The partial purification and properties of thyroxine dehalogenase

Abstract

A method for a 60-90-fold purification of the enzyme, thyroxine dehalogenase from rabbit skeletal muscle, has been described. Enzymic deiodination and debromination of I131- and Br82-labelled thyroxine and some iodo- and bromothyronines, thyroacetic acids and tyrosines have been described. The enzyme was unstable on storage and was not resistant to freezing and thawing or to freeze-drying. Thyroxine dehyalgenase was activated by Fe2+ ions and by flavins (flavin mononucleotide, flavinadenine dinucleotide and ribo-flavin); the activation by these factors appears to be additive, but the activators acting singly lead to formation of different products. Relationship between amount of enzyme and initial velocity of deiodination was linear only if a correction were applied for the "instability" of thyroxine observed with small amounts of the enzyme. Inhibition of the enzyme by Hg2+ ions could be reversed to about 40% of the original activity by the addition of excess of flavin mononucleotide. The pH optima were around 6.4 for o-dihalogenated phenols and around 7.3 for o-monohalogenated phenols. Of all the compounds tested the enzyme had the highest affinity for thyroxine, which was about four to five times that for tri-iodothyronine and 20 times that for 3.5-di-iodotyrosine. D-Thyroxine was readily deiodinated. Km values have been given for some iodinated and brominated thyronines, thyroacetic acids and tyrosines. The relative affinities of thyroxine dehalogenase for thyroxine and related compounds closely resemble those of serum thyroxine-binding protein. Addition of blood serum suppressed enzymic dehalogenation of thyroxine. The extent of inhibition thus caused was equal to the fraction of thyroxine bound to serum thyroxine-binding protein and albumin. Thyroxine dehalogenase can attack only free or non-protein-linked substrate. A new method based on this property for studying binding of thyroxine to proteins is proposed. The major product of deiodination was iodide. About 20-25% of the iodine released was incorporated into proteins present in the enzyme preparation; the formation of iodinated proteins required flavin but not Fe2+ ions. 3:5:3''-Triiodothyronine was not one of the important products of deiodination of thyroxine. The findings are discussed in the light of enzyme mechanism and some physiological implications.