Biochimica et Biophysica Acta, 453 (1976) 365-373 © Elsevier/North-Holland Biomedical Press BBA 37523 CONFORMATIONAL AND BIOLOGICAL PROPERTIES OF A COVALENTLY LINKED DIMER OF GLUCAGON REACTION OF MONO- AND B1FUNCTIONAL SULFENYL HALIDES R. M. EPAND and T. E. COTE Department of Biochemistry, McMaster University Health Sciences Centre, Hamilton, Ontario L8S 4J9 (Canada) (Received July 1st, 1976) SUMMARY The tryptophan residue of glucagon was modified by reaction with a mono- functional sulfenyl chloride (2-nitrophenylsulfenyl chloride) and with a bifunctional sulfenyl chloride (2,4-dinitro-l,5-phenyldisulfenyl chloride) to produce a monomeric form of glucagon with a modified tryptophan, glucagon-nitrophenylsulfenyl and a dimeric form (glucagon)z-dinitrophenyldisulfenyl respectively. The dimeric form was isolated by chromatography on Sephadex G-50. The circular dichroism spectra of the derivatives suggest an increased content of secondary structure, especially at low pH and low temperature. The derivatives activated adenylate cyclase from rat liver to an extent comparable to that of the native hormone, indicating that a glucagon dimer is capable of biological activity and that an intact tryptophan residue is not essential for biological response. INTRODUCTION Glucagon is a hormone which stimulates hyperglycemia and is thought to have an important role in diabetes mellitis [1]. The structural features of the hormone which are responsible for its biological activity are currently being elucidated. In this study we have investigated the importance of the tryptophan residue to the biological activity of the hormone. Glucagon is a 29 amino acid polypeptide hormone with a single tryptophan residue at position 25 [2]. Tryptophan residues of proteins not con- taining sulfhydryl groups have been shown to react specifically with 2-nitrophenyl- sulfenyl chloride [3]. In the case of adrenocorticotropic hormone this reaction leads to enhanced melanocyte-stimulating activity [4] although diminished ability to activate adrenal adenylate cyclase has been reported [5]. In addition, a bifunctional reagent, 2,4-dinitro-l,5-phenyldisulfenyl chloride has been developed based on the reactivity of nitrophenylsulfenyl chloride with tryptophan. It has been shown to be capable of forming covalently linked dimers with glucagon [6]. As glucagon attains a higher degree of secondary structure upon non- covalent association [7], one might also expect that the conformation of the hormone