Cleavage of phycocyanobilin from C-phycocyanin. Separation and mass spectral identification of the products.

The chromophore of C-phycocyanin, phycocyanobilin, was cleaved from the protein with methanol, concentrated hydrochloric acid, or subtilisin BPN'. The pigments obtained were converted to their dimethyl esters and purified by preparative high pressure liquid chromatography and examined for purity by analytical high pressure chromatography on silica gel. They were characterized by proton transfer and electron impact mass spectroscopy. The principal product obtained by the three cleavage procedures was phycocyanobilin. Methanol and hydrochloric acid adducts of phycocyanobilin were obtained with methanol and concentrated hydrochloric acid cleavages, respectively. Methanol adduct formation of phycocyanobilin can occur subsequent to cleavage and requires acid catalysis. No adduct formation was observed with mesobiliverdin under similar conditions. These results and mass spectral data support the conclusion that adduct formation takes place at the exocyclic olefin linkage of ring A in phycocyanobilin. The ease of co-valent adduct formation strongly suggests that the ethylidene side chain is an important binding site of phycocyanobilin to the polypeptide chain.