A spectrophotometric study on the interaction of hemoglobin in a diazo coupling reaction of neonatal bilirubin: II. Interaction of bilirubin and hemoglobin in the Jendrassik-Grof diazo coupling reagents

The foregoing experiments have shown some of the difficulties in establishing a mechanism of action for hemoglobin in diazo coupling reactions for bilirubin. All attempts to understand the reaction are complicated by a variety of factors, including the diversity of the reactions generally referred to as Jendrassik-Grof, the heterogeneity of the reaction matrix, and the potential multiple interactions of any generated intermediate or product with other constituents present in the diazo reagents. Probing of the reaction mechanism with an enzyme such as catalase seems to be of questionable value at this point. Because of the unusually high concentrations of enzyme required to inhibit azobilirubin fading, there is a greater possibility for side reactions in the diazo media. The possibility of the occurrence of a Fenton reaction with the spontaneous generation of a superoxide ion or OH radical by a chelated ferrous species cannot be conclusively defined from the several experiments described. However, a hint involving peroxide formation is suggested by the catalase and SOD experiments.