Open AccessSpecific covalent modification of the tryptophan residues in murine interleukin‐6
Author(s) -
ZHANG JianGuo,
REID Gavin E.,
MORITZ Robert L.,
WARD Larry D.,
SIMPSON Richard J.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18217.x
Subject(s) - tryptophan , chemistry , edman degradation , circular dichroism , peptide , urea , recombinant dna , covalent bond , trypsin , biochemistry , amino acid , peptide sequence , chromatography , stereochemistry , enzyme , organic chemistry , gene
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 [ΔΔG(H 2 O) = 3.98 kJ/mol] than unmodified mIL‐6 as measured by urea‐gradient gel electrophoresis.