Purification and iodinating activity of hog thyroid peroxidase.

Abstract 1. A peroxidase was purified from the particulate fraction of hog thyroid tissue by solubilization with trypsin, chromatography on diethylaminoethyl cellulose, and filtration through Biogel P-100. The final product, although not homogeneous, represented about a 400-fold purification over the starting particulate fraction. 2. The molecular weight of thyroid peroxidase was estimated by gel filtration studies. Elution volumes of proteins of known molecular weight were compared with the elution volume of the peroxidase, the latter being localized by its enzyme activity. The average molecular weight in three determinations was 64,000. 3. An absorption maximum was observed at 410 mµ, suggestive of a hemoprotein. The progressive increase in A410:A280 during the purification procedure suggested that the enzyme activity was associated with a hemoprotein. Inhibitor studies provided further support for this view. 4. When supplemented with H2O2 or, more effectively, with the H2O2-generating system, glucose-glucose oxidase, purified thyroid peroxidase readily catalyzed the iodination of protein, free tyrosine, or free monoiodotyrosine. Iodination was catalyzed over a wide range of iodide concentrations, from l10-7 m to g5 x 10-3 m. In an incubation system containing 1.2 µg of peroxidase, 0.5 mg of purified thyroglobulin, 1.0 mg of glucose, 0.83 µg of glucose oxidase, and I- varying from 50 µm to 4 mm, in 1.0 ml of 0.05 m phosphate buffer, pH 7.0, Vmax was 1.2 mµmoles of I- per min per µg of enzyme protein and Km was 2.5 x 10-4 m I-. 5. No consistent effect of pH on iodination was observed in the range, pH 5 to 7 (the only range tested). Iodination occurred about as readily at pH 5 as at pH 7. 6. During the peroxidase-catalyzed iodination of thyroglobulin or other protein, iodide was rapidly bound as 3-monoiodotyrosine, 3,5-diiodotyrosine, and thyroxine. The best yields of thyroxine were obtained with thyroglobulin as acceptor. The formation of 3',3,5-triiodothyronine was not observed. 7. The iodination of protein by thyroid peroxidase was markedly inhibited by low concentrations of antithyroid drugs. This was true for compounds of the aromatic inhibitor group—resorcinol, p-aminobenzoic acid, sulfathiazole, and others—as well as those of the thioamide group—thiourea, thiouracil, and 1-methyl-2-mercaptoimidazole. Glutathione and thiocyanate were also effective inhibitors, but perchlorate was not. Inhibition by 1-methyl-2-mercaptoimidazole and by thiocyanate was competitive with iodide under certain conditions. 8. Cyanides, and especially azide, were potent inhibitors of thyroid peroxidase, supporting the view that enzyme activity was associated with a hemoprotein. 9. At pH 5.0, iodination of thyroglobulin or of bovine serum albumin with thyroid peroxidase was inhibited at concentrations of I- greater than 1 x 10-4 m. This suggests a possible mechanism for the antithyroid action of excess iodide. However, for reasons which are not yet clear, this inhibition was not observed at pH 7.0. 10. Results obtained with purified thyroid peroxidase support the view that it is unnecessary to postulate the existence of a separate "tyrosine iodinase" in the thyroid gland. Moreover, the findings suggest that a peroxidase may be involved in the coupling reaction as well as in iodination of protein in the thyroid. 11. No evidence was obtained that 3,5-diiodo-4-hydroxyphenylpyruvic acid is an intermediate in thyroxine formation during the iodination of thyroglobulin with thyroid peroxidase. The results of this study favor the view that the coupling of diiodotyrosine to form thyroxine occurs within the matrix of the protein molecule.