values of 2.9 × 10 -9 M and 5.5 × 10 -9 M have been determined for the two IgGi-2. Materials and methods Fe(ToCPP) complexes. Those values are the best ones ever reported for iron-porphyrin-antibody complexes. UV-vis. studies have shown that the two IgGI-Fe(ToCPP) complexes were high- l. Synthesis ~!/' meso-o;,o;,ct,~-tetrakis-orthoearhox.vphenyl-porphvrin 2 and its iron(Ill) complexes 1spin hexacoordinate iron(Ill) complexes, with no amino acid Condensation of ortho-carbomethoxybenzaldehyde with pyrrole acresidue binding the iron, whereas the IgG2a-Fe(ToCPP)complex cording to an already described procedure [21] gave a mixture of 4 was a low-spin hexacoordinate iron(III) complex with two strong atropoisomers of meso-tetrakis-ortho-carbomethoxyphenyl-porphyrligands binding the iron atom. Both IgGI-Fe(ToCPP) complexes ins [22]. The ct,~,t~,[I atropoisomer was isolated by column chromawere found to catalyze the oxidation of 2,2'-azinobis (3-tography on silica gel (eluent: CH2C1.2/Et,_O: 80/20) and identified by ethylbenzothiazoline-6-sulfonic acid (ABTS) 5-fold more effiits UV-visible and 1H NMR spectra which were identical to those ciently than Fe(ToCPP) alone whereas the binding of lgG2a to reported in the literature [22]. 2 was then obtained after hydrolysis this iron-porphyrin had no effect on its catalytic activity, kcat of the methyl esters at 40°C in 50% H2SO4 (v/~ Preparation ~[ monoelonal antibodies1 was activated by N-hydroxy-succinimide and covalently attached to KLH (keyhole limpet hemocyanin) and to BSA (bovine serum albumin) in PBS pH 7.5. The conjugates were then purified by column 1. Introduction chromatography on Biogel P10. Hapten-carrier protein ratios determined spectrophotometrically were in the range of 15/1 to 20/1. Two In the last few years, many monoclonal antibodies elicited 5 week old, female BALB/c mice were immunized with the haptenagainst ca...

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-A,A,A,β-meso-tetrakis(ortho-carboxyphenyl)porphyrin {A,A,A,β-Fe [(o-COOHPh) 4 -porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H 2 O 2 . In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.