IDENTIFICATION OF HORMONOGENIC TYROSINES IN FRAGMENT-1218-1591 OF BOVINE THYROGLOBULIN BY MASS-SPECTROMETRY - HORMONOGENIC ACCEPTOR TYR-1291 AND DONOR TYR-1375

A fragment of bovine thyroglobulin encompassing residues 1218-1591 was prepared by limited proteolysis with thermolysin and continuous-eluti on polyacrylamide gel electrophoresis in SDS. The reduced and carboxym ethylated peptide was digested with endoproteinase Asp-N and fractiona ted by reverse-phase high performance liquid chromatography. The fract ions were analyzed by electrospray and fast atom bombardment mass spec trometry in combination with Edman degradation. The post-translational modifications of all seven tyrosyl residues of the fragment were char acterized at an unprecedented level of definition. The analysis reveal ed the formation of: 1) monoiodotyrosine from tyrosine 1234; 2) monoio dotyrosine, diiodotyrosine, triiodothyronine (T-3), and tetraiodothyro nine (thyroxine, T-4) from tyrosine 1291; and 3) monoiodotyrosine, dii odotyrosine, and dehydroalanine from tyrosine 1375. Iodothyronine form ation from tyrosine 1291 accounted for 10% of total T-4 of thyroglobul in (0.30 mol of T-4/mol of 660-kDa thyroglobulin), and 8% of total T-3 (0.08 mol of T-3/mol of thyroglobulin). This is the first documentati on of the hormonogenic nature of tyrosine 1291 of bovine thyroglobulin , as thyroxine formation at a corresponding site was so far reported o nly in rabbit, guinea pig, and turtle thyroglobulin. This is also the first direct identification of tyrosine 1375 of bovine thyroglobulin a s a donor residue. It is suggested that tyrosyl residues 1291 and 1375 may support together the function of an independent hormonogenic doma in in the mid-portion of the polypeptide chain of thyroglobulin.