Degradation of [125I]iodoglucagon by normal rat plasma in radioimmunoassay mixture containing aprotinin and its prevention by p-chloromercuriphenyl sulfonate and leupeptin.

Degradation of [125I]iodoglucagon during RIA of glucagon would result in erroneously high values for immunoreactive glucagon (IRG). Normal rat plasma was found to have high activity for glucagon degradation that was not suppressed by the aprotinin and EDTA routinely added to the RIA system. About 30% of the added radioactive glucagon was degraded during RIA in assay mixture at pH 7.4 containing 0.2 ml normal rat plasma, and the IRG value was calculated to be 150-180 pg/ml plasma. The degradation was completely inhibited by addition of 2 mM p-chloromercuriphenyl sulfonate (PCMS) plus 0.25 mM leupeptin as protease inhibitors, and in their presence the IRG value was about 80 pg/ml. The glucagon-degrading activity was about half as much in assay mixture at pH 8.8 as in that at pH 7.4, but the degradation still affected the accuracy of IRG values. When rat plasma was incubated with [125I]iodoglucagon in the assay conditions used for RIA and then subjected to Bio-Gel P-6 filtration, three new peaks of radioactivity were found in low mol wt fractions, with decrease in the peak corresponding to [125I]iodoglucagon, whereas on similar treatment in the presence of PCMS and leupeptin all the radioactivity was recovered in the glucagon fraction. The average recoveries of authentic glucagon as IRG in the absence and presence of the inhibitors were less than 60% and more than 90%, respectively. These findings indicate that determination of plasma IRG in rats by RIA with assay mixture containing aprotinin gives spuriously high values owing to degradation of the radiotracer, and that PCMS and leupeptin should be added to the sample and assay mixture to prevent this degradation.