title: Structural transition of glucagon in the concentrated solution observed by electrophoretic and spectroscopic techniques.
abstract: Glucagon, a polypeptide hormone consisting of 29 amino acid residues, tends to form gel-like fibrillar aggregates, and the glucagon fibril, as well as other pathologically related fibrils including prion, amylin, and beta-amyloid, have been found to be cytotoxic through the activation of apoptotic signaling pathways. To understand the aggregation properties of glucagon fibril, we have characterized and compared the physicochemical properties of glucagon, secretin, a member of the glucagon superfamily, and amylin using analytical techniques including capillary electrophoresis (CE), circular dichroism (CD), FT-IR, FT-Raman, transmission electron microscopy (TEM), and beta-sheet-imaging probe. Aging treatment of glucagon resulted in the formation of fibrillar aggregates in time- and concentration-dependent manner, and FT-IR and FT-Raman analyses showed the spectral shift of amide I band, suggesting the conformational changes from alpha-helix to beta-sheet structure. Interestingly, secretin, having high sequential and secondary structural homology with glucagon, did not generate the fibrillar aggregates at the conditions tested. In addition, we evaluated the association state of glucagon at various pHs raging from pH 2.0 to 3.5 using CE. Based on the CE data, the rate constants of glucagon aggregation were calculated to be 0.002 +/- 0.004/h and 0.080 +/- 0.011/h for aging at pH 2.0 and 3.5, respectively, suggesting the pH dependence of self-association. CE showed the potential to separate and detect the glucagon aggregates and intermediates during aging process.
authors.affiliation: Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan. Satomi.Onoue@pfizer.com
authors.forename: Satomi
authors.initials: S
authors.lastname: Onoue
authors.forename: Sumiko
authors.initials: S
authors.lastname: Iwasa
authors.forename: Takashi
authors.initials: T
authors.lastname: Kojima
authors.forename: Fumie
authors.initials: F
authors.lastname: Katoh
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Debari
authors.forename: Keitatsu
authors.initials: K
authors.lastname: Koh
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Matsuda
authors.forename: Takehiko
authors.initials: T
authors.lastname: Yajima
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005934:Glucagon / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods*
chemical_list: D012996:Solutions; D005934:Glucagon