Further chromatographic studies on beef thyrotropin and a comparison of beef, sheep, and whale thyrotropins.

The partial purification of beef thyrotropin with the cation exchangers Amberlite IRC-50 (2, 3) and carboxymethyl (CM) cellulose (3, 4) has been previously reported. The preceding paper (5) describes the rechromatography on diethylaminoethyl (DEAE) cellulose of thyrotropin preparations assaying 20 U.S.P. units per mg which resulted in fractions apparently containing only those glycoproteins with the biological activities ascribed to thyrotropin. By means of the starch-gel electrophoresis technique (6, 7) six active components designated as thyrotropins a to f were recognized to have been eluted progressively from the columns, and two of these components were obtained as what appeared to be essentially homogeneous preparations by electrophoretic criteria. The present paper reports further experiments to test the possibility that the biological activity could be separated from the glycoproteins of molecular weight of approximately 28,000 (8), and to determine if the same bands observed after anion exchange could be observed in the starch-gel patterns after cation exchange chromatography. Thyrotropin preparations which had been once chromatographed on DEAE-cellulose were rechromatographed on the cation exchanger CM-cellulose (3, 4, 9, 10) or on hydroxyl apatite (11-13). Chromatography on these media allowed a greatly improved separation of the inactive components h, i, and j (5) from what still proved to be the active components, a to f. It is of interest that the active components were found to emerge in reverse order from both CMcellulose and the hydroxyl apatite than from the anion exchanger (5), thus offering strong evidence that a principal difference between the active components is a difference of charge. The carbohydrate contents of fractions obtained both by anion exchange chromatography and by chromatography on hydroxyl apatite are presented. This paper also describes a comparative study of beef, sheep, and whale thyrotropins utilizing DEAE-cellulose chromatography (9, 14, 15), starch-gel electrophoresis (5-7, 15), and the starch-gel electrodialysis method for determination of approximate molecular weight (8).