SELECTIVITY IN TYROSYL IODINATION SITES IN HUMAN THYROGLOBULIN

Previous studies indicate that when low iodine thyroglobulin (Tg) is i odinated enzymatically with thyroid peroxidase (TPO), the tyrosyl resi dues that are used for the formation of thyroid hormone (hormonogenic sites) are selected for early iodination. The aim of the present study was to assess the relative importance of the substrate (Tg) and the e nzyme (TPO) in the selection of the early tyrosyl sites that undergo i odination. For this purpose, low iodine human Tg (2.0 atoms I per 660, 000 dimer) was iodinated chemically with I-125(3)- and enzymatically w ith TPO + I-125(-) to a matched low level of iodination (similar to 8 added I atoms per molecule). After reduction and allkylation, the two Tg preparations were digested with trypsin, and the tryptic digests we re separated by reverse-phase HPLC into 10 I-125-containing pools. Eac h pool was further fractionated by HPLC to provide purified I-125-pept ides suitable for sequence analysis. From the sequence information and the known amino acid sequence of Tg, it was possible to define the lo cation of the iodinated tyrosyl residues. Surprisingly, almost identic al results were obtained with chemically and enzymatically iodinated T g. Not only were the I-125-peptide maps very similar, but all of the r ecovered I-125 in the purified peptides from both samples was located in only three different tyrosyl sites, 5, 2553, and 2520. Tyr 5 and Ty r 2553 are well-established sites of thyroxine formation, while Tyr 25 20 has previously been proposed by us to be a donor site. Our observat ion that the same hormonogenic tyrosyl sites are iodinated by chemical as web as enzymatic iodination indicates that preferential iodination of hormonogenic sites is dependent primarily on the native structure of Tg. TPO plays a minor role, if any, in the selection of early tyros yl iodination sites in Tg. Consistent with this conclusion was our fin ding that chemical iodination, as well as enzymatic iodination, led to formation of uniformly iodinated Tg, as determined by isopycnic centr ifugation in rubidium chloride. However, we observed a slightly higher diiodotyrosine (HIT) content and a correspondingly lower monoiodotyro sine content in enzymatically iodinated Tg, compared to matched chemic ally iodinated Tg. This was not observed with two other proteins, bovi ne serum albumin and trypsinogen, or with free tyrosine, as substrates for iodination. The same preferential formation of DIT in Tg was, how ever, observed when lactoperoxidase was substituted for TPO. Preferent ial formation of DIT, therefore, appears to involve interaction betwee n Tg and the peroxidase. (C) 1996 Academic Press, Inc.