蛋白内切酶 Glu-C 来源于金黄色葡萄球菌 V8

Protease S. aureus, V8 (endoproteinase-Glu-C) specifically cleaves peptide bonds on the COOH-terminal side of either aspartic or glutamic acids (Drapeau et al. 1972). Houmard and Drapeau (1972) report that in the presence of ammonium buffers the enzyme specificity can be limited to glutamoyl bonds. Its rather unique specificity, which can be considered as opposite to that of trypsin, makes it a useful tool for protein chemistry and peptide mapping studies (Cleveland et al. 1977) and (Hall et al. 1978). Characteristics of Protease from S. aureus, V8: Characteristics of Protease, Staph aureus (Endoproteinase Glu-C) Molecular Weight 27,000 (Drapeau 1978). Optimal pH 4.0 and 7.8 with hemoglobin substrate. (Drapeau et al. 1972). Extinction Coefficient extinction coefficient = 4.26 (Houmard 1976). Inhibitors Diisopropyl fluorophosphate (DFP) and monovalent anions such as F-, Cl-, Br-, CH3COO-, and NO3- (Houmard 1976). Method Enzyme activity is determined by the casein digestion assay described by Drapeau (1976). Since substrate grade caseins can vary from lot to lot and according to the manner produced, the standardization of casein digestion assays has been difficult to achieve. The use of a reference enzyme preparation is suggested. One unit is that amount of enzyme which releases acid soluble fragments equivalent to 0.001 A280 per minute at 37°C and pH 7.8 under the specified conditions. Reagents 1% Casein in 0.05 M Tris⋅PO4 buffer, pH 7.8. (Dissolve 1 gram "Hammersten" casein in 50 ml 0.01 N NaOH with gentle heating and stirring. Add 40 ml reagent grade water and 5.0 ml 1.0 M Tris. Adjust pH to 7.8 with H3PO4 and q.s. to 100 ml.) 10% Trichloroacetic acid (TCA) Enzyme Dissolve at 1 mg/ml in reagent grade water. Procedure Equilibrate a series of tubes with 5.0 ml of 1% casein at 37°C for 5 minutes. At zero time add 10 ul or 20 ul of enzyme. Mix. Include a reagent blank. Exactly ten minutes after adding sample, stop reaction by adding 5.0 ml TCA. Mix. Allow tubes to stand ten minutes and then filter. Read A280 of the filtrate. Calculation

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