Disulfide assignments in recombinant mouse and human interleukin 4

Biochemistry. 1991 Feb 12;30(6):1515-23. doi: 10.1021/bi00220a011.


The disulfide pairings of mouse and human interleukin 4 (IL-4) proteins have been determined. The purified proteins, synthesized by recombinant DNA technology, are fully active as judged by their ability to stimulate an appropriate biological response in a variety of functional assays. Peptide maps were produced by digesting the proteins with pepsin and separating the resulting fragments by reverse-phase HPLC using linear acetonitrile-TFA gradients. Cystine-containing peptides were identified by determining which reverse-phase peaks showed an altered elution pattern after reduction. These peptides were purified further and defined by composition and sequence analysis. Three sets of disulfide-linked peptides were consistently identified for each protein. For mouse IL-4, the first and fifth, second and fourth, and third and sixth cysteines are joined. The disulfide bonds in human IL-4 are between the first and sixth, second and fourth, and third and fifth cysteines. A large double-loop region within the central three-fifths of each protein is stabilized by these bonds. Sequence analysis of the peptides containing the third and fifth cysteines of human IL-4 also demonstrated that only one of the potential N-glycosylation sites is used by C127 mammary tumor cells. Complete alkylation of mouse IL-4 under mild conditions completely destroyed its biological activity in a hematopoietic precursor cell proliferation assay.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Division / drug effects
  • Cell Line
  • Cloning, Molecular
  • Disulfides / analysis*
  • Dithiothreitol / pharmacology
  • Escherichia coli / genetics
  • Humans
  • Interleukin-4 / chemistry*
  • Interleukin-4 / genetics
  • Interleukin-4 / isolation & purification
  • Interleukin-4 / pharmacology
  • Mice
  • Molecular Sequence Data
  • Protein Conformation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology
  • Sequence Homology, Nucleic Acid


  • Disulfides
  • Recombinant Proteins
  • Interleukin-4
  • Dithiothreitol