440 Biochimica et Biophysica Acta, 372 (1974) 440--449 © Elsevier Publishing Company, Amsterdam -- Printed in The Netherlands BBA 27542 NON-EQUIVALENCE OF THE CARBOXYL GROUPS OF GLUCAGON IN THE CARBODIIMIDE-PROMOTED REACTION WITH NUCLEOPHILES AND THE ROLE OF CARBOXYL GROUPS IN THE ABILITY OF GLUCAGON TO STIMULATE THE ADENYL CYCLASE OF RAT LIVER* GRAHAM E. WHEELER, RICHARD M. EPAND** AND DALE BARRETT Department of Chemistry, University of Guelph, Guelph, Ontario NIG 2W1 (Canada) (Received June 4th, 1974) Summary The polypeptide hormone glucagon can react with the nucleophiles; gly- cinamide, taurine or ethylenediamine in the presence of 1-ethyl-3-(3-dimethyl- aminopropylcarbodiimide). The number of carboxyl groups which are modified depend on the concentration of guanidine hydrochloride in the reaction media. These results demonstrate an additional property which glucagon possesses in common with larger globular proteins and suggests that the hormone has a specific, folded structure in dilute aqueous solution. In the absence of guanidine hydrochloride only one taurine residue is incorporated into the ter- minal carboxyl group of the peptide. In 7 M guanidine hydrochloride all four of the carboxyl groups react with glycinamide or taurine while only two and a half residues of ethylenediamine are incorporated. All of these derivatives and glucagon have identical circular dichroism spectra in dilute aqueous solution. The taurine modified derivative has greatly enhanced solubility compared with glucagon but still associates to structures of higher helical content. Both of the taurine derivatives of glucagon have the ability to stimulate the adenyl cyclase of rat liver membranes but at concentrations several fold higher than is needed for the native hormone. It is suggested that each carboxyl group contributes to the binding of the hormone to the specific membrane receptor sites. Introduction Glucagon a polypeptide hormone of 29 amino acids has four carboxyl groups, three from the side chains of the acidic amino acid aspartic acid, at * This work was presented in part at the Biochemistry/Biophysics 1974 Meeting. Minneapolis. ** To whom requests for reprints should be addressed; and present address: McMaster University, Health Sciences Centre, Department of Biochemistry, Hamilton, Ontario Canada, LSS 4J9.