Lin J J, Patino M M, Gaffield L, Walden W E, Smith A, Thach R E
Department of Biology, Washington University, St. Louis, MO 63130.
Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6068-71. doi: 10.1073/pnas.88.14.6068.
Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. (We refer to this interaction as a "crosslink," without intending to imply knowledge as to its chemical nature.) Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. [14C]Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H2O2 is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.
将一种90千道尔顿的铁蛋白阻遏蛋白（FRP）与少量放射性标记的血红素一起温育，导致两者之间形成了一种对SDS/PAGE稳定的强相互作用。（我们将这种相互作用称为“交联”，并不意味着了解其化学性质。）在单独测试的其他七种蛋白质中，只有脱辅基血红素结合蛋白和牛血清白蛋白表现出类似的交联能力，尽管程度要低得多。在总小麦胚芽蛋白过量50倍的情况下，[14C]血红素特异性地与FRP交联。加入过氧化氢酶并不能阻止血红素与FRP的反应，这表明H2O2不参与其中。随后加入化学计量的脱辅基血红素结合蛋白并不能使反应逆转。用金黄色葡萄球菌V8蛋白酶彻底消化该复合物产生了一个17千道尔顿的主要标记肽段。这些结果表明FRP上存在一个高度特异性、独特反应性的血红素结合位点。
