Extraction and Characterization of High-Molecular-Weight CPIs from Silver Carp Liver

Abstract:

In this study，different concentrations of the serine protease inhibitors phenylmethanesulfonyl fluoride（PMSF），different heat treatment and different pH were conducted to treat on the crude extraction of high-molecular-weight cysteine proteinase inhibitors（CPIs） from Silver carp liver，and we investigated their effect on the protein response values and specific activity of CPIs by HPLC with column of TSK-GEL G2000SW. The optimum extraction conditions were as follows：the condition of extraction buffer is containing 5 mmol/L PMSF，100 mmol/L Tris，and 3 mmol/L EDTA at pH 7.5，followed by pH readjustment to 7.0 after alkali（pH 8.7） treatment at 90 ℃ for 5 min. Then，the high-molecular-weight active CPI was obtained by chromatography on a Sephacryl S-200 column，and it was identified as a high-molecular-weight CPI by the reverse zymography method. Therefore，it may be conjugated with other protein or a dimeric protein.