HYDROLYSIS OF I131-THYROPROTEIN BY PANCREATIC ENZYMES

Abstract

Until about 5 years ago, treatment with hot alkali was the most widely used method for hydrolyzing the iodoprotein of thyroid tissue. By such a procedure, Kendall first isolated thyroxine from the thyroid glands of oxen (1). Proteolytic enzymes have also been used for this purpose with varying degrees of success, since the early work of Hutchison (a), Oswald (3), and Harington (4). More recently, the usefulness of enzymic hydrolysis has been extended considerably by combining this method for fragmenting the thyroid protein with the very sensitive techniques of chromatography and radioisotopic labeling for the isolation and identification of the products of hydrolysis. Although this combinat,ion of procedures has been used in several laboratories (5-la), a thorough study of the hydrolysis with proteases has not been reported. In our investigation of this procedure we found, in agreement with Roche et al. (7), that the iodotyrosines are released quite rapidly from the thyroprotein, whereas the release of thyroxine proceeds more slowly. In addition, we found that the yield of thyroxine can be increased by performing the hydrolysis in the presence of certain metal cations (Mn++, Mg++, Ca++). These findings are described below, along with a procedure for the analysis of 1131-labeled thyroid tissue. This procedure requires 10 to 20 hours of hydrolysis, yields approximately 95 per cent recovery of 1131 protein in the form of 1131-amino acids, and results in little release of inorganic I131.