Etter-kjelsaas H, Kuenzle C C
Biochim Biophys Acta. 1975 Jul 21;400(1):83-94. doi: 10.1016/0005-2795(75)90129-4.
An alkali-stable bilirubin conjugate has been obtained from human T-tube bile as its phenylazo derivative. The conjugate consists of a polypeptide probably of molecular weight 7000 to which the azo pigment of bilirubin is linked covalently through its carboxyl group. It thus constitutes the first biliprotein found in mammals. It is not known whether both carboxyl groups of native bilirubin participate in the binding of the conjugating protein, nor has it been possible to determine the number of pigment moieties occurring on a single polypeptide chain. The isolation makes use of the tendency of the conjugate to form large aggregates and involves the following steps: azo coupling of the native bile, (NH4)2S04 precipitation of macromolecules and aggregates, removal of low molecular weight contaminants by dialysis and gel filtration (first on Sepharose 6B IN 6 M guanidine, then on Sephadex LH-20 in 50% acqueous 2-chloroethanol) and a concluding purification by chromatography on p-aminobenzyl cellulose using a PH gradient. The final preparation appeared to be homogeneous on polyacrylamide electrophoresis.
已从人T形管胆汁中获得一种碱稳定的胆红素共轭物，即其苯偶氮衍生物。该共轭物由一种分子量可能为7000的多肽组成，胆红素的偶氮色素通过其羧基与之共价连接。因此，它构成了在哺乳动物中发现的第一种胆蛋白。尚不清楚天然胆红素的两个羧基是否都参与结合共轭蛋白，也无法确定单条多肽链上色素部分的数量。该分离利用了共轭物形成大聚集体的倾向，包括以下步骤：天然胆汁的偶氮偶联、大分子和聚集体的硫酸铵沉淀、通过透析和凝胶过滤去除低分子量污染物（先在6M胍中的琼脂糖6B上，然后在50%含水2 - 氯乙醇中的葡聚糖凝胶LH - 20上），最后在对氨基苄基纤维素上使用pH梯度进行色谱纯化。最终制剂在聚丙烯酰胺电泳上似乎是均一的。
