The Diazo Reaction of Bilirubins and Phycorubins: A Quantitative Study*

A quantitative study o f the diazo reaction has been made with several bilirubins including a 2,3dihydrobilirubin and several phycorubins. The latter have been prepared from the integral phycocyanin of two cyanobacteria (Mastigocladus laminosus and Spirulina platensis), and from the phycocyanin subunits. These phycorubins have been subjected to the diazo reaction in order to test for the presence o f a second covalent chromophore protein bond in the latter. 1) The diazo reaction o f unsymmetrically substituted bilirubins, mesobilirubin (3) and bilirubin IX a (13) in aqueous solution yields four products, two isomeric 9-azo-dipyrrom ethenones, and two isomeric 9-hydroxymethyl-dipyrromethenones. The maximum total yield is > 97% with diazotized sulfanilic acid, and < 60% with diazotized ethylanthranilate. 2) The diazo reaction of the 2,3-dihydrobilirubin (16) yields likewise four products, two o f them containing the 2,3-dihydrogenated ring A/B fragment. The attack is regioselective at C l l (6:4). 3) The diazo reaction o f the phycorubins with ethylanthranilate yields two peptide bound products, containing the ring A/B fragment, and two low molecular weight products. The latter correspond to the C/D fragment and are identical with the respective products derived from mesobilirubin 3. The attack is preferential at C-9 (> 4:1 with < 1 mole reagent added). These results show, that there is no second covalent chromophore peptide bond in the PC investigated at the ring C/D fragment.