Purification and characterization of proteases from hepatopancreas of crawfish (Procambarus clarkii)

Four trypsin-like enzymes (CP-I, II, III and IV in order of elution on DEAE -Sepharose chromatography), purified from the hepatopancreas of crawfish, w ere inhibited by protease inhibitors such as phenyl methyl sulfonyl fluorid e (PMSF), soybean trypsin inhibitor (SBTI), aprotinin and tosyl lysine chlo romethyl ketone (TLCK). The molecular weights of CP-I, II, III and IV were determined to be 35.0, 41.2, 37.9 and 39.5 kDa, respectively, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These protea ses had optimal esterase activity at pH 8.0-8.5 and showed the highest acti vity at 60-70C. Crawfish proteases were rich in acidic amino acids. Activat ion energies for hydrolysis of tosyl arginine methyl ester (TAME) by these proteases were 6.98 - 8.34 kcal/mole. Unlike other serine proteases, the ac tivities of CP-I and CP-II were activated by mercury while CP-III and IV we re inhibited.