Evidence that thyroglobulin contains nonidentical half molecule subunits.

Bovine thyroglobulin was extracted from unfrozen glands, purified by sucrose gradient centrifugation, and fractionated into a narrow range in iodine content by RbCl isopycnic centrifugation. The subunit composition of these preparations was studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The extent of dissociation of 19 S into 12 S half-molecules followed the known relationship with iodine, i.e. decreased dissociability of 19 S with increased iodine content. The undissociated 19 S band always consisted of three closely spaced, equidistant bands. Reduction of the disulfide bonds of thyroglobulin by mercaptoethanol in SDS solution resulted in the formation of two major and one minor components (S, F, and A). The concentration of A was always less than 10% of the total. The ratio of S to F was, however, about equal in thyroglobulin preparations which ranged in iodine content from 0.2 to 1%. The final ratios were obtained before all the disulfides were reduced. The relative mobilitis of S, F, and A, decreased with increasing extent of reduction. Fully reduced S and F, but not A, migrated slower than unreduced 12 S. The three reduced alkylated polypeptides were purified by preparative SDS-polyacrylamide gel electrophoresis and their molecular weights were determined by sedimentation equilibrium in 8 M urea. Their Mw and Mz values agreed closely with that of the unreduced 12 S half-molecule subunit, thus indicating that reduction of the disulfide bonds changes the shape but not the molecular weights of the subunits.