藻蓝蛋白酶解肽的分离纯化及其细胞毒活性

Purification and Cytotoxicity of C-Phycocyanin(C-PC) from Spirulina platensis and Its Tryptic Peptides

研究钝顶螺旋藻(Spirulina platensis)藻蓝蛋白(C-phycocyanin,C-PC)及其胰蛋白酶水解肽的分离纯化。采用反复冻融和超声破碎法破碎细胞,用28～55g/100mL硫酸铵沉淀反复盐析获得纯度(A620nm/A280nm)为2.19的藻蓝蛋白,再通过羟基磷灰石(HA)柱层析和Sephacryl S-200 HR凝胶层析对其进行纯化,得到纯度(A620nm/A280nm)为3.89藻蓝蛋白。纯化后的藻蓝蛋白在40℃条件下经胰蛋白酶酶解60min后,用DEAE-Sepharose Fast Flow柱层析对酶解肽产物进行分离,收集得到4组藻蓝蛋白酶解肽。采用MTT方法,研究藻蓝蛋白、酶解混合液、分离的4个酶解肽组分对肿瘤细胞系HeLa和293T增殖的影响。结果显示:肽组分1和4对HeLa细胞的抑制率分别为37.71%和47.04%,而混合肽和藻蓝蛋白抑制率分别为34.02%和26.03%,因此活性肽组分1和4显示出较好的肿瘤抑制效果,而这两种活性肽组分对正常细胞293T并无细胞毒活性,特别是组分4效果最明显,是极具开发潜力的保健产品。

This paper deals with the separation,purification and cytotoxicity of C-phycocyanin（C-PC） from Spirulina platensis and its tryptic peptides.Repeated freezing and thawing coupled with ultrasonic treatment was used for disrupting the cell wall of Spirulina platensis.The purity（A620nm/A280nm） of C-PC was 2.19 after fractional precipitation by 28-55 g/100 mL（NH4）2SO4 and could reach 3.89 after further purification by sequential chromatography on hydroxylapatite（HA） column and Sephacryl S-200 HR gel column.The purified C-PC was hydrolyzed by trypsin at 40 ℃ for 60 min.Four peptides were obtained from the hydrolysate of C-PC by DEAE-Sepharose Fast Flow column chromatography.The cytotoxicity of C-PC and its hydrolysate as well as the 4 peptides on HeLa and 293T cells was evaluated by MTT assay.The results showed that the inhibition rates of peptide fractions I and IV,C-PC hydrolysate and C-PC on the growth of HeLa cells were 37.71%,47.04%,34.02% and 26.03%,respectively.Therefore,peptide fractions I and IV revealed obvious suppressive effect on the proliferation of cancer cells,while neither of them had cytotoxicity on 293T cells.Moreover,peptide fraction IV had the strongest tumor suppression activity,indicating a great potential to be developed as health-care products.