Transcript Lab Activity 10 Enzymes

Lab Activity 10
Enzymes
IUG, Spring 2013
Dr. 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
Oxidases or Dehydrogenases
Type of Reaction catalyzed
Oxidation-reduction reactions
Transferases
Transfer of functional groups
Hydrolases
Hydrolysis reactions
Lyases
Isomerases
Ligases or Synthetases
Addition to double bonds or its reverse
Isomerization reactions
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 1
Comparison 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 2
Identification 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 Aldehyde oxidase HCOOH + FADH2
• FADH2 + O2
Aldehyde oxidase
FAD + H2O2
• MB as a model hydrogen acceptor, on its addition to
the system studied, is converted to a reduced form
(leucoform), MBH2: Aldehyde oxidase
• H2C=O + H2O + MB
HCOOH +MBH2
FAD
FADH2
• The colorless methylene Blue solution on vigorous
shaking regains the initial blue color.
• MBH2 + O2
MB + H2O2
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Procedure
1. Transfer 5 ml of fresh milk to 2 test tubes
2. 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.
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