Oxidation of recombinant methionyl human granulocyte colony stimulating factor.
The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of this system was confirmed with micellar electrokinetic capillary chromatography. In the pH range 1.9-7.5, the kobs value for the oxidation process is constant. Above pH 7.5, kobs tends to increase with increasing pH. In the pH range 1.9-11.8, four oxidation products were detected in RP-HPLC. Mass spectrometric analysis revealed that one mono-, one di- and two trioxidation products were formed. Using the cyanogen bromide cleavage method the nature of the oxidation products was determined. The mono-oxidation product is the protein with Met121 oxidized, while the dioxidation product has oxidized Met121 and Met126 residues. The trioxidation products are the proteins with Met121, Met126 and Met137 or Met0, Met121 and Met126 oxidized.
Reubsaet
J L
JL
Department of Pharmaceutical Analysis, Faculty of Pharmacy, Utrecht University, The Netherlands.
Beijnen
J H
JH
Bult
A
A
Hop
E
E
Scholten
S D
SD
Teeuwsen
J
J
Underberg
W J
WJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000737:chemistry; D000069585:Filgrastim; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D008715:Methionine; D006861:Hydrogen Peroxide; D003488:Cyanogen Bromide; D000069585:Filgrastim