Purification, Characterization and Radioimmunoassay of Thyrocalcitonin from Rat Thyroid Glands¹

Abstract

A highly purified preparation of rat thyrocalcitonin (TCT) has been obtained from lyophilized thyroid glands by gel chromatography following acid-acetone extraction. Biological activity of Sephadex G-50 eluates appeared in two peaks. The TCT in the major peak was concentrated, and applied to a Bio-Gel P-6 column, and a major protein peak was eluted which coincided with TCT activity. Potency, estimated by bioassay in rats, increased approximately 3500-fold from 0.075 MRC U/mg lyophilized glands to 250–400 MRC U/mg in the final product. The overall yield of TCT activity was about 36%. The purified product was characterized by chemical procedures and evaluated for its antigenic properties and use for radioimmunoassay. The purified rat TCT was used both labeled with l25I and as unlabeled standard. The following results were obtained: 1) Guinea pig antisera to either human or rat TCT were capable of binding l25I-rat TCT or l25I-human TCT; 2) Using either l25I-human or l25I-rat TCT and antisera to either TCT, pg amounts of rat and human TCT reacted in the assay while ng to ¼g amounts of salmon calcitonin or porcine TCT failed to react; 3) Using l25I-rat TCT and antisera to human or rat TCT, synthetic C-terminal (10–32 or 22–32) fragments of human TCT reacted well, while N-terminal (1–18) or desamide (1–32) derivatives reacted poorly or not at all; 4) Rat TCT was easily detected in normal thyroid venous plasma (5–10 ng/ml) and thyroid gland extracts (∼1 ¼g/gland) but not in peripheral blood; 5) Bioassay and radioim-munoassay of rat thyroid extracts (N = 18) showed good agreement (r = 0.86, p < 0.001). The results support the idea that rat TCT is closely related to human TCT, indicate that major antigenic determinants reside in the C-terminal portion of the molecule, and show that antisera to either human or rat TCT can be used to measure rat TCT. (Endocrinology96: 340, 1975)