BILIRUBIN-PROTEIN LINKAGES IN SERUM AND THEIR RELATIONSHIP TO THE VAN DEN BERGH REACTION 1

Abstract

By means of filter-paper electrophoresis is was shown that most, if not all, of the bilirubin in serum is bound to albumin irrespective of its van den Bergh reaction. Evidence presented to show that indirect bilirubin is not a glo-bin complex include the demonstration that (a) the electrophoretic mobility of native globin differs from that of serum bilirubin, (b) the direct-and indirect-reacting pigments cannot be sep-erated at pH 6.0, a level at which albumin and globin migrate toward opposite poles, and (c) the solubility and bilirubin-bind-ing capacity of globin at the normal pH of blood are so low that a globin complex, if present, could not account for more than 1 mg of bound pigment per 100 ml of serum. Attempts to corroborate previous reports that the 2 types of pigment in serum can be separated by ammonium sulfate fractionation have failed. It was found that both pigments remain firmly bound to albumin between pH 6.0 and 9.0, and separate below pH 5.0. At pH 5.0 the indirect type separates, while the direct remains bound to albumin, suggesting a difference in protein linkage. Since the van den Bergh reaction is usually carried out at pH 2.0 to 4.0, it must be assumed that both types of bilirubin are in the unbound state when they react so that the type of reaction cannot be dependent on differences in protein linkage. The implications of these observations are discussed and the possibility considered that the variable behavior of bilirubin on diazotiza-tion may be related to differences in the solubility of 2 closely related but structurally different pigments.