改进SDS-底物-PAGE技术鉴定淡水鱼消化酶

Characterization of Freshwater Fish Digestive Proteinases by Modified SDS-Substrate-PAGE

改进SDS-底物-PAGE技术以用于分析检测淡水鱼中的消化蛋白酶.主要改进了胶浓度、反应温度和时间以及底物蛋白质浓度和处理时间等因素.试验表明:用150 g/L的分离胶,胶片在4℃30 g/L的酪蛋白溶液中浸泡90 min,然后在35℃保温120 min,结合专一的蛋白酶抑制剂,使用SDS-底物-PAGE技术可以检测出草鱼和青鱼肠道中碱性白蛋酶的种类、数量,并推算出它们的分子质量.草鱼肠道中有3种胰蛋白酶,1种非丝氨酸蛋白酶,它们的分子质量分别为:26.4 kDa3、0.75 kDa、40.5 kDa和约105 kDa.青鱼肠道中有2种胰蛋白酶,2种胰凝乳蛋白酶,1种非丝氨酸蛋白酶,它们的分子质量分别为:27.5 kDa、30.1 kDa、40.5 kDa、42.5 kDa和78.5 kDa.

SDS-substrate-PAGE method was modified and used to characterize the proteinases from the digestive organs of freshwater fish. The concentration of separating gel, reaction temperature, reaction time, substrate （protein） solution concentration and gel soaking time in the substrate solution were modified. Using 15 % of separating gel and specific proteinase inhibitors, soaking gel into 3 % of casein at 4 ℃ for 90 min and then incubating gel at 35 ℃ for 120 min, SDS-substrate-PAGE could be used to characterize types, number and molecular mass of the active component of alkaline proteinases from grass carp Ctenopharyngodon idella （Val.） and black carp Mylopharyngodon piceus viscera. There were three types of trypsins and an un-serine proteinase with molecular masses of 26.4 kDa, 30.75 kDa, 40. 5 kDa and 105 kDa, respectively in the grass carp digestive track. There were two types of trypsins, two types of chymo- trypsins and an un-serine proteinase with molecular masses of 27. S kDa, 30.1 kDa, 40. S kDa, 42.5kDa and 78.5 kDa, respectively in the black carp digestive track.