Zhang J G, Reid G E, Moritz R L, Ward L D, Simpson R J
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Parkville, Australia.
Eur J Biochem. 1993 Oct 1;217(1):53-9. doi: 10.1111/j.1432-1033.1993.tb18217.x.
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.
在温和酸性条件（0.1 M醋酸钠，pH 3.5）下，用色氨酸特异性试剂2-硝基苯基亚磺酰氯修饰重组鼠白细胞介素-6（mIL-6），得到一种衍生物，其含2.02摩尔2-硝基苯基亚磺酰色氨酸/摩尔蛋白质。修饰位点鉴定为Trp36和Trp160。如内蛋白酶Asp-N肽图谱分析、埃德曼降解和电喷雾质谱联用所示，分子中的其他氨基酸未发生可检测到的副反应。mIL-6中两个色氨酸残基的亚磺酰化导致在使用7TD1细胞的鼠杂交瘤生长因子测定中的生物活性以及与mIL-6受体的受体结合亲和力均降低50%。通过远紫外圆二色性测量以及与中和性抗mIL-6单克隆抗体6B4的结合情况表明，mIL-6的亚磺酰化对蛋白质的整体构象没有显著影响。然而，通过尿素梯度凝胶电泳测量，亚磺酰化蛋白质的稳定性明显低于未修饰的mIL-6 [ΔΔG(H2O) = 3.98 kJ/mol]。
