Ultraviolet-Visible Spectral Characteristics and Mechanism Analysis of Purification of Phycobiliprotein by Column Chromatography
WU Kang1, WANG Jia-quan1，ZHAO Bing-bing2, FANG Yan1, ZHANG Fa-yu3*
1. School of Resources and Environmental Engineering, Hefei University of Technology, Hefei 230009, China
2. College of Civil Engineering, Hefei University of Technology, Hefei 230009, China
3. School of Electronic Science and Applied Physics, Hefei University of Technology, Hefei 230009, China
Abstract：UV-Vis absorption spectroscopy can be applied not only to the analysis of phycobiliprotein species and purity, but also to the analysis and guidance of its extraction and purification processes. In this paper, the fresh cyanobacteria of Chaohu Lake were used as experimental material, and the phycocyanin and allophycocyanin were refined by column chromatography using Cellufine A-500 and hydroxyapatite as fillers. According to the characteristics of the elution peaks corresponding to the two kinds of fillers on the elution curve, making full use of the difference in the UV-visible spectral characteristics of phycoerythrin, phycocyanin, allophycocyanin and nucleic acid, carotenoids，general proteins, so the dynamic change of the elution peak composition and content can be judged. UV-Visible absorption spectroscopy was used to study the spectral characteristics and variation of phycobiliprotein elution peaks by two kinds of packed column chromatography, the change of composition and content of each elution peak can be qualitatively and quantitatively determined; combining the characteristics of the two kinds of fillers, the charge characteristics and coordination ability of phycocyanin, allophycocyanin, phycoerythrin, etc. can be analyzed, revealing the intrinsic mechanism and essence of the fractional elution of the two column chromatography materials. During the purification of phycobiliprotein by Cellufine A-500, four elution peaks appeared on the elution curve with the replacement of the eluate. After scanning the ultraviolet-visible absorption spectrum of the sampling point, it was found that: The main component of peak I is positively charged or electrically neutral hetero protein and carotenoid; the main component of peak Ⅱ is phycoerythrin, heteroprotein and nucleic acid with a small amount of negative charge; the main component of the peak Ⅲ is high-purity phycocyanin with a large negative charge and a small amount of allophycocyanin, and the further improvement of the purity of phycocyanin is restricted due to the incomplete separation of phycocyanin and allophycocyanin; The main component of the peak Ⅳ is a hetero protein and a low-purity phycocyanin with a large amount of negative charge. In the process of purifying phycobiliprotein by hydroxyapatite, three elution peaks appeared on the elution curve with the replacement of the eluate. After scanning the ultraviolet-visible absorption spectrum of the sampling point, it was found that: The main component of the peak I iscationic or basic protein, such as hetero protein，nucleic acid, carotenoid, or the like; The main component of the peak Ⅱ is high-purity phycocyanin which combines with calcium ions to form a weak coordination bond, and the phycocyanin and the allophycocyanin can be completely separated, which is beneficial to further improvement of the purity of the phycocyanin; The main component of the peak Ⅲ is a high-purity allophycocyanin which combines with calcium ions to form a strong coordinate bond.
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