Abstract: 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 water-soluble 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.

Key words: Eubranchipus vernalis, water-soluble protein, purification, antioxidant activity