Studies on the bioactivity of radioiodinated highly purified bovine thyrotropin: analytical polyacrylamide gel electrophoresis.
Highly purified bovine TSH (stored in solution at -70 C) was radioiodinated by the stoichiometric chloroamine-T method. The iodinated material ws subjected to analytical polyacrylamide disc gel electrophoresis. TSH was eluted from gel slices (1 mm width) and was analyzed for radioactivity and bioactivity. The latter was determined using the cultured thyroid cell cAMP response assay. Radioactivity in the TSH preparation migrated separately from bioactivity, but concordant with the protein bands observed in gels run in parallel. Further studies performed on bovine TSH purified in our laboratory, as well as on a different TSH preparation of exceptionally high potency (both stored as lyophilized powder) revealed a different pattern, with TSH bioactivity and radioactivity eluting concurrently. Iodination of TSH did not alter its electrophoretic migration on disc gel electrophoresis. In all preparations polymorphism of TSH bioactivity was observed, with at least four separate protein bands containing TSH bioactivity being present in our preparation. The relationship between the degree of iodination and retention of TSH bioactivity was examined. Incorporation of 125I into TSH was greatly different at two different concentrations of chloramine-T. Despite this, however, the progressive loss of TSH bioactivity was similar at both concentrations, indicating that incorporation of iodine into the TSH molecule is not itself responsible for the decrease in bioactivity. These studies indicate variability among different TSH preparations in terms of their retention of bioactivity. Significant loss of TSH bioactivity appears to occur during storage in solution. The damage to the biological activity of TSH during the iodination procedure is more likely related to the oxidation process than to the incorporation of iodine.
D000236:Adenoma / Q000503:physiopathology; D000818:Animals; D001681:Biological Assay; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D004285:Dogs; D004589:Electrophoresis, Disc; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods; D013961:Thyroid Gland / Q000187:drug effects / Q000502:physiology*; D013964:Thyroid Neoplasms / Q000503:physiopathology; D013972:Thyrotropin / Q000494:pharmacology*
D007457:Iodine Radioisotopes; D013972:Thyrotropin; D000242:Cyclic AMP