Study On Characterization And Bovine Myofibrillar Degradation Effect Of Cathepsin L From Bovine Pancreas

Tenderness,determined by the structure and biochemical properties of myofibrils and intramuscular connective tissue,has been considered as the most important indicator for beef edible quality.In this study,cathepsin L from bovine pancreas was isolated and purified through column chromatography technology,and characterized.Structural effect and proteolytic changes of bovine myofibrils were observed through protein gel electroforesis,transmission electron microscope and phase-contrast microscopy.Potential mechanisms for beef tenderization of purified cathepsin L was discussed,providing theoretical basis for its role in tenderization during postmortem ageing.The main contents and results of this study were showed as follow:Purification and identification of cathepsin L from bovine pancreas.0.58 mg purified enzyme was prepared from acidified crude extraction of fresh bovine pancreas homogenate followed by purification method of salting out,dialysis,concentration,and column chromatography,including DEAE Sephacel anion exchange,Sephacryl S-100 size exclusion,SP Sepharose F.F.cation exchange,Blue Sepharose 6 F.F.dye affinity and ConA Sepharose affinty.The purification fold was 530.54.The molecular weight of the purified enzyme was identified as 29.1 kDa on SDS-polyacrylamide gel electrophoresis(SDS-PAGE).Characterization of bovine pancreas cathepsin L.Results showed that the optimum reaction temperature for bovine pancreas cathepsin L was 50 ?,with an active temperature range of 20-50 ?,and its thermal stability decreased sharply as the ascention of reaction temperature.The optimum reaction pH was 6.5,with an active pH range of 4.0-7.5,and the enzyme activity remained over 70%after treated in pH 3.0-8.0 for 1 h.Bovine pancreas cathepsin L was efficiently activated by dithiothreitol(DTT)and L-cysteine(L-Cys),while it could be completely inhibited by 10?mol/L E-64.1 mmol/L Zn2+ could evidently inhibit cathepsin L activity.The purified enzyme could hydrolyze Z-Phe-Arg-MCA with the Km value of 3.52 ?mol/L and Vmax of 500 U/mg,and had no hydrolytic activity on Z-Arg-Arg-MCA and L-Arg-MCA.Multiple bovine myofibrillar proteins could be degraded by bovine pancreas cathepsin L,which also disrupted the structure of Z-line.Structrual effect and proteolytic degradation of bovine myofibrils by purified bovine pancreas cathepsin Lwas investigated.Myosin heavy chain(MHC),myosin light chain 1(M LC1),actin,troponin T,troponin I,troponin C,tropomyosin and a-actinin were hydrolyzed by the enzyme showed on SDS-PAGE.Phase contrast microscopy and transmission electron microscopy showed that,bovine pancreas cathepsin L could cause a structural failure on Z-line and have a certain hydrolysis on A-band.Cathepsin L may contribute to post mortem tenderization due to the similarity between its degradation pattern and changes happening in post mortem ageing.