Purification and Properties of Whale Thyroid-stimulating Hormone<subtitle>II. Isolation of Multiple Components</subtitle>

Abstract

In view of the finding that whale thyroid-stimulating hormone (TSH) contains at least four biologically active components with different isoelectric points (2), attempts were made to purify these four components in a homogeneous state. Crude preparations of whale TSH obtained by acetone precipitation were purified by large-scale zone electrophoresis, and the TSH-rich fraction thus prepared was fractionated by isoelectric focusing into four components having the isoelectric point at pH 8.1, 8.3, 8.5 and 8.7, respectively. These fractions still showed the presence of two bands in disc electrophoresis; only the faster band had TSH activity. Each fraction of TSH obtained by isoelectric focusing was subjected to chromatography on TEAE-cellulose to remove the slower-migrating protein, and further purified by gel filtration on Sephadex G-100. These highly active preparations (TSH I–IV) were found to be homogeneous in disc electrophoresis, immunodiffusion and immunoelectrophoresis, except for one preparation which showed signs of the presence of a small amount of contaminant in immunoelectrophoresis. The slower-migrating component in each TSH fraction obtained by isoelectric focusing was also purified. These preparations, which gave a single band in disc electrophoresis, showed a strong luteinizing hormone (LH) activity comparable to that reported for purified LH from other species.