HIGH MOLECULAR THYROTROPHIN ("BIG"-TSH) FROM HUMAN PITUITARIES: PREPARATION AND PARTIAL CHARACTERIZATION

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Abstract
Homogenates of human pituitaries were centrifuged at 30,000 .times. g and the supernatant chromatographed on Sephadex G-100. Approximately 1% of the radioimmunologically measured total activity of TSH [thyrotropin] 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: The molecular weight was approximately 200,000 by comparison with bovine catalase on Sephadex G-200. Immunoidentity as compared with standard-TSH (M.R.C. [Metabolic Research Council] 68/38) was shown by parallel dilution curves in the radioimmunoassay. Concanavalin-A-Sepharose adsorbed big-TSH, which could be eluted with .alpha.-methyl-D-mannoside, indicating the glycoprotein nature of big-TSH. 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. Big-TSH, in contrast to regular TSH, was remarkably stable against 6 mol/l guanidine hydrochloride, suggesting a covalently linked (aggregate) structure. Mercaptoethanol (1%) destroyed the immunological activity of both regular and big-TSH. 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. 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.

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