The purification and refolding of the recombinant horseradish peroxidase produce by E. coli transformants are described. The recombinant enzyme is of 34 kDa and has an isozyme spectrum similar to Sigma type VI horseradish peroxidase. The specific activity of the refolded peroxidase is of about 2000 U/mg with ABTS as a substrate. The recombinant and native enzyme are similar with respect to their catalytic properties in the reaction of enhanced chemiluminescence. Operational and thermal stability of the refolded peroxidase is two to three times lower than for the native one.

Peroxidase-dependent cooxidation of 4-iodophenol and 4-aminoantipyrine (AAP), and 4-iodophenol and luminol has been studied The influence of polyclonal and monoclonal antibodies (polyAb and monoAb) to horseradish peroxidase on the kinetics of the reaction was investigated over a wide range of H2O2 concentrations (0.01-10.0 Mm). Three types of MonoAb (2C, 3E, and 9D) in the concentration range 0.05-500 Nm inhibited the cooxidation of AAP and 4-iodophenol at H2O2 concentrations above 1.0 Mm but activated the cooxidation of luminol and 4-iodophenol. Binary and ternary mixtures of monoAb activated the cooxidation of AAP and 4-iodophenol at the same H2O2 concentrations but inhibited or did not affect the cooxidation of luminol and 4-iodophenol. PolyAb activated the cooxidation of AAP and 4-iodophenol more effectively and only slightly activated (or inhibited) the cooxidation of luminol and 4-iodophenol. PolyAb diminished rate constants for the interaction of peroxidase active intermediates, E1 and E2, with 4-iodophenol, AAP, and luminol. Possible mechanisms for the influence of the antibodies on the cooxidation of the substrate pairs are discussed

Binding of the opioid antagonist [H-3]naloxone to immunocompetent cells of Fl(CBA x C57Bl/6) mice has been studied The ligand binding in medium 199 was found to be reversible, saturable, maximal within 15-20 min at 37-degrees-C, and stereospecific for mu-type opioid receptors. The isotherm for specific ligand binding on lymphocytes appears to be positively cooperative (n(H) = 2.3, IC50 = 5 nM). Mitogenic activation (concanavalin A, staphylococcal enterotoxin A, Phytolacca mitogen) increases the number of binding sites, and antigenic activation by ovalbumin changes the binding parameters. The distribution of [H-3]naloxone binding sites on cells of different immune system organs has been investigated, their maximal number being found on lymphocytes of lymphatic nodes and marrow cells. Both T- and non-T-cells play a role in ligand binding.

Properties of four types of monoclonal antibodies to horse-radish peroxidase were investigated. The dissociation constants and molecular-weight composition of the immune complexes were determined. The antibodies are shown to be directed to different epitopes on the polypeptide chain. Results of the theoretical prediction of the epitope localisation are presented. The interaction between the antibodies and peroxidase isozymes were studied.

Some biochemical and catalytic properties of peroxidase from Arthromyces ramosus (EC 1.11.7.1) in chemiluminescent reaction of luminol oxidation by hydrogen peroxide were investigated. The second order rate constants were determined by the stopped-flow technique. Optimal conditions to quantity the enzyme were found, the detection limit being 5.10(-13) M. The peroxidase was used as a marker in the human IgG immunoassay.

In this communication some of the advantages and constraints in the use of ELISA (enzyme-linked immunosorbent assay) procedures to evaluate antigen-antibody dissociation constants (Kd) are discussed and experimental conditions under which the effective Kd is close to the true value are proposed. Interactions between horseradish peroxidase (POD), human myoglobin and insulin with mono- and polyclonal antibodies (McAb and PcAb) were used to demonstrate that ELISA can be used to determine the average Kd, characterizing the interaction between antigens and PcAb. The Kd values obtained by ELISA were similar to those determined by luminescent immuno-cofactor analysis (LICA).