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Lab Activity 10 Enzymes

Lab Activity 10 Enzymes. IUG, Spring 2014 Dr. Tarek Zaida. Background. An enzyme is a protein molecule that is a biological catalyst with three characteristics:

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Lab Activity 10 Enzymes

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  1. Lab Activity 10Enzymes IUG, Spring 2014 Dr. TarekZaida

  2. Background • An enzyme is a protein molecule that is a biological catalyst with three characteristics: • First, the basic function of an enzyme is to increase the rate of a reaction. Most cellular reactions occur about a million times faster than they would in the absence of an enzyme. • Second, most enzymes act specifically with only one reactant (called a substrate) to produce products. • Third and most remarkable characteristic is that enzymes are regulated from a state of low activity to high activity and vice versa.

  3. Classes of Enzymes

  4. Comparative Activity of Enzymes and Nonbiological Catalysts • Enzymes are different from other nonbio-logical catalysts (metals, acids, and salts) in the fact that they exhibit a high catalytic efficiency, specificity of action, and ability to accelerate reactions under mild conditions.

  5. Experiment 1Comparison of Action Exerted by Salivary α-Amylase and Hydrochloric Acid on Starch Hydrolysis Reaction • Reagents & Materials • 1% solution of Starch in 0.3% aqueous NaCl solution • Iodinated potassium iodide solution, • Benedict’s solution. • Test tube stand with a set of test tubes, a funnel, glass rod, eye pipettes, a thermometer, pipettes of 5 ml capacity.

  6. Preparation of dilute Saliva • Rinse your mouth thoroughly to remove eventually food remnants. • Take a portion of distilled water (about 20 ml) in your mouth and keep it in for about 2 min. to allow it to mix the salivary secretion; use your tongue as a stirrer. Let the salivary liquid out into a beaker • Pour the contents into a funnel with a cotton wad in it for a filter and filter off the liquid. • Set aside the filtrate to be used in further exp.

  7. Procedure • Transfer 1 ml of distilled water to a test tube, 1 ml of hydrochloric acid solution to another test tube, 1 ml of dilute saliva to a third test tube. • Add 5 ml of starch solution to each of the three test tubes, stir the contents with a glass rod. • Place the first and the third test tubes in a water bath at 38 C, and the second test tube in a boiling water bath.

  8. 4. In 15 min let the test tubes cool. 5. Sample 5 drops from each test tube into three clean test tubes. 6. Add 1-2 drops of iodine solution and compare the coloration developed in the samples. 7 . To test for maltose sample 3 ml from each test tube, add 1 ml of Benedict’s solution and heat the upper layer of the mixture to boiling. 8. Note the formation of a red cuprous oxide precipitate in the samples.

  9. Experiment 2Identification of Enzymes of Different Groups • Identification of Oxidoreductases in Biological Material • Identification of AldehydeOxidase (Aldehyde: Oxygen Oxidoreductase; EC1.2.3.1) in milk

  10. Identification of Oxidoreductases in Biological Material • For most enzymes of this group, the recommended names are dehydrogenases and reductases. • When O2 is the acceptor, the term oxidase is used; • If the oxygen is involved in the reaction, makes part of the substrate, the enzyme is named oxygenase. • Peroxidase is an enzyme that utilizes H2O2 as an acceptor, and catalase is an enzyme capable of catalyzing the reaction in which a donor-acceptor pair is involved, which is composed of 2 H2O2 molecules

  11. Reagents & Materials • 0.4% aqueous solution of Formaldehyde, • 0.01% aqueous solution of Methylene Blue • Test tube stand with test tubes • Water bath • Thermometer • Pipettes of 1 and 5 ml capacity.

  12. Identification of AldehydeOxidase (Aldehyde: Oxygen Oxidoreductase; EC1.2.3.1) in milk • The method is based on visual observation of Methylene Blue (MB) decoloration by binding the hydrogen abstracted from the substrate through the aid of aldehydeoxidase • AldehydeOxidase is a catalyst for the dehydrogenation reaction of a variety of aldehydes, for example formaldehyde. • Hydrogen is transferred onto FAD which is a coenzyme for the given enzyme, and then onto the final acceptor (oxygen) according to the scheme.

  13. Aldehydeoxidase Aldehydeoxidase • H2C=O + H2O + FAD HCOOH + FADH2 • FADH2 + O2 FAD + H2O2 • MB as a model hydrogen acceptor, on its addition to the system studied, is converted to a reduced form (leucoform), MBH2: • H2C=O + H2O + MB HCOOH +MBH2 • The colorless methylene Blue solution on vigorous shaking regains the initial blue color. • MBH2 + O2 MB + H2O2 Aldehydeoxidase FAD FADH2

  14. Procedure • Transfer 5 ml of fresh milk to 2 test tubes • Add 2 ml of distilled water to one test tube and an equal volume of formaldehyde solution to the other test tube. • Pour 0.5 ml of Methylene Blue into each test tube, mix the contents with shaking and add 3 to 4 drops of vaseline oil (or paraffin oil) to prevent contact of liquid mixture with ambient oxygen.

  15. 4. Place the test tubes in a water bath at 37C. Within 10 – 15 min note a change in sample color. Shake vigorously the test tubes and observe again a change in color.

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