Separation and identification of CPIs from grass carp hepto-pancreas and its activity assay

The applicability of the methods of azocasein and fluorescence synthetic peptide in monitoring the inhibitory activity of CPIs(cysteine proteinase inhibitors) from grass carp hepto-pancreas was firstly evaluated.Then the molecular weight distribution and the possible superfamily classification of CPIs were identified by gel filtration HPLC combining with reversed-phase enzyme spectrometry and western blotting methods. The results showed that after adding serine protease inhibitors(40 mmol/L PMSF or 0.4 mmol/L Pefabloc SC) into the crude extract or heating treatment at 90 ℃ for 5 min,the inhibitory activity of CPIs could accurately monitored only by the fluorescence synthetic peptide method. Furthermore,the results of HPLC disclosed that there were several forms of active CPIs with different molecular weight(98,26,12,5~1 ku) in grass carp hepto-pancreas.However,only the CPIs more than 20 ku could be determined by the reverse zymography method. In addition,the results from western blotting revealed the existence of CPIs belonging to Cystatin( family II) with the molecular weight about 12 ku and the developing band less than 12 ku was speculated to be the active fragment degraded from the Cystatin protein.