Kinetics of the reactions of unconjugated and conjugated bilirubins with p-diazobenzenesulfonic acid.

We report the kinetics of the reactions of unconjugated bilirubin and conjugated bilirubin with p-diazobenzene sulfonic acid in aqueous media. Our studies confirm that each reaction proceeds in two steps and that the second step is catalyzed by sulfanilic acid. In the presence of an excess of p-diazobenzenesulfonic acid and in the absence of sulfanilic acid, the reaction for either unconjugated or conjugated bilirubin proceeds in two successive first-order steps, the second step being much the slower. This study emphasizes the first step in the reaction for each species and includes data on the effects of p-diazobenzenesulfonic acid, albumin, benzoate, and caffeine concentrations, pH in the range from 4 to 12, and temperature. Mechanisms proposed for reactions with and without caffeine are used to develop rate equations, and the kinetic data are used to evaluate rate constants, acid dissociation constants for the different bilirubin species, and formation constants for bilirubin-caffeine complex species that are proposed.