ABSTRACT
Peroxidase activity of a complex of water-soluble cationic metalloporphyrin with anti-cationic porphyrin antibody is reported. Antibody 12E11G, which was prepared by immunization with a conjugate of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridyl)porphine iodide (3MPy1C), bound to tetramethylpyridylporphyrin iron complex (FeIII-TMPyP) with the dissociation constant of 2.6 x 10(-7) M. The complex of antibody 12E11G with FeIII-TMPyP catalyzed oxidation of pyrogallol, catechol, and guaiacol. A Lineweaver-Burk plot for the oxidation of pyrogallol catalyzed by the FeIII-TMPyP-antibody complex showed Km=8.6 mM and kcat=680 min(-1). Under the same conditions, Km and kcat for horseradish peroxidase (HRP) were 0.8 mM and 1750 min(-1), respectively. Although the binding interaction of the antibody to the substrates was one order lower than that of native HRP, the peroxidase activity of this system was in the same order of magnitude as that of HRP.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigen-Antibody Complex/chemistry , Metalloporphyrins/chemistry , Peroxidases/chemistry , Antibodies, Monoclonal/metabolism , Antibody Specificity , Antigen-Antibody Complex/metabolism , Catalysis , Cations , Kinetics , Metalloporphyrins/immunology , Molecular Structure , Oxidation-Reduction , Peroxidases/metabolism , Pyrogallol/chemistryABSTRACT
In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)-alpha,alpha,alpha,beta-mesotetrakis-orthocarboxypheny l-porphyrin (Fe-(ToCPP))-KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG1 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 Kd values of 2.9 x 10(-9) M and 5.5 x 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 high-spin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2a-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 (3-ethylbenzothiazoline-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. kcat values of 100 min(-1) and 63 min(-1) and kcat/Km values of 105 M(-1) s(-1) and 119 M(-1) s(-1) have been found respectively for the two IgG1-Fe(ToCPP) complexes.
Subject(s)
Antibodies, Catalytic/metabolism , Heme/metabolism , Hemeproteins/metabolism , Metalloporphyrins/metabolism , Peroxidases/metabolism , Animals , Antibodies, Monoclonal , Benzothiazoles , Female , Haptens/immunology , Hemeproteins/chemical synthesis , Hemeproteins/immunology , Hydrogen-Ion Concentration , Immunoglobulin G , Indicators and Reagents , Kinetics , Metalloporphyrins/chemical synthesis , Metalloporphyrins/immunology , Mice , Mice, Inbred BALB C , Models, Chemical , Oxidation-Reduction , Sulfonic Acids/metabolismABSTRACT
Monoclonal antibodies against Pd-coproporphyrin I have been obtained. The antibody specificity for free as well as for conjugated Pd-coproporphyrin I is characterized. Affinity constants are estimated for 3 monoclonal antibodies effectively interacting with free Pd-coproporphyrin I. A comparative study on the binding of monoclonal antibodies with analogues and derivatives of Pd-coproporphyrin I has revealed that the antigen is mainly located inside the antibody paratope. The protein adjoins complementary to the metalloporphyrin in such a manner that antibodies obtained discern only isomer I, and to some degree, isomer III of coproporphyrin.
