Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1996 Jun;5(6):1165–1173. doi: 10.1002/pro.5560050619

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

Y R Hsu 1, L O Narhi 1, C Spahr 1, K E Langley 1, H S Lu 1
PMCID: PMC2143427  PMID: 8762148

Abstract

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.

Full Text

The Full Text of this article is available as a PDF (775.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amiconi G., Ascoli F., Barra D., Bertollini A., Matarese R. M., Verzili D., Brunori M. Selective oxidation of methionine beta(55)D6 at the alpha 1 beta 1 interface in hemoglobin completely destabilizes the T-state. J Biol Chem. 1989 Oct 25;264(30):17745–17749. [PubMed] [Google Scholar]
  2. Anderson D. M., Lyman S. D., Baird A., Wignall J. M., Eisenman J., Rauch C., March C. J., Boswell H. S., Gimpel S. D., Cosman D. Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell. 1990 Oct 5;63(1):235–243. doi: 10.1016/0092-8674(90)90304-w. [DOI] [PubMed] [Google Scholar]
  3. Arakawa T., Yphantis D. A., Lary J. W., Narhi L. O., Lu H. S., Prestrelski S. J., Clogston C. L., Zsebo K. M., Mendiaz E. A., Wypych J. Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure. J Biol Chem. 1991 Oct 5;266(28):18942–18948. [PubMed] [Google Scholar]
  4. Coltrera M., Rosenblatt M., Potts J. T., Jr Analogues of parathyroid hormone containing D-amino acids: evaluation of biological activity and stability. Biochemistry. 1980 Sep 2;19(18):4380–4385. doi: 10.1021/bi00559a035. [DOI] [PubMed] [Google Scholar]
  5. Guptasarma P., Balasubramanian D., Matsugo S., Saito I. Hydroxyl radical mediated damage to proteins, with special reference to the crystallins. Biochemistry. 1992 May 5;31(17):4296–4303. doi: 10.1021/bi00132a021. [DOI] [PubMed] [Google Scholar]
  6. Langley K. E., Wypych J., Mendiaz E. A., Clogston C. L., Parker V. P., Farrar D. H., Brothers M. O., Satygal V. N., Leslie I., Birkett N. C. Purification and characterization of soluble forms of human and rat stem cell factor recombinantly expressed by Escherichia coli and by Chinese hamster ovary cells. Arch Biochem Biophys. 1992 May 15;295(1):21–28. doi: 10.1016/0003-9861(92)90482-c. [DOI] [PubMed] [Google Scholar]
  7. Lu H. S., Chang W. C., Mendiaz E. A., Mann M. B., Langley K. E., Hsu Y. R. Spontaneous dissociation-association of monomers of the human-stem-cell-factor dimer. Biochem J. 1995 Jan 15;305(Pt 2):563–568. doi: 10.1042/bj3050563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Manning M. C., Patel K., Borchardt R. T. Stability of protein pharmaceuticals. Pharm Res. 1989 Nov;6(11):903–918. doi: 10.1023/a:1015929109894. [DOI] [PubMed] [Google Scholar]
  9. Martin F. H., Suggs S. V., Langley K. E., Lu H. S., Ting J., Okino K. H., Morris C. F., McNiece I. K., Jacobsen F. W., Mendiaz E. A. Primary structure and functional expression of rat and human stem cell factor DNAs. Cell. 1990 Oct 5;63(1):203–211. doi: 10.1016/0092-8674(90)90301-t. [DOI] [PubMed] [Google Scholar]
  10. McNiece I. K., Langley K. E., Zsebo K. M. Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages. Exp Hematol. 1991 Mar;19(3):226–231. [PubMed] [Google Scholar]
  11. McNiece I. K., Langley K. E., Zsebo K. M. The role of recombinant stem cell factor in early B cell development. Synergistic interaction with IL-7. J Immunol. 1991 Jun 1;146(11):3785–3790. [PubMed] [Google Scholar]
  12. Nocka K., Buck J., Levi E., Besmer P. Candidate ligand for the c-kit transmembrane kinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors. EMBO J. 1990 Oct;9(10):3287–3294. doi: 10.1002/j.1460-2075.1990.tb07528.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Reddy V. Y., Desorchers P. E., Pizzo S. V., Gonias S. L., Sahakian J. A., Levine R. L., Weiss S. J. Oxidative dissociation of human alpha 2-macroglobulin tetramers into dysfunctional dimers. J Biol Chem. 1994 Feb 11;269(6):4683–4691. [PubMed] [Google Scholar]
  14. Strandberg L., Lawrence D. A., Johansson L. B., Ny T. The oxidative inactivation of plasminogen activator inhibitor type 1 results from a conformational change in the molecule and does not require the involvement of the P1' methionine. J Biol Chem. 1991 Jul 25;266(21):13852–13858. [PubMed] [Google Scholar]
  15. Teh L. C., Murphy L. J., Huq N. L., Surus A. S., Friesen H. G., Lazarus L., Chapman G. E. Methionine oxidation in human growth hormone and human chorionic somatomammotropin. Effects on receptor binding and biological activities. J Biol Chem. 1987 May 15;262(14):6472–6477. [PubMed] [Google Scholar]
  16. Vale W., Spiess J., Rivier C., Rivier J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science. 1981 Sep 18;213(4514):1394–1397. doi: 10.1126/science.6267699. [DOI] [PubMed] [Google Scholar]
  17. Zsebo K. M., Wypych J., McNiece I. K., Lu H. S., Smith K. A., Karkare S. B., Sachdev R. K., Yuschenkoff V. N., Birkett N. C., Williams L. R. Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver--conditioned medium. Cell. 1990 Oct 5;63(1):195–201. doi: 10.1016/0092-8674(90)90300-4. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES