Determination of proteolytic hydrolysis of thyroglobulin

A method for the determination of the free thyronine- and tyrosine-like amino acids in the thyroidal protein thyroglobulin is presented. The compounds of interest are monoiodotyrosine, diiodotyrosine, thyronine, diiodothyronine, triiodothyronine and tetraiodothyronine. The extent of proteolysis was followed by high-performance liquid chromatographic monitoring of both the remaining peptides and the formation of the free thyroidal amino acids. Total hydrolysis was achieved by a combination of proteolytic enzymes. A number of enzymes were tested, such as trypsin, chymotrypsin, pronase, aminopeptidase-M, carboxypeptidase-A, carboxypeptidase-P and carboxypeptidase-Y. The best combination turned out to be pronase followed by aminopeptidase-M. The relative amounts of the enzymes, with respect to the substrate thyroglobulin, and the time of incubation were optimized to achieve total proteolysis in 4 h. The method was applied successfully to samples from a toxicological experiment with sodium bromide.