Specific covalent modification of the tryptophan residues in murine interleukin-6. Effect on biological activity and conformational stability.
Modification of recombinant murine interleukin-6 (mIL-6) with the tryptophan-specific reagent 2-nitrophenylsulfenyl chloride under mild acidic conditions, 0.1 M sodium acetate, pH 3.5, yielded a derivative containing 2.02 mol 2-nitrophenylsulfenyl tryptophan/mol protein. The sites of modification were identified as Trp36 and Trp160. No detectable side reactions occurred on other amino acids in the molecule, as indicated by the combination of endoproteinase Asp-N peptide mapping, Edman degradation and electrospray mass spectrometry. Sulfenylation of the two tryptophan residues in mIL-6 caused a 50% reduction in both the biological activity in the murine-hybridoma-growth-factor assay using 7TD1 cells and receptor-binding affinity to mIL-6 receptors. Sulfenylation of mIL-6 did not significantly affect the overall conformation of the protein as measured by farultraviolet circular dichroism and binding to the neutralizing anti-mIL-6 mAb 6B4. The sulfenylated protein was, however, significantly less stable [delta delta G(H2O) = 3.98 kJ/mol] than unmodified mIL-6 as measured by urea-gradient gel electrophoresis.
Zhang
J G
JG
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Parkville, Australia.
Reid
G E
GE
Moritz
R L
RL
Ward
L D
LD
Simpson
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D002942:Circular Dichroism; D004355:Drug Stability; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015850:Interleukin-6 / Q000031:analogs & derivatives / Q000737:chemistry* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
D015850:Interleukin-6; D011994:Recombinant Proteins; C083928:interleukin-6, 2-nitrophenylsulfenyl-Trp(36,160)-; D014364:Tryptophan