In most highly structured native proteins, as well as in thyroglobulin, the reactivity in vitro of the various tyrosyl residues toward iodine is widely different. The present work demonstrates that of nearly 70 tyrosyl residues present in rat thyroglobulin, there is one, residue number 5 from the NH2-terminal end, which has in vivo the highest affinity toward iodine, being the first one to be iodinated. In fact, when 6-(n-propyl)-2-thiouracil (PTU)-treated, iodine-deficient animals were injected with 125I and killed shortly after, we isolated from thyroid glands poorly iodinated thyroglobulin (about 1 iodine atom/thyroglobulin molecule), nearly 90% of the radioactivity of which was found as monoiodotyrosine. Although CNBr cleavage of this protein gave several fragments after gel electrophoresis only one of these, with apparent mass 27,000 Da, contained 125I. This fragment was isolated and fully characterized. Twelve cycles of automated Edman degradation were performed; the sequence found, i.e. N-I-F-E-X-Q-V-X-A-Q-X-L, indicated that the 27,000-Da fragment is the NH2 terminus of thyroglobulin. This portion of the polypeptide chain contains several tyrosyl residues which may well all be potentially involved in the early iodination of the protein. The observation that the removal of seven amino acids from the NH2 terminus is accompanied (at the fifth step) by the total disappearance of radioactivity in the resulting shortened peptide suggested that the fifth residue was the only one iodinated under these conditions. A second, more quantitative experiment was performed on thyroglobulin obtained from 6-(n-propyl)-2-thiouracil-treated animals whose death was postponed 24 h after the injection of 125I. In this case the radioactivity was found not only in a single CNBr fragment (27,000 Da) but also in other discrete species of lower molecular mass. The mixture of these peptides was subjected to seven steps of manual Edman degradation. Fragments before and after partial degradation were run in parallel on a polyacrylamide gel and the distribution of 125I compared. Besides some change in the background, the two profiles were identical except for the absence of the 27,000-Da species. This proves that all the 125I present in the 27,000-Da species was localized at the fifth residue, the same site at which the hormone molecule is preferentially synthesized under normal conditions. This result is not unexpected and is in accord with the known properties of thyroglobulin which has a polypeptide chain designed for efficient synthesis of the hormone even at low levels of iodination.
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