Human pituitary thyrotropin. Isolation and recombination of subunit isoforms

The alpha and beta subunits of human pituitary thyrotropin were prepared by a process developed earlier. Each subunit preparation contained four isoforms which were isolated by preparative agarose-suspension electrophoresis. The alpha subunit isoforms were homogeneous upon examination by analytical electrophoresis in polyacrylamide gel. Contrarily, the isolated beta subunit isoforms were always to some extent contaminated by the adjacent more acidic component. This was considered to show a transformation of these forms favoured by hydroxyl ions. Amino acid analysis revealed no differences between the alpha isoforms nor between the beta isoforms, and the amino acid compositions were in very good agreement with those earlier obtained for multi-alpha and multi-beta preparations. Attempts to recombine different alpha and beta isoforms were made at pH 7.0. The activities of recombined thyrotropin (in terms of MRC Research Standard A) ranged from 1.1 unit/mg (recombination of two minor components) to 3.5 units/mg (recombination of two major components). The activities of native and recombined thyrotropins as well as the subunit activities were followed by radioimmunoassays. The activity of native thyrotropin as a function of time was studied under different storage conditions. The subunit activities were also measured. All conditions resulted in a decrease in the thytrotropin activity (about 40% in 10 weeks). This decrease was not due to dissociation as the subunit activities remained approximately constant.