Subunit Heterogeneity of Thyroglobulin

Abstract

The subunit structure of thyroglobulin has been investigated by gel filtration and polyacrylamide electrophoresis in sodium dodecyl sulfate (SDS) of the native and the S-carboxymethylated proteins. When the thyroglobulins from several species (calf, sheep, pig, man, rat, and guinea pig) were examined by polyacrylamide electrophoresis in SDS in the absence of reducing agents, most of the protein was distributed between two components which appeared to be the half- and the undissociated molecule. However, smaller components with molecular weights ranging down to 20,000 were also detected in the polyacrylamide gels. These represented about 20% of the total protein and could be separated from the larger subunits by filtration on agarose columns (Bio-Gel A-15m), which also succeeded in separating the intact and half-molecules. Polyacrylamide electrophoresis of the thyroglobulins in the presence of both SDS and mercaptoethanol indicated the existence of a large number of subunits. The two most prominent components of the calf protein had molecular weights of 215,000 and 190,000, but others with molecular weights from 235,000 to 30,000 were also found. Bio-Gel A-15m filtration of the S-carboxymethylated protein in SDS permitted separation of the subunits with molecular weights of approximately 200,000 from those of smaller size, which after disulfide bond cleavage represented approximately 40% of the protein. Separation on Bio-Gel A-5m of the 27 S and 19 S forms of calf thyroglobulin which are present in the protein purified by phosphate buffer fractionation made it possible to demonstrate that both had similar subunit patterns when examined by SDS-polyacrylamide electrophoresis in the presence of mercaptoethanol. The distribution of the protein among the components seen on SDS-polyacrylamide electrophoresis was determined from the total amino acids present in the separated bands, and the relative number of moles of each was calculated. This indicated a nonstoichiometric relationship among the subunits. NH2-terminal analyses performed on the fractions obtained by Bio-Gel A-15m filtration of the SDS-treated native calf thyroglobulin and the reduced-alkylated protein showed that the disulfide-bonded molecules present in the native protein contain almost as many NH2-terminal amino acids as the intact protein. In contrast, the 200,000 molecular weight subunits of the reduced-alkylated protein have insufficient NH2-terminal amino acids for their molecular weight, suggesting the presence in them of blocked terminal residues, while the small molecular weight components are enriched in this respect compared to the original thyroglobulin. The nonstoichiometry of both the subunits and the NH2-terminal amino acids may be explained by the presence in this large molecule of fragments of varying length both noncovalently and disulfide-bonded to the larger subunits. 14C-Labeled rat thyroglobulin was shown to have radioactivity in both large and small subunits and the distribution was similar when the protein was isolated at 1 and 3 hours after the injection of labeled leucine. Comparable findings were obtained with 14C-labeled calf thyroglobulin prepared by incubation of thyroid slices with radioactive leucine for varying periods of time. This suggested that the cleavage of thyroglobulin to yield the diversity of subunits observed occurs at some stage during the synthesis or maturation of the molecule rather than during its storage. The possibility of endogenous proteolysis during thyroglobulin preparation as a cause of subunit heterogeneity was considered unlikely, since incubation of the thyroid slices or of the thyroglobulin itself at pH 6.8 had no effect on the polyacrylamide electrophoretic pattern observed. The free mobilities of the thyroglobulin subunits during SDS-polyacrylamide electrophoresis were shown to be somewhat lower than those of the standard proteins used and the molecular weights calculated for some of the subunits depended on the gel concentration employed. Molecular weights for these components were therefore calculated from the asymptotic region of the plots of molecular weight versus gel concentration.