Subunit heterogeneity of thyroglobulin.

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 halfand 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 halfmolecules. 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 com.ponents 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