Monoiodoglucagon: Synthesis, Purification by High Pressure Liquid Chromatography, and Characteristics as a Receptor Probe*

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Abstract
The synthesis of [125I-Tyr10]monoiodoglucagon from glucagon and carrier-free 125I using l,3,4,6-tetrachloro-3-6-diphenylglycouril (Iodogen) and its separation in pure form by reverse phase high pressure liquid chromatography (HPLC) over C18-μBondapak columns using two consecutive linear gradients between solvent A [40:60 mixture of methanol and 10 mM H3PO4 in H2O (pH adjusted to 3.0 with triethylamine)] and solvent B (50:50 mixture of acetonitrile and 0.1 M Tris-HCl, pH 9.0) is reported. The newly synthesized [125I] monoiodoglucagon is shown to activate adenylyl cyclase in liver membranes with an EC50 between 5- and 8-fold lower than that of native glucagon. Further, it binds specifically to sites on liver plasma membranes that have the characteristics of glucagon receptors in terms of gua-nine nucleotide sensitivity and rates of reaction. It is suggested that [125I-Tyr10]monoiodoglucagon is a suitable probe for studying structural and functional properties of glucagon receptors. (Endocrinology113: 711, 1983)