Tritium-labelled Peptide Hormones with High Specific Radioactivity

Abstract

PRESENT interest in the assay of peptide hormones has led to the development of radio-immunoassay methods. These are based on the competition for antibody by labelled and unlabelled hormones, and the subsequent separation of antibody-bound from ‘free’ radioactivity¹. ¹³¹I-labelled hormones have the practical disadvantage of the short eight-day half-life of ¹³¹I, and the theoretical disadvantage that iodination tends to destroy the biological potency of the hormones. With these considerations in mind, we have prepared tritium-labelled hormones of high specific activity using the technique of acetylation. In general, between 0.2 and 1.0 µmoles of hormone are mixed with 25 me. (7 µmoles) tritiated acetic anhydride (3,570 mc./mmole from Nuclear Chicago Corporation) in 0.25 ml. water saturated with sodium acetate and adjusted to pH 8.0–8.5 with 0.1 N NaOH. The acetylated hormone is then separated from the free acetate by passing the mixture through a 2 × 15 cm ‘Sephadex G-25’ column. With this technique we have prepared tritium-labelled human growth hormone (3.2 acetyl/mole, 275 µc./mg) tritium-labelled bovine growth hormone (12 acetyl/mole, 570 µc./mg), and tritium-labelled pork insulin (127 µC./mg, 0.5 acetyl/mole). Human and bovine growth hormone acetylated to this extent with ¹⁴C-labelled acetic anhydride have previously been demonstrated to be biologically and antigenically comparable with unlabelled hormone^2,3.