Peroxydase particulaire thyroïdienne

Abstract

Summary o 1. A procedure for the solubilization and purification of a peroxidase from hog thyroid tissue is described. The particulate fraction is previously treated with trypsin under conditions wich do not release the enzyme activity. Then the activity is released by digitonin treatment. Purifications by DEAE-cellulose column chromatography yields a final product which, although not homogenous, represented an about 700 fold purification over the starting material. Purer preparations (1500 fold purification) are obtained but are quite unstable. 2. The molecular weight of thyroid peroxidase, estimated by gel filtration techniques, is 103 000. The sedimentation coefficient by sucrose gradient ultracentrifugation is 5.2 S. 3. The heme content, estimated by the ratio A.410/A.280 (Rz), varies from 0.04 to 0.11 for the purest preparations. The Rz of this peroxidase appears therefore to be very low. 4. When supplemented with a H2O2 generating system, glucose/glucose-oxidase, the purified enzyme catalyzes the iodination of free tyrosine, monoiodotyrosine, lysozyme and thyroglobulin ; iodide oxidation by this enzyme is linear, in the absence of serum-albumin, for at least two minutes. 5. « km(M, I−) in these reactions was measured. The values obtained for the iodination reactions are about 60 to 600 times lower (0,8 × 10−4 to 0,6 × 10−5 M) than those measured for I2 formation (0,6 × 10−2 M). These results agree with the possibility that, for the reactions of iodination of free or linked tyrosine residues only one iodide molecule is linked to the enzyme, whereas for I2 formation the peroxidase forms a ternary complex with two halogen molecules. 6. « km(M, I−) for lysozyme and thyroglobulin iodination and for I2 formation were also measured with horse radish peroxidase. The value of these « km , when compared with those obtained for the thyroid enzyme, show that the thyroid peroxidase is relatively much more specific for protein iodination than for I2 formation.