Phycocyanin 645. The chromophore assay of phycocyanin 645 from the cryptomonad protozoa Chroomonas species.

Phycocyanin 645 was isolated and purified from the cryptomonad Chroomonas species. Its chromophore content was obtained from absorption spectra in acidic 8.0 M urea for both whole protein and the separated subunits. The principal method used to separate the alpha and beta subunits was gel filtration through a Sephacryl S-200 column in acidic urea. The subunits were shown to be completely separated during this procedure by sodium dodecyl sulfate-gel electrophoresis. Spectra were analyzed by three component Beer's law equations. The whole protein was found to consist of four phycocyanobilins (Amax at 662 nm), two cryptoviolins (Amax at 590 nm), and two unnamed bilins with an Amax at 697 nm. The separated subunits were analyzed, and the beta subunit was shown to have two phycocyanobilins for each cryptoviolin and alpha was composed of the 697-nm bilin exclusively. A comparison of the total amounts of alpha and beta from the Sephacryl columns showed that the molar ratios of phycocyanobilin on beta to the 697-nm bilin on alpha was 2:1, and the ratio of cryptoviolin on beta to 697-nm bilin on alpha was 1:1. We therefore propose that, assuming a symmetrical distribution, each beta subunit on the alpha 2 beta 2 protein has two phycocyanobilins and one cryptoviolin and each alpha subunit has one 697-nm bilin. This chromophore distribution differs from one previously reported in which the subunits were separated on a BioRex 70 cation exchange resin in 12% formic acid via a 4-10 M urea gradient.