Drug Discov Ther. 2025;19(5):304-313. (DOI: 10.5582/ddt.2025.01053)
Purification and characterization of a novel antioxidant protein from Arca subcrenata Lischke
Yan Q, Shi H, Gao Y, Li CL, Dong L, Zhu JH, Yu RM
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 diethylaminoethanol (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.
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