Superoxide anion-scavenging activity of alkaline protease hydrolysate from sardine meat

Superoxide anion-scavenging activity (SOSA) of alkaline protease hydrolysate from sardine meat (A-1) was measured by colorimetry using water-soluble tetrazolium salt (WST-1). A-1 showed low activity with IC ₅₀ value of 9.21 mg powder/ml. The hydrolysate was separated into F-1-F-6 on an ODS column. The most, active fraction (F-4) was fractionated by ion exchange chromatography on SP-Sephadex C-25 into three active fractions (1/SP-3/SP). The first fraction, 1/SP, showed the most potent activity (IC ₅₀=4.11 mg powder/ml). By elution with a stepwise gradient of acetonitrile containing 0.1% TFA on Sep-pak plus C18 cartridge, A/SE (IC ₅₀=2.12 mg powder/ml) and B/SE (IC₅₀=7.14 mg powder/ml) were obtained from 1/SP. A/SE was extracted with water or 20% acetonitrile. A/ SE W, the water extract, showed high SOSA with an IC₅₀ value of 0.51 mg powder/ml. The amino acid analysis of each active fraction indicated that five amino acid residues (Leu, Phe, Pro, Met, Tyr) played important roles in the activity. Moreover, it was suggested that the potent scavenger contained few histidine residue.