OXIDATION OF RECOMBINANT METHIONYL HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR

The oxidation of methionine residues in recombinant methionyl human gr anulocyte colony stimulating factor with hydrogen peroxide has been in vestigated. Kinetic data of the oxidation were obtained by using rever sed phase-high performance liquid chromatography. The stability-indica ting capability of this system was confirmed with micellar electrokine tic capillary chromatography. In the pH range 1.9-7.5, the k(obs) valu e for the oxidation process is constant. Above pH 7.5. k(obs) 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 reveal ed 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 wi th Met(121) oxidized, while the dioxidation product has oxidized Met(1 21) and Met(126) residues. The trioxidation products are the proteins with Met(121), Met(126) and Met(137) or Met(0), Met(121) and Met(126) oxidized. (C) 1998 Elsevier Science B.V. All rights reserved.