Resolution of radioiodinated thyrotropin into receptor active and inactive components by column chromatography.
Following radioiodination by the lactoperoxidase method and subsequent purification on Sephadex G100, it was found that [125I]TSH exhibited varying degrees of binding activities to the thyrotropin receptor. In order to further purify the radiolabeled hormone, the [125I]TSH preparation was chromatographed on Sepharose 6B. Two peaks of radioactive material (Peaks I and II) were recovered, containing approx. 60% of the applied radioactivity. Upon elution with Mg2+, the remainder of the radiolabeled material was recovered as a single peak (Peak III). Characterization of these 3 peaks by radioimmunoassay demonstrated that all 3 were immunocompetent, although Peaks I and III were 3-4-fold more immunoreactive than Peak II. Analysis by radioreceptor assay indicated that Peak III showed an increase in receptor-binding capacity (in comparison with the [125I]TSH preparation purified by Sephadex G100 alone), while both Peaks I and II exhibited significantly reduced binding activity. In contrast, human TSH (NIH) chromatographed mainly as a receptor inactive peak, although it was fully immunocompetent. Scatchard analysis of receptor binding to bovine [125I]TSH from Peak III yielded a curvilinear plot with affinities similar to those we have previously reported for [125I]TSH purified by Sephadex G100 chromatography. The total number of binding sites, however, increased proportionally with the active fraction of the [125I]TSH preparation. Since the mass of bound hormone is calculated from the percent bound of total radioactivity and only a fraction of the measured total participates in the binding, it is therefore necessary to correct for the inactive fraction when calculating the total receptor number.
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007457:Iodine Radioisotopes; D007525:Isoelectric Focusing; D007553:Isotope Labeling; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011989:Receptors, Thyrotropin; D013972:Thyrotropin / Q000302:isolation & purification* / Q000378:metabolism
D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011989:Receptors, Thyrotropin; D013972:Thyrotropin