Separation and quantitation of phycobiliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection

The similar electrophoretic mobilities and sizes of several of the phycobiliproteins, which are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae, render their separation and quantitation a challenging problem. However, we have developed a suitable capillary electrophoresis (CE) method that employs a phytic acid–boric acid buffer and laser-induced fluorescence (LIF) detection with a single 594 nm He–Ne laser. This method takes advantage of the remarkably high quantum yields of these naturally fluorescent proteins, which can be attributed to their linear tetrapyrrole chromophores covalently bound to cysteinyl residues. As such, limits of detection of 1.18·10−14, 5.26·10−15, and 2.38·10−15 mol/l were obtained for R-phycoerythrin, C-phycocyanin, and allophycocyanin proteins, respectively, with a linear dynamic range of eight orders of magnitude in each case. Unlike previously published CE–LIF methods, this work describes the separation of all three major classes of phycobiliproteins in under 5 min. Very good recoveries, ranging from 93.2 to 105.5%, were obtained for a standard mixture of the phycobiliproteins, based on seven-point calibration curves for both peak height and peak area. It is believed that this development will prove useful for the determination of phycobiliprotein content in naturally occurring cyanobacteria populations, thus providing a useful tool for understanding biological and chemical oceanographic processes.