Separation of Thyroidal Iodoproteins and Purification of Thyroglobulin by Gel Filtration and Density Gradient Centrifugation.

Thyroglobulin, the major protein component of normal thyroid glands, contains more than 80% of the thyroidal iodine and has been considered the only source of thyroid hormones (1). Aqueous thyroid extracts contain, besides thyroglobulin, other proteins in variable amounts. Essentially, four methods have been used for the characterization of the various components in such extracts and for the purification of thyroglobulin: salting out, electrophoresis, column chromatography, and ultracentrifugation. Salting out with ammonium sulfate or potassium phosphate (2) is the classical method for preparing thyroglobulin. However, salting out preparations are always contaminated with compounds that sediment faster than thyroglobulin, the 19 S ultracentrifugal component of thyroid extracts (3-5). Electrophoretic methods likewise yield preparations of thyroglobulin known to be heterogeneous when analyzed in the analytical ultracentrifuge (4, 6-9). Column chromatography on DEAEcellulose gives highly purified thyroglobulin (4, 10) but the yield is rather poor. The main disadvant.age of this method is that thyroglobulin itself is fractionated on these columns according to its iodine content. The ultracentrifugally homogeneous fraction obtained by this method has a considerably lower iodine content than the whole thyroglobulin (4, 11-13). Ultracentrifugation is one of the most useful analytical procedures, but preparative moving boundary ultracentrifugation (3) requires multiple runs to obtain good yields of highly purified thyroglobulin. All the methods now available for the purification of thyroglobulin and for the analysis of crude or partially purified thyroid estracts therefore present major shortcomings. The need for suitable methods for the analysis of thyroid extracts, as well as for the preparation of highly purified thyroglobulin, led to the work to be reported here. Methods of fractionation based essentially on molecular size were found to be most useful. Filtration through granulated agar or dextran gel columns was used for the purification of thyroglobulin; ultracentrifugation in a linear density gradient was employed for both analytical and preparative purposes.