Protocol for determining the properties of an enzyme

Protocol for determining the properties of an enzyme - 5...

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Protocol for determining the properties of an enzyme Weigh 1 to 10g of peeled turnip, horseradish, or potato tissue. Homogenize the tissue by adding it to 100ml of cold 0.2M phosphate buffer at pH7 Grind mixture Prepare a solution of peroxidase Label four 50mL beakers as follows: turnip extract, buffer, 10mM H2O2, and 25mM guaiacol. Fill each half full with appropriate stock solution. Label four pipettes to correspond with the beaker Number seven test tubes as following: 1. Control with all reactants except enzyme to be used in calibrating the spectrophotometer 2. Substrate and indicator dye 3. Dilute extract 4. Substrate and indicator dye
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Unformatted text preview: 5. Medium concentration of extract 6. Substrate and indicator dye 7. Concentrated extract (see table 7.1) Add stock solution ot each tube using the corresponding graduated 5 ml pipette or dispensing device. Adjust spectrophotometer to zero absorbance at 500nm Use test tube 1 as the blank for the spectrophotometer Mix tubes 2 and 3 within ten seconds Place mixture in the spectrophotometer and read absorbance at 20 second intervals from start of mixing. Mix contents of tubes 4 and 5 and repeat measurements for two minutes at 20 second intervals Mix contents of tubes 6 and 7 repeat measuring steps...
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