Further studies on acetamidination as a technique for preparation of a biologically valid 3-H-labeled tracer for parathyroid hormone.

The technique of acetamidination of amino groups in parathyroid hormone (PTH) for the purpose of preparation of a tritiated, biologically valid tracer for this hormone, has been more extensively studied. It was found that eight of the ten amino groups in PTH are readily reactive but that two appear unreactive. The kinetics of labeling suggest that preparations of PTH which are labeled to this level of 80% of theoretical consist of a homogeneous population of molecules in which each PTH molecule contains eight tritiated acetamidino groups. There is no question of the presence of unlabeled hormone in such preparations. Eighty per cent labeled acetamidino-PTH is identical, qualitatively and quantitatively, in its biological activity with native PTH as shown by three accepted bioassays: serum calcium elevation, urine phosphate excretion, and activation of kidney cortex plasma membrane adenylate cyclase in vitro. The dose-response relationships are identical for labeled and native hormone in all three systems. Tritiated acetamidino-PTH tends to lose biological activity on storage but full activity can be regained by reduction of the hormone with excess cysteine. However, cysteine reduction conducted at 80 degrees for 2 hours causes some loss of tritium from the hormone. Therefore, dithiothreitol reduction at room temperature was utilized to maintain biological activity. Eighty per cent acetamidino-PTH can be further purified by ion exchange chromatography on carboxymethylcellulose using a continuous gradient of sodium acetate in 8 M urea. Such chromatography reveals the presence of isohormones in both cold and radioactive PTH. Co-chromatography of tritiated acetamidino-PTH with a cold isohormone of PTH shows that while the isohormones can be separated from one another, the acetamidino derivatives of each isohormone elute in a virtually identical position with their parent unlabeled PTH molecule.