Reduction of Cys36-Cys42 and Cys64-Cys74 disulfide bonds in recombinant human granulocyte colony stimulating factor.
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.
Léon
J
J
Department of Pharmaceutical Analysis, Faculty of Pharmacy, Utrecht University, The Netherlands.
Reubsaet
E
E
Beijnen
J H
JH
van Bennekom
W P
WP
Bult
A
A
Hoekstra
A J
AJ
Hop
E
E
van Os
P J
PJ
Teeuwsen
J
J
Underberg
W J
WJ
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
D004220:Disulfides; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D003545:Cysteine