High molecular thyrotrophin ("big"-TSH) from human pituitaries: preparation and partial characterization.

Abstract

ABSTRACT Homogenates of human pituitaries were centrifuged at 30 000 × g and the supernatant chromatographed on Sephadex G-100. Approximately 1 % of the radioimmunologically measured total activity of TSH was eluted in the void volume. Rechromatography of this material on Sephadex G-200 usually showed TSH-activity at Kav = 0.5 (regular TSH), Kav = 0 (void volume-TSH) and at Kav = 0.3 ("big"-TSH). "Big"-TSH was extracted from the corresponding fractions by affinity-chromatography with solid phase anti-TSH. It was eluted with 5 mol/l ammonium thiocyanate and further characterized: 1. The molecular weight was approximately 200 000 by comparison with bovine catalase on Sephadex G-200. 2. Immunoidentity as compared with standard-TSH (M. R. C. 68/38) was shown by parallel dilution curves in the radioimmunoassay. 3. Concanavalin-A-Sepharose adsorbed "big"-TSH, which could be eluted with α-methyl-D-mannoside, indicating the glycoprotein nature of "big"-TSH. 4. On polyacrylamide-gel-electrophoresis pH 7.5, "big"-TSH migrated faster (RF = 0.32) than regular TSH (RF = 0.1), indicating a more negatively charged molecule. 5. "Big"-TSH, in contrast to regular TSH, was remarkably stable against 6 mol/l guanidine hydrochloride, suggesting a covalently linked (aggregate) structure. 6. 1 % mercaptoethanol destroyed the immunological activity of both regular and "big"-TSH. 7. "Big"-TSH was digested by trypsin, under mild conditions, to radioimmunologically active products with molecular weights between "big"- and regular TSH, but practically no regular TSH was formed. 8. "Big"-TSH and guanidine-treated "big"-TSH, as well as regular TSH and TSH from the void volume of Sephadex G-200 columns, exhibited biological activity in a cytochemical bioassay in good agreement with the respective immunological activities.