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Lab Activity 10Enzymes

IUG, 2015Dr. Tarek Zaida

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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.

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Classes of Enzymes

IEC Classification of Enzymes

Group Name Type of Reaction catalyzed

Oxidases or Dehydrogenases Oxidation-reduction reactions

Transferases Transfer of functional groups

Hydrolases Hydrolysis reactions

Lyases Addition to double bonds or its reverse

Isomerases Isomerization reactions

Ligases or Synthetases Formation of bonds with ATP cleavage

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Comparative Activity of Enzymes and Nonbiological Catalysts

• Enzymes are different from other nonbiological 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.

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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.

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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.

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Procedure

1. 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.

2. Add 5 ml of starch solution to each of the three test tubes, stir the contents with a glass rod.

3. 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.

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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.

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Experiment 2Identification of Enzymes of Different Groups

• Identification of Oxidoreductases in Biological Material

• Identification of Aldehyde Oxidase (Aldehyde: Oxygen Oxidoreductase; EC1.2.3.1) in milk

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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

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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.

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Identification of Aldehyde Oxidase (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 aldehyde oxidase

• Aldehyde Oxidase 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.

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• 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

Aldehyde oxidase

Aldehyde oxidase

Aldehyde oxidase

FAD FADH2

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Procedure

1. Transfer 5 ml of fresh milk to 2 test tubes2. Add 2 ml of distilled water to one test tube

and an equal volume of formaldehyde solution to the other test tube.

3. 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.

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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.