Lauff J J, Kasper M E, Wu T W, Ambrose R T
Clin Chem. 1982 Apr;28(4 Pt 1):629-37.
We have isolated from pathological sera a bilirubin fraction (delta) that is very tightly, if not covalently, bound to protein, most likely albumin. This delta fraction absorbed at a lambda max of 433 nm in the visible spectrum, between the lambda max of unconjugated (alpha) and that of conjugated (Bc) bilirubin when measured in solutions containing albumin. However, unlike the other bilirubin species, this fraction could not be separated from the proteins in serum by exhaustive ultrafiltration in the presence of caffeine/benzoate solution. In the Jendrassik-Grof diazo procedure for bilirubin analysis, the delta fraction gave a large direct reaction (76-89% of the total reaction). Yet, when relatively hydrophobic azo dyes were formed by reaction of the delta fraction with the diazonium salt of dichloroaniline, only 50% of the dyes were extractable from aqueous solution. On chromatography the rest remained associated with protein. Of the extractable dye, more than 70% was accounted for by two liquid-chromatographic peaks with retentions identical with those of azo dyes formed from unconjugated bilirubin. This delta fraction was not appreciably separated from protein by treatment with strong acid or base, or by prolonged digestion with various enzymes. Finally, in a highly denaturing solvent (urea/mercaptoethanol), this fraction was not dialyzable through a membrane with a 12 000-dalton cutoff.
我们从病理性血清中分离出一种胆红素组分（δ），它即便不是通过共价键，也是与蛋白质紧密结合，很可能是与白蛋白结合。在含有白蛋白的溶液中进行测量时，这种δ组分在可见光谱中的最大吸收波长为433nm，处于未结合胆红素（α）和结合胆红素（Bc）的最大吸收波长之间。然而，与其他胆红素种类不同的是，在咖啡因/苯甲酸盐溶液存在的情况下，通过彻底超滤无法将该组分与血清中的蛋白质分离。在用于胆红素分析的Jendrassik-Grof重氮法中，δ组分产生很大的直接反应（占总反应的76 - 89%）。然而，当δ组分与二氯苯胺重氮盐反应形成相对疏水的偶氮染料时，只有50%的染料可从水溶液中萃取出来。在色谱分析中，其余的染料仍与蛋白质结合。在可萃取的染料中，超过70%由两个液相色谱峰组成，其保留时间与由未结合胆红素形成的偶氮染料相同。用强酸或强碱处理，或用各种酶长时间消化，都不能明显地将这种δ组分与蛋白质分离。最后，在一种高度变性的溶剂（尿素/巯基乙醇）中，该组分不能透过截留分子量为12000道尔顿的膜进行透析。
