Studies on the Biosynthesis of Thyroid Hormone: Bulk Fractionation Procedures and Absorbency Spectra of Calf Thyroid, Iodide Peroxidase-Tyrosine Iodinase; Stabilization of Enzyme and Polymeric Structure

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Abstract
Purification and properties of calf thyroid iodide peroxidase-tyrosine iodinase have been investigated. The enzyme, solubilized from cell particles by trypsin-deoxycholate treatment, can be concentrated by (NH4)2SO4 precipitation, by precipitation at pH 4.8, by differential precipitation with detergents, or by sucrose density gradient centrifugation. Heme protein absorbing maximally at 409 xμ is present in the partially purified preparations of the soluble iodinating enzyme. The heme protein forms carbon monoxide (420–421 mμ) and cyanide (427 mμ) complexes, and the Soret band is moved to 423 mμ by reducing agents. A variety of compounds that inhibit iodotyrosine formation by the enzyme shift the Soret band to the reduced form. H2O2, which inactivates the enzyme, obliterates the Soret band. The absorbency spectrum of the enzyme is distinct from hemoglobin, and neither hemoglobin nor hematin is significantly active in the iodinating assay. Although the most purified preparations contain heme protein, the ratio O.D. 410/O.D. 280 is low. The enzyme is active in peroxidation of I- to I8-. The I3-, or I3 in equilibrium with I3, reacts quantitatively with tyrosine to form iodotyrosine. Partially purified soluble calf thyroid iodide peroxidase-tyrosine iodinase is gradually inactivated in low molarity (0.005M) sodium phosphate buffer, but is protected by higher concentrations (0.1M) of this buffer and several other solutes. Enzyme activity lost during storage in low strength buffer is partially restored by addition of higher strength buffer. During chromatography of enzyme in 0.005M sodium phosphate buffer, enzyme activity is resolved into a peak appearing with the void volume, and 2 retarded fractions which have approximate molecular weights of 90,000 and 17,000. Addition of 0.01 % Triton increases the proportion of enzyme of the smaller size. Both forms of enzyme contain heme protein and are active in iodotyrosine formation. The data indicate the enzyme exists as a polymer (possibly tetramer) and monomer. Conditions favoring formation of the monomer are associated with enzyme inactivation, indicating the monomeric form is more labile than the polymeric form. (Endocrinology88: 470, 1971)

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