The effects of in vitro methionine oxidation on the bioactivity and structure of human keratinocyte growth factor

Abstract

Publisher Summary Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. The molecule is expressed by stromal fibroblasts and is involved in the proliferation and differentiation of epithelial cells in a paracrine mode. E.coli -derived human KGF is biologically active and may be clinically useful. In this chapter, hydrogen peroxide oxidation has been performed on E.coli -derived human KGF at pH 5.0 to understand the structure and function of the protein. Reverse-phase high performance liquid chromatography (RP-HPLC), peptide mapping, protein sequencing, and mass spectrometry have been used to separate and identify the different methionine oxidized KGF species. Cation exchange HPLC is utilized to remove hydrogen peroxide from the different modified forms of the protein prior to mitogenic bioassay and circular dichroism (CD) analysis. Preferential oxidation of the methionine residues to methionine sulfoxide has enabled to determine that Met 160 may play an important role in the biological function of KGF. In vitro, hydrogen peroxide oxidation of the methionine residues of KGF to their sulfoxide derivatives demonstrate that Met 28, Met 60, and Met 160 are preferentially oxidized. Met 28 and Met 60 appear to be solvent-accessible and exposed on the surface of the protein, while Met 160 appears to be partially solvent-accessible. In contrast, Met 67, Met 90, and Met 132 oxidize much slower, which suggests that they are located in a buried, less solvent accessible environment. Oxidation of these buried residues results in the generation of an extremely unstable form of KGF that readily forms aggregates.