| Preparation and Properties of A Chloroperoxidase-like CatalyticAntibody Doctor Candidate: Qi ChaoAdvisor: Prof. Zhang Yu-jinglodination of free tyrosine and tyrosine in protein is mediated by an enzyme present in thyroid homogenates and subcellular fractions. Alexander has demonstrated an iodide poroxidase in animal thyroid tissue and believes this enzyme is responsible for the iodination reactions through formation of an oxidized iodinating intermediate. This enzyme is chloroperoxidase(CPO)(EC1. 11. 10). It is a heme protein of Mr -42000Da containing one ferriprotoporphyrin IX per molecule. The enzyme uses hydrogen peroxide as the oxidant for the peroxidative formation of a carbon-halogen bond between Cl, Br-, Y and a suitable halogen acceptor. But it is unable to utilize F for this reaction.lodination of tyrosine residues in thyroglobulin, or in its subunits, is one of the essential steps in the formation of thyroxine in the thyroid gland. Iodide can be rapidly bound as 3-iodotyrisine, 3, 5-diiodotyrosine, and thyroxine during the chloroperoxidase-catalyzed iodination. Thus, theenzyme plays an important role in the metabolism and physiological function of thyroxine. Deficiency of chloropeeroxidase will result in some serious thyroid diseases.Because of the low abundance of chloroperoxidase in thyroid tissue and the instability of the catalytic activity in detergent, etc, attempts to isolate and purify chloroperoxidase by conventional techniques of protein chemistry, have been difficult. For this reason, the studies of biochemical properties and structure-function of the enzyme have been impeded. Therefore, it will be of considerable interest to prepare a catalytic antibody with chloroperoxidase activity for mechanistic studies and therapeutic application. In addition, the antibody-catalyzed selective halogenation also will have broad prospects for organic synthesis, etc.In short, condensation of o-nitrobenzaldehyde and pyrrolefollowed by reduction of the meso-tetra(o-nitrophenyl)porphyrin led to a satisfactory yield of meso-tetra(o-aminophenyl) porphyrin, H2TamPP. Thin-layer chromatography on silica gel gave excellent separation of four components which, in ratio 1:2:4:1, were presumed to be the four atropisomers in statistical abundance. With Rf values based on their expected polarity and considering the relative amounts of the individual atropisomers these are provisionally assigned as , with the most polar tetra- a -atropisomer, moving most slowly. Isolation of a, a , a , a -H2TamPP by silica gel '.column chromatography afforted gram quantities of product. The mixture containing the remaining three atropiaomers was reequilibrated in boiling tobuene followed by chromatography to isolate more of the a , a , a , a -atropisomer. Repetition of these steps leads to ultimate conversion of nearly all of the H2TamPP into the desired a , a , a , a - H2TamPP. The four amino atropisomers are rather stable in solution at 25?C. The amino groups of H2TamPP are also more reactive. The a , a , a , a -atropisomer of H2TamPP could be further modified and the configurational energy barrier raised through reaction with phenylacetyl chloride forming amide. Phenylacetyl chloride gave 72% yield of meso-tetra( a , a , a , a -o- phenylacetylamide phenyl)porphyrin. McAb was raised against meso-Tetra( a , a , a , a -o-phenylacetylamide phenyl)porphyrin through hybridoma methology.Small molecule meso-Tetra (a , a-, a, a -0-phenylacetylamide phenyl)porphyrin'could be used as complete antigen to immunize Balb/c mice and induce McAb 1F2. The purity of McAb 1F2 was indicated very high by MALDI/TOF MS and SDS-PAGE. The subtype of McAb 1F2 is IgG2a. A relative molecular weight of McAb 1F2 was determined to be 156678.8Da by MALDI/TOF MS. The intensities of UV and CD spectra over a pH range between 6 and 12 almost remain constant, which reveals that the tight binding of Fe porphyrin and McAb 1F2 and the high stability of abzyme. The chloroperoxidase activity of McAb lF2-Fe porphyrin complex appears thermostable until 60C...
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