Peroxidase

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Publication No. 11137

Enzyme Activity Guided Inquiry Lab Turnip Peroxidase
Introduction
Peroxidase enzymes are widely distributed in plants and animals, including bacteria, to protect cells against the effects of oxidative stress and cell damage due to hydrogen peroxide. Peroxidases are easily extracted from turnips and other root vegetables and provide a model enzyme for studying enzyme activity—how the rate of an enzyme-catalyzed reaction depends on biotic and abiotic factors. Enzyme activity studies reflect enzyme structure and function and provide the foundation for understanding the mechanism or theory of enzyme action.

Background
The term peroxidase refers to both a class of oxidoreductase enzymes and to specific enzymes within that class. As a general class of enzymes, peroxidases catalyze the oxidation−reduction decomposition reaction of hydrogen peroxide. There are two general types of peroxidases—catalase and peroxidase. Catalase catalyzes the disproportionation reaction of hydrogen peroxide to water and oxygen gas (Equation 1). In reactions mediated by catalase, hydrogen peroxide substrate molecules act as both oxidizing agent (electron acceptor) and reducing agent (electron donor). In contrast, peroxidase acts in the presence of other naturally occurring organic reducing agents, such as ascorbic acid and glutathione, to catalyze the decomposition of hydrogen peroxide. Organic reducing agents, abbreviated AH2, transfer hydrogen atoms and electrons to hydrogen peroxide, resulting in the formation of water and oxidized organic substrates such as A2 in Equation 2. Catalase-catalyzed reaction Peroxidase-catalyzed reaction 2H2O2 → 2H2O + O2 2H2O2 + 2AH2 → 4H2O + A2 Equation 1 Equation 2

The differences in the two equations shown above provide a basis for studying the enzyme activity of turnip peroxidase in this guided-inquiry laboratory investigation. Many endogenous organic compounds may be used as reducing agents in Equation 2. One of the most common and convenient reducing agents for this purpose is guaiacol, a colorless compound having the formula C7H8O2. Oxidation of guaiacol according to Equation 2 converts it to a dark orange compound called tetraguaiacol. The rate of the reaction may be followed by measuring the absorbance or color intensity of the orange product as a function of time.

Materials
Buffer capsules, pH 3−8, 100 mL each Distilled or deionized water Guaiacol, C7H8O2, 1 mL Hydrogen peroxide, H202, 3%, 3 mL Isopropyl alcohol, (CH3)2CHOH 70%, 100 mL Phosphate buffer, pH 7, 300 mL, NaH2PO4 and Na2 HPO4, 0.05 M Turnip (root/tuber) Ice and water baths Blender Erlenmeyer flask, 500-mL Filter paper and funnel Hot plate Knife, paring pH paper, narrow range Pipets, serological, 2- and 5-mL Spectrophotometer Test tubes, 13 × 100 mm, 6, and rack Thermometer Timer

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Safety Precautions
Guaiacol is toxic by ingestion. It has an aromatic, creosote-like odor and may be irritating to the nose and throat. Isopropyl rubbing alcohol (70%) is a flammable liquid. Keep away from heat, flames, and other sources of ignition. Dilute hydrogen peroxide solution (3%) may be irritating to the eyes and skin. Exercise care when using a knife to peel and cut the turnip. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Please review current Material Safety Data Sheets for additional safety, handling, and disposal information.

Preparation
1. Extraction Buffer: Prepare 500 mL pH 7 phosphate buffer by mixing equal volumes, 150 mL each, of 0.1 M sodium phosphate monobasic and sodium phosphate dibasic solutions. Verify pH using narrow-range pH paper; it should be between 6.8 and 7.2. 2. Reaction Buffers: Dissolve one each pH 3−8 buffer capsules in 100 mL distilled or deionized water according to instructions. 3. Guaiacol Solution: Dissolve 0.2 mL guaiacol in 100...
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