title: Reduction of Cys36-Cys42 and Cys64-Cys74 disulfide bonds in recombinant human granulocyte colony stimulating factor.
abstract: The Cys36-Cys42 and Cys64-Cys74 disulfide bonds in recombinant methionyl human granulocyte colony-stimulating factor were reduced to sulfhydryls with dithiothreitol or mercury. Both reduction reactions are dependent on the pH. The reduction reaction with dithiothreitol increased in rate with increasing pH; between pH 7-9 and above pH 10.5 this increase was less than in other regions. These observations are explained by repulsive forces between dithiothreitol and regions in granulocyte colony-stimulating factor which intensify in these pH-regions. The hydroxyl catalysis causes the overall increase in k(obs) in the pH-region studied. The reduction of the disulfides with mercury is, as could be expected from the Nernst equation for disulfide reduction, also pH dependent: the half-wave potential decreases with increasing pH as predicted by theory.
authors.affiliation: Department of Pharmaceutical Analysis, Faculty of Pharmacy, Utrecht University, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Léon
authors.forename: E
authors.initials: E
authors.lastname: Reubsaet
authors.forename: J H
authors.initials: JH
authors.lastname: Beijnen
authors.forename: W P
authors.initials: WP
authors.lastname: van Bennekom
authors.forename: A
authors.initials: A
authors.lastname: Bult
authors.forename: A J
authors.initials: AJ
authors.lastname: Hoekstra
authors.forename: E
authors.initials: E
authors.lastname: Hop
authors.forename: P J
authors.initials: PJ
authors.lastname: van Os
authors.forename: J
authors.initials: J
authors.lastname: Teeuwsen
authors.forename: W J
authors.initials: WJ
authors.lastname: Underberg
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D003545:Cysteine