Stability of the Hypocalcemic Activity of Porcine Thyrocalcitonin

Abstract

Porcine thyrocalcitonin was modified by enzymatic and chemical treatment for the purpose of studying the inter-relationships between structure and hypocalcemic activity. Enzymatic treatment with trypsin, chymotrypsin, pepsin and polyphenyl oxidase led to rapid and complete inactivation of the hypocalcemic activity. We conclude from these experiments that thyrocalcitonin either is a simple polypeptide or contains a peptide moeity which is absolutely essential for hypocalcemic activity. Incubation of thyrocalcitonin in dilute HC1 or neutral Tris buffer at elevated temperature was accompanied by gradual loss of activity. Inactivation was also seen after treatment with hydrogen peroxide, photooxidation and treatment with n-bromosuccinimide, results demonstrating that there is at least one site in the thyrocalcitonin molecule that is sensitive to oxidation. The hypocalcemic activity which was lost during incubation with hydrogen peroxide could not be recovered by treatment of the oxidized products with cysteine, 2-mercaptoethanol or dithiothreitol. There were no detectable changes in hypocalcemic activity either after enzymatic treatment with carboxypeptidase A, leucine aminopeptidase and neuraminidase, or after chemical treatment with 2-OH-5-nitro-benzyl bromide, N-acetylimidazole, p-OH-mercuribenzoate, iodo-acetate and cyanogen bromide. Because thyrocalcitonin has not yet been purified, the sites of the modifications produced by various treatments cannot be shown. Results of experiments in which no loss of hypocalcemic activity was detected must be viewed with caution since it was not possible to show that amino acid residues in thyrocalcitonin were modified. However, these data may help to explain losses of hypocalcemic activity which occur during handling and to suggest new approaches to purification and characterization of thyrocalcitonin.