ACTIVE-SITE TOPOLOGY OF ARTIFICIAL PEROXIDASE-LIKE HEMOPROTEINS BASEDON ANTIBODIES CONSTRUCTED FROM A SPECIFICALLY DESIGNED ORTHO-CARBOXY-SUBSTITUTED TETRAARYLPORPHYRIN

The topology of the binding site has been studied for two monoclonal a ntibodies 13G10 and 14H7, elicited against ,alpha-meso-tetrakis(ortho- carboxyphenyl)porphyrin {alpha,alpha,alpha,beta-Fe [(o-COOHPh)(4)-porp hyrin]}, and which exhibit in the presence of this alpha,alpha,alpha,b eta-Fe[(o-COOHPh)(4)-porphyrin] cofactor a peroxidase activity. A comp arison of the dissociation constants of the complexes of 13G10 and 14H 7 with various tetra-aryl-substituted porphyrin has shown that: (a) th e central iron(III) atom of alpha,alpha,alpha,beta-Fe[(o-COOHPh)(4)-po rphyrin] is not recognized by either of the two antibodies; and (b) th e ortho-carboxylate substituents of the meso-phenyl rings of alpha,alp ha,alpha,beta-Fe[(o-COOHPh)(4)-porphyrin] are essential for the recogn ition of the porphyrin by 13G10 and 14H7. Measurement of the dissociat ion constants for the complexes of 13G10 and 14H7 with the four atropo isomers of (o-COOHPh)(4)-porphyrinH(2) as well as mono- and di-ortho-c arboxyphenyl-substituted porphyrins suggests that the three carboxylat es in the a, alpha, beta position are recognized by both 13G10 and 14H 7 with the two in the alpha, beta positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G 10 and 14H7 has roughly two-thirds of the alpha,alpha,alpha,beta-Fe[(o -COOHPh)(4)-porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts a s an axial ligand to the iron atom. Chemical modification of lysine, h istidine, tryptophan and arginine residues has shown that only modific ation of arginine residues causes a decrease in both the binding of al pha,alpha,alpha,beta-Fe[(o-COOHPh)(4)-porphyrin] and the peroxidase ac tivity of both antibodies. Consequently, at least one of the carboxyla tes of the hapten is bound to an arginine residue and no amino acids s uch as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H2O2. In addition, the am ino 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 ac ids in the complementary determining regions which could bind other ca rboxylates through a network of H bonds.