Acetylated derivatives of glucagon have been prepared by reacting this hormone under various conditions with acetic anhydride. They have been chemically characterized by the use of a 14C‐labeled reagent, by peptide mapping techniques following hydrolysis by pronase and chymotrypsin, and by spectroscopy. Acetylation in sodium acetate (pH 5.5) results in a full substitution of the α‐amino group of the N‐terminal histidyl residue, but in a partial (about 0.3 acetyl group per residue) substitution of the ɛ‐amino group of the lysyl residue 12. The monosubstituted (on the α‐amino group) and the disubstituted (on both amino groups) acetylated components have been separated by chromatography on DEAE‐cellulose and CM‐cellulose. Acetylation in sodium bicarbonate (pH 8.0) results in a complete substitution of both amino groups and of the hydroxyl groups of the tyrosyl residues 10 and 13. Complete deacetylation of the O‐acetyltyrosyl residues occurs upon treatment with hydroxylamine. Mono, di and tetraacetylglucagon are homogeneous when analyzed by disc gel electrophoresis; di and tetrasubstituted derivatives show an increased mobility towards the anode. 125I‐labeled derivatives of acetylgluca
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