Perdew G H, Poland A
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
J Biol Chem. 1988 Jul 15;263(20):9848-52.
The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.
芳烃受体的光亲和配体[125I]-2-叠氮基-3-碘-7,8-二溴二苯并-对-二恶英，先前已被证明可选择性标记C57BL/6J小鼠肝脏胞质溶胶组分中的两种肽：一种95 kDa的肽，即芳烃受体的配体结合部分，以及由较大肽形成的70 kDa蛋白水解片段（波兰，A.，格洛弗，E.，埃贝蒂诺，F.H.，和肯德，A.S.（1986年）《生物化学杂志》261，6352 - 6365）。通过以下方案将这两种肽部分纯化至约20,000倍富集，产率为15 - 20%：1）对肝脏胞质溶胶35 - 55%硫酸铵级分进行光亲和标记；2）在低电荷密度偶联的聚乙烯亚胺 - 琼脂糖上进行色谱分离，并用肝素/Mn2 +沉淀稀释的柱洗脱液；3）用DEAE - 琼脂糖色谱法去除肝素；4）在肝素 - 琼脂糖上进行色谱分离；5）进行制备性十二烷基硫酸钠 - 聚丙烯酰胺平板凝胶电泳，然后对蛋白质进行电洗脱和离子对萃取以去除十二烷基硫酸钠；6）在反相C - 4柱上进行高效液相色谱。在聚乙烯亚胺琼脂糖上进行初始色谱分离后，发现只有在变性条件下才能实现大量的后续纯化。
