Purification and characterization of a novel antioxidant protein from Arca subcrenata Lischke.

Abstract

A novel protein, G1H2GC2, was isolated from Arca subcrenata Lischke using homogenization and ammonium sulfate precipitation, and further purified by several column chromatography techniques including diethyl-aminoethanol (DEAE) Sepharose Fast Flow anion exchange, gel filtration chromatography (Sephadex G-25), Phenyl Sepharose CL-4B hydrophobic chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). The purity of G1H2GC2 was over 97% in RP-HPLC, and its high purity was further verified by the appearance of a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein content of G1H2GC2 was found to be over 99% by Bradford assay. The molecular weight was determined as 25.6 kDa by electrospray ionization-mass spectrometry (ESI-MS/MS). The isoelectric point of G1H2GC2 was measured to be 7.71 by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) was employed to detect and identify the protein by mass fingerprinting coupled with fragmentation patterns. No matched protein was found, confirming that G1H2GC2 was a novel protein. An attempt was made to detect the N-terminal amino acid sequence of G1H2GC2 by Edman degradation, but the sequence of G1H2GC2 was found to be blocked. The scavenging percentage of G1H2GC2 at 15 mg/mL against 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+) was 52.84%. The median effective concentration (EC₅₀) value of G1H2GC2 against ABTS^•+ was 17.67 mg/mL. The results showed that G1H2GC2 might be developed as a potential food additive agent.