Antioxidant and antidiabetic activities of vanadium binding proteins purified from the sea squirt Halocynthia roretzi

Minoli Anuththara Gunasinghe, Sang Moo Kim

Sea squirts accumulate vanadium compounds with potent antidiabetic activity, which are involved in immune defense. In this study, vanadium concentrations of fresh blood plasma, intestine, and muscle of the sea squirt Halocynthia roretzi were 6.3, 3.7 and 2.1 mg/kg respectively. Two vanadium binding proteins (VBPs) from blood plasma and intestine were purified through (NH4)2SO4 precipitation, and DEAE-Sepharose ion exchange and Sephacryl S-200 HR gel filtration chromatography, in that order. The purity and yield of the intestine and blood plasma vanadium binding proteins, VBPintestine and VBPblood plasma, were 13.4 folds and 7.1%, and 20.9 folds and 6.8%, respectively. There were two protein bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 24.3 and 68.8 kDa and one with 96.7 kDa on the native-PAGE of VBPblood plasma, whereas only one protein band of VBPintestine on the SDS-PAGE with 26.5 kDa. Antioxidant activities of VBPs were lower than that of ascorbic acid. Both VBPs exerted strong inhibitory activity against Saccharomyces cerevisiae and mild against Bacillus stearothermophilus and rat intestinal α-glucosidase. IC50 values of VBPintestine and VBPblood plasma against S. cerevisiae α-glucosidase were 28.34 and 12.60 μg/ml, respectively. The K m , V max , k cat , and k cat /K m values of VBPintestine and VBPblood plasma were 4.29, 0.036, 6.58 and 1.53 × 103, and 7.63 mM, 0.057 mM/min, 10.41 s-1 and 1.36 × 103 (M sec)-1, respectively. There was a synergistic interaction between VBPblood plasma and VBPintestine on rat intestinal α-glucosidase inhibitory activity.