Synthesis of tritiated rat insulin with high specific activity: A method for radiolabeling the active site of insulin

Abstract Insulin was synthesized by rat islets from tritiated amino acids under conditions designed to achieve high specific activity. Islets were isolated by the collagenase method. Stores of unlabeled insulin were depleted by culturing them for 40 hours in the presence of 3-isobutyl-1-methylxanthine. The islets were then incubated for 22 hours in the presence of [ 3 H]Isoleucine or [ 3 H]Phenylalanine. These amino acids were chosen because they are specific markers from the A and B chains of insulin respectively. Labeled insulin was extracted from the islets and purified by gel filtration. Its biological activity was indistinguishable from monoiodinated insulin as assessed by binding to receptors on cultured human lymphocytes and by precipitation by anti-insulin antibodies. The specific activity was (18 Ci/mmole) and (37 Ci/mmole) for [ 3 H]Ile and [ 3 H]Phe insulin respectively.