[17] Covalent Labeling of the Hepatic Glucagon Receptor

The procedure for covalent labeling the hepatic glucagon receptor utilizes the light-sensitive heterobifunctional cross-linker hydroxysuccinimidyl-p-azidobenzoate (HSAB) to link the bound [¹²⁵I-Tyr^l0]monoiodoglucagon ([¹²⁵I]MIG) to the receptor protein. The procedure was described to covalently attach ¹²⁵I -labeled glucagon to its liver membrane receptor. The method involves first the binding of the labeled hormone to its receptor and the removal of the excess unbound label. This is then followed by incubation with the crosslinker, in the dark and then under ultraviolet illumination to covalently couple the bound [¹²⁵I] MIG. The chapter also explains that HSAB contains an amino reactive group and an aryl azide, which, upon light activation, is converted to an aryl nitrene that reacts in a chemically unspecific manner. The chapter discusses various methods for covalent labeling of the hepatic glucagon receptor such as [¹²⁵I] MIG binding to liver membranes, cross-linking of bound [¹²⁵I] MIG to liver membranes, and SDS-gel electrophoretic analysis of covalently labeled membranes.