丰年虫水溶性蛋白的分离纯化及体外抗氧化性研究

目的:对丰年虫水溶性蛋白进行分离纯化,获得体外抗氧化活性最好的水溶性蛋白组分,并测定其分子质量。方法:丰年虫水溶性粗蛋白经硫酸铵沉淀、透析、DEAE-Sepharose FF离子交换层析和Sephadex G-100凝胶层析分离纯化,对纯化后的蛋白质进行体外抗氧化活性的研究,利用SDS-PAGE电泳测定蛋白质分子质量。结果:丰年虫水溶性蛋白经DEAE-Sepharose FF离子交换层析,梯度洗脱得到4个洗脱峰(峰1～4),峰2的体外抗氧化活性最强。峰2经Sephadex G-100凝胶层析分离纯化得到3个洗脱峰(峰2-1～2-3),峰2-1的体外抗氧化活性最强。利用SDS-PAGE对峰2-1进行鉴定,得到单一蛋白条带,纯度较高,蛋白分子质量为82.47kD。体外抗氧化活性比较,峰2-1远远强于水溶性粗蛋白。结论:蛋白组分峰2-1体外抗氧化活性最强,为单一蛋白组分,其分子质量为82.47kD。

Antioxidant Activity of Water-Soluble Protein Purified from Eubranchipus vernalis

Objective: To isolate and purify water-soluble protein with the highest antioxidant activity from Eubranchipus vernalis and determine its molecular weight.Methods: Crude water-soluble protein from Eubranchipus vernalis was extracted and purified by ammonium sulfate precipitation,salting out,ion-exchange column chromatography,DEAE-fast-flow Sepharose anion-exchange chromatography and Sephadex G-100 column chromatography.The antioxidant activities of different protein fractions were compared and their molecular weights were determined by SDS-PAGE.Results: The prepared crude watersoluble protein was mainly composed of 4 fractions named as Peak-1,Peak-2,Peak-3 and Peak-4 in DEAE-Sepharose fast-flow column chromatography and Peak-2 revealed higher antioxidant activity than other 3 fractions.In addition,Peak-2 was mainly composed of 3 sub-fractions including Peak 2-1,Peak 2-2 and Peak 2-3 in Sephadex G-100 gel-permeation column chromatography.Peak 2-1 exhibited higher antioxidant activity than other 2 sub-fractions.Peak 2-1 was identified by SDS-PAGE to reveal a molecular weight of 82.47 kD.The antioxidant activity of Peak 2-1 was much higher than that of the crude protein extract.Conclusion: Peak 2-1 has obtained from Eubranchipus vernalis as the most antioxidant protein with a molecular weight of 82.47 kD.