A novel method of single step hydrophobic interaction chromatography for the purification of phycocyanin from Phormidium fragile and its characterization for antioxidant property

Phycocyanin--a major phycobiliprotein constitutively produced by many cyanobacteria--holds several promising applications in diagnostics, biomedical research, and therapeutics. This paper discusses a novel rapid method for the purification of cyanobacterial phycocyanin (C-PC) from Phormidium fragile using hydrophobic interaction chromatography. The protein was extracted and concentrated by grinding under liquid nitrogen and ammonium sulfate fractionation. C-PC was purified by single step hydrophobic interaction chromatography. Purified phycocyanin showed absorbance maximum (lambda(max)) at 624 nm. The criterion of purity (R) achieved was 4.52. Phycocyanin to phycoerythrin and phycocyanin to allophycocyanin purity ratio were 3.85 and 7.49, respectively. The purified protein showed a pI of 5.2 and has two subunits with molecular mass of 19 and 20 kDa each, corresponding to its highly reported alpha and beta subunits. The subunits of phycocyanin were confirmed by their bilin fluorescence using zinc assisted fluorescence enhancement technique. Intact C-PC was of 125 kDa as determined by HPLC, suggested the (alphabeta)(3) subunit assembly. Results obtained by this method in terms of purity, recovery, process time, simplicity, and efficacy are much better than previous methodologies. Purified phycocyanin was further scrutinized for its antioxidant capacity and judged against five non-enzymatic antioxidants by FRAP assay.