Thyroglobulin fractionation on diethylaminoethyl cellulose columns.

Thyroglobulin may be defined as the iodine-containing protein of the thyroid gland which has a sedimentation coefficient of 4o.w = 19 S, an electrophoretic mobility at pH 8.6 of approximately 5 x 10F5 cm2 volt-l set-1, and a steep salting out curve at about 38% ammonium sulfate saturation at neutral pH (1). Although other iodoproteins, both soluble in neutral salt and attached to cell particles, as well as minute amounts of nonprotein-bound iodine, can be found in normal thyroid tissue, thyroglobulin contains most of the thyroidal iodine. This iodine is in the form of peptide-linked iodoamino acids. Approximately one-third of the iodine is thyroxine, which, in this state, is the storage form of the thyroid hormone. There is also a smaller amount of triiodothyronine, but most of the thyroglobulin iodine is present as monoiodotyrosine and diiodotyrosine. It has long been known that the iodine content of thyroglobulin may be variable within certain limits (2), and it has been suggested that even thyroglobulin molecules stored in a single thyroid gland may contain different amounts of iodine. Until recently, no methods were available to separate such thyroglobulins; the fractions obtained by salting out by Derrien, Michel, and Roche (3) were found to have identical iodine to nitrogen ratios. Ion exchange chromatography of sheep thyroglobulin on diethylaminoethyl (DEAE) cellulose was first described in 1959 by Ingbar, Askonas, and Work (4). Although they presented evidence for heterogeneity of thyroglobulin, especially with respect to newly synthesized, I 131-labeled thyroglobulin compared to total protein, 1127 to nitrogen ratios of various portions of the gradient eluates were the same. Subsequent experiments in our own and other laboratories (5, 6) showed that beef and hog thyroglobulin could be divided by stepwise elution from DEAE-cellulose into three or six arbitrarily selected fractions. Ui et al. (6) found that the fractions eluted at higher ionic strength contained higher I127 to nitrogen ratios than early fractions. Using a more complicated elution procedure, Shulman and Stanley (7) found hog thyroglobulin subfractions differing in iodoamino acid composition, but they did not report iodine to protein ratios. In this report, detailed studies are presented on three subfractions of beef thyroglobulin obtained by the stepwise elution chromatography scheme described earlier (5). The results clearly show that these fractions differ in iodine content as well as in iodoamino acid composition.