Freezing-Induced Alterations in Thyroglobulin from Iodine Deficient and Iodine Sufficient Rats1

Soluble thyroid extracts from iodine sufficient and iodine deficient rats, labeled with radioiodine for varying periods, were frozen for 2 hr or more in the presence of 0.05m methylmercaptoimidazole (MMI), and after thawing were analyzed by sucrose gradient density centrifugation. The freezing-MMI procedure produced 2 types of alteration in the sedimentation pattern: a) a decrease in the sedimentation constant of thyroglobulin, indicating some conformational change in the protein, and b) dissociation of thyroglobulin into 11–12S subunits. With thyroid glands from iodine sufficient rats labeled for a few hr or more, the former change predominated, and there was minimal dissociation. With thyroid glands from iodine deficient rats, however, even after equilibrium labeling, the freezing-MMI procedure resulted in extensive dissociation, as well as in a marked downward shift in the S value of the remaining undissociated thyroglobulin. Labeled prethyroglobulin (14.4S) displayed practically complete dissociation to 10.8S subunits. A single, acute injection of only 3 jug of stable iodide into iodine deficient rats restored within a few hr the normal resistance of thyroglobulin to dissociation by the freezing-MMI procedure. These findings indicate that the freezing-MMI procedure can be used to detect subtle differences in thyroglobulin structure, and they support the conclusion stated in the preceding paper that the conformation of thyroglobulin is altered with increasing degree of iodination. The surprising observation was also made in the course of these studies that freezing of soluble thyroid protein or of thyroid extracts in the presence of 0.05m MMI before proteolytic digestion results in loss of T4* and T3*, not attributable to any impairment in the efficiency of enzymatic digestion. Possible implications of this finding are discussed. (Endocrinology83: 833, 1968)