柱层析法纯化藻胆蛋白的紫外-可见光谱特征研究与机理分析

紫外-可见吸收光谱法不仅可以应用于分析藻胆蛋白种类以及纯度,还可以用于分析并指导其提取纯化过程。以巢湖新鲜蓝藻为实验原料,以CellufineA-500与羟基磷灰石为填料,运用柱层析法精致纯化藻蓝蛋白和别藻蓝蛋白;根据两种填料对应的洗脱峰在洗脱曲线上的特点,充分利用藻红蛋白、藻蓝蛋白、别藻蓝蛋白与核酸、类胡萝卜素、一般蛋白质的紫外-可见光谱特性的差别,研判洗脱峰组分与含量的动态变化。通过紫外-可见吸收光谱法分段研究两种填料柱层析法精致纯化藻胆蛋白洗脱峰的光谱学特征及其变化规律,能够定性定量地判断出各洗脱峰的组分和含量变化;结合两种填料的特性,能够分析出藻蓝蛋白、别藻蓝蛋白、藻红蛋白等的电荷特性与配位能力强弱,从而揭示两种柱层析填料分段洗脱的内在机理和本质。在CellufineA-500纯化藻胆蛋白过程中,随着洗脱液的更换,洗脱曲线上会出现4个洗脱峰,经扫描取样点的紫外-可见吸收光谱分析后发现:Ⅰ峰主要成分为带正电荷或电中性的杂蛋白与类胡萝卜素;Ⅱ峰主要成分为带少量负电荷的藻红蛋白、杂蛋白与核酸;Ⅲ峰主要成分为带有较多负电荷的高纯度藻蓝蛋白及少量别藻蓝蛋白,且由于藻蓝蛋白与别藻蓝蛋白未能完全分离,制约了藻蓝蛋白纯度的进一步提高;Ⅳ峰主要成分为带有大量负电荷的杂蛋白与低纯度藻蓝蛋白。在羟基磷灰石纯化藻胆蛋白过程中,随着洗脱液的更换,洗脱曲线上出现3个洗脱峰,经扫描取样点的紫外-可见吸收光谱后发现:Ⅰ峰主要表征为阳离子或碱性蛋白质的杂蛋白、核酸与类胡萝卜素成分等;Ⅱ峰主要表征为与钙离子结合生成较弱配位键的高纯度藻蓝蛋白成分,且由于藻蓝蛋白与别藻蓝蛋白能完全分离,有利于藻蓝蛋白纯度的进一步提升;Ⅲ峰主要表征为与钙离子结合生成较强配位键的高纯度别藻蓝蛋白成分。

Ultraviolet-Visible Spectral Characteristics and Mechanism Analysis of Purification of Phycobiliprotein by Column Chromatography

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 peakⅡis phycoerythrin,heteroprotein and nucleic acid with a small amount of negative charge;the main component of the peakⅢis 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 peakⅣis 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 peakⅡis 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 peakⅢis a high-purity allophycocyanin which combines with calcium ions to form a strong coordinate bond.