Purification and characterization of calpastatin from grass prawn muscle (Penaeus monodon)

Calpastatin, a specific calpain inhibitor was purified to electrophoretical homogeneity from grass prawn (Penaeus monodon) muscle by 100 degrees C heat-treatment, DEAE-Sephacel, and Q-Sepharose chromatographs. No significant change in the inhibitory activity of crude calpastatin was observed even after 20 min incubation at 100 degrees C, pH 7.0. The purified prawn calpastatin had a molecular weight (M(r)) of 80 and 88.7 kDa determined by SDS-PAGE and Sephacryl S-200 HR gel filtration, respectively. According to the active site titration, the purified calpastatin revealed four beef mu-calpain and two beef m-calpain binding domains, respectively. It was stable during 1 h of incubation at 30 degrees C under pH 4.5-10.0 and shown to be a highly specific inhibitor for calpain.