Simultaneous Identification of Different Domains of Calpain from Blood and Turkey Meat Samples Using Casein Zymography

Abstract

The aim of this study was to develop a simple method for simultaneous identification of two different domains of calpains (μ- and m-calpains) in blood and turkey meat samples. The method is based on Tris-based extraction techniques followed by casein zymography detection. The extracts were purified by dialysis, and target compounds were separated on Diethylaminoethyl (DEAE)-Sephacel anion-exchange chromatography. Results indicated that a buffer pH of 6.7 produced optimum extraction efficiency. The separation of calpastatin, μ- and m-calpains on column chromatography was carried out using stepwise elution profiles of 100, 200, and 400 mM NaCl solutions, respectively. Casein zymography study clearly demonstrated the presence of μ- and m-calpains in the crude extracts of breast and blood samples; however, μ-calpain was absent in the thigh sample. Similarly, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis identified both the calpains from muscle and blood samples, but presence of calpastatin was observed in both the breast and thigh muscle samples only. The activity analysis of purified fractions indicated that both the μ- and m-calpain concentrations were differed significantly (P < 0.05) amongst breast, thigh and blood samples. Collectively, it was concluded that Tris buffer (pH 6.7) and DEAE-Sephacel column for extraction and separation of μ- and m-calpains and casein zymography and SDS-PAGE detection methods were most suitable for accurate identification of calpains and calpastatin from blood and turkey meat samples.