A polypeptide conjugate of bilirubin from human bile.

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.