Abstract
Glucagon is a vital peptide hormone involved in the regulation of blood sugar under fasting conditions. Although the processes underlying glucagon production and secretion are well understood, far less is known about its degradation, which could conceivably be manipulated pharmacologically for therapeutic benefit. We describe here the development of novel assays for glucagon degradation, based on fluoresceinated and biotinylated glucagon (FBG) labeled at the N- and C-termini, respectively. Proteolysis at any peptide bond within FBG separates the fluorescent label from the biotin tag, which can be quantified in multiple ways. In one method requiring no specialized equipment, intact FBG is separated from the cleaved fluoresceinated fragments using NeutrAvidin agarose beads, and hydrolysis is quantified by fluorescence. In an alternative, high-throughput-compatible method, the degree of hydrolysis is quantified using fluorescence polarization after addition of unmodified avidin. Using a known glucagon protease, we confirm that FBG is cleaved at similar sites as unmodified glucagon and use both methods to quantify the kinetic parameters of FBG degradation. We show further that the fluorescence polarization-based assay performs exceptionally well ( Z'-factor values >0.80) in a high-throughput, mix-and-measure format.