Difference in susceptibility of tyrosine residue to oxidative iodination between a thioredoxin box region and a hormonogenic region

Peptide fragments, isolated from proteolytic cleavage of thyroglobulin at s pecific sites, were examined for the iodination of tyrosine residues. The 5 0 kDa polypeptide, which was prepared from digestion of bovine thyroglobuli n and continuous preparative SIDS-PAGE, was subjected to reduction with DTT and alkylation with iodoacetic acid to generate S-carboxymethylated peptid e derivative, which was further hydrolysed by endoproteinase-Asp N. Peptide products were separated by RP-HPLC, and each fraction was analyzed by LC/E SI-MS and MALDI-MS analyses. Based on the specificity of endoproteinase Asp -N and the mass spectra data, a peptide fragment turned out to correspond t o a peptide, DALGCVKCPEGSYFQ (1438-1452), characterized by the presence of a thioredoxin box (CVKC) and a tyrosine residue. In addition, another pepti de fragment (1453-1465) containing a thioredoxin box (CIPC) and a tyrosine residue was also observed. However, any evidence of iodination of the tyros ine residue present in these peptides was not provided. Meanwhile, tyrosine residues in the peptides, DVEEALAGKYLAGRFA (1366-1381) and DYSGLLLAFQVFLL (1290-1303) were found to be iodinated; mono- or diiodinated tyrosine resid ues, characteristic of a hormogenic site, existed in both peptides. in addi tion, the tyrosine residue in the peptide (1218-1252), corresponding to a h ormonogenic site was also iodinated. Thus, there was a sharp difference of the susceptibility to oxidative iodination between the tyrosine residue in a hormonogenic site and that in a thioredoxin region. From these results, i t is suggested that polypeptide region adjacent to tyrosine residues may go vern the susceptibility of tyrosine to oxidative iodination.