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Volume 272, Number 1, Issue of January 3, 1997 pp. 639-646
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of Hormonogenic Tyrosines in Fragment 1218-1591 of Bovine Thyroglobulin by Mass Spectrometry
HORMONOGENIC ACCEPTOR TYR-1291 AND DONOR TYR-1375

(Received for publication, September 11, 1996)

Fabrizio Gentile , Pasquale Ferranti ^¶ , Gianfranco Mamone ^¶ , Antonio Malorni ^¶ and Gaetano Salvatore

From the  Centro di Endocrinologia e Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, University of Naples "Federico II", 80131 Naples, Italy and the ^¶ Servizio di Spettrometria di Massa del Consiglio Nazionale delle Ricerche, 80131 Naples, Italy

A fragment of bovine thyroglobulin encompassing residues 1218-1591 was prepared by limited proteolysis with thermolysin and continuous-elution polyacrylamide gel electrophoresis in SDS. The reduced and carboxymethylated peptide was digested with endoproteinase Asp-N and fractionated by reverse-phase high performance liquid chromatography. The fractions were analyzed by electrospray and fast atom bombardment mass spectrometry in combination with Edman degradation. The post-translational modifications of all seven tyrosyl residues of the fragment were characterized at an unprecedented level of definition. The analysis revealed the formation of: 1) monoiodotyrosine from tyrosine 1234; 2) monoiodotyrosine, diiodotyrosine, triiodothyronine (T₃), and tetraiodothyronine (thyroxine, T₄) from tyrosine 1291; and 3) monoiodotyrosine, diiodotyrosine, and dehydroalanine from tyrosine 1375. Iodothyronine formation from tyrosine 1291 accounted for 10% of total T₄ of thyroglobulin (0.30 mol of T₄/mol of 660-kDa thyroglobulin), and 8% of total T₃ (0.08 mol of T₃/mol of thyroglobulin). This is the first documentation of the hormonogenic nature of tyrosine 1291 of bovine thyroglobulin, as thyroxine formation at a corresponding site was so far reported only in rabbit, guinea pig, and turtle thyroglobulin. This is also the first direct identification of tyrosine 1375 of bovine thyroglobulin as a donor residue. It is suggested that tyrosyl residues 1291 and 1375 may support together the function of an independent hormonogenic domain in the mid-portion of the polypeptide chain of thyroglobulin.

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