Effect of specific trinitrophenylation of the lysine epsilon amino group of glucagon on receptor binding and adenylate cyclase activation.

The trinitrophenyl group was specifically introduced into the epsilon-amino group of glucagon by reaction of N alpha-citraconyl glucagon with trinitrobenzenesulfonic acid. The N alpha-citraconyl blocking group was subsequently removed by acid treatment yielding N epsilon-trinitrophenyl glucagon which was purified by anion-exchange chromatography. The derivative showed less secondary structure as measured by circular dichroism than the native hormone at pH 8.0 and at pH 2.0 in the presence of sodium dodecyl sulfate. The analog possessed 4-5% the potency of glucagon in stimulating adenylate cyclase with 90% maximal stimulation and possessed 30% the potency of glucagon in competing for glucagon-specific receptor sites in hepatic plasma membranes. Although the structure of N epsilon-trinitrophenyl glucagon is very similar to N epsilon-4-azido-2-nitrophenyl glucagon, the photoaffinity antagonist synthesized by M.D. Bregman and D. Levy [(1977) Biochem. Biophys. Res. Commun. 78, 584-590.], the biological activities of the two are different. Possible explanations for these differences are discussed.