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Lab Activity 10

This document describes two lab experiments involving enzymes. The first compares the activity of salivary amylase and hydrochloric acid in breaking down starch. Samples treated with each are tested with iodine to detect starch and Benedict's solution to detect maltose. The second experiment identifies different classes of enzymes in biological materials. It detects oxidoreductases that use oxygen as an electron acceptor, like the aldehyde oxidase found in milk, which can be identified by its ability to reduce methylene blue in the presence of formaldehyde and oxygen.

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Benroi Queqquegan
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17 views

Lab Activity 10

This document describes two lab experiments involving enzymes. The first compares the activity of salivary amylase and hydrochloric acid in breaking down starch. Samples treated with each are tested with iodine to detect starch and Benedict's solution to detect maltose. The second experiment identifies different classes of enzymes in biological materials. It detects oxidoreductases that use oxygen as an electron acceptor, like the aldehyde oxidase found in milk, which can be identified by its ability to reduce methylene blue in the presence of formaldehyde and oxygen.

Uploaded by

Benroi Queqquegan
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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Lab Activity 10

Enzymes

IUG, Spring 2013


Dr. Tarek Zaida
1

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.

Classes of Enzymes
IEC Classification of Enzymes
Group Name

Type of Reaction catalyzed

Oxidases or Dehydrogenases
Transferases
Hydrolases
Lyases

Oxidation-reduction reactions
Transfer of functional groups
Hydrolysis reactions
Addition to double bonds or its
reverse

Isomerases

Isomerization reactions
Formation of bonds with ATP
cleavage

Ligases or Synthetases

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.

Experiment 1
Comparison of Action Exerted by Salivary -Amylase
and Hydrochloric Acid on Starch Hydrolysis Reaction

Reagents & Materials


1% solution of Starch in 0.3% aqueous
NaCl solution
Iodinated potassium iodide solution,
Benedicts solution.
Test tube stand with a set of test tubes,
a funnel, glass rod, eye pipettes, a
thermometer, pipettes of 5 ml capacity.
5

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.
6

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.
7

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 Benedicts solution and
heat the upper layer of the mixture to boiling.
8. Note the formation of a red cuprous oxide
precipitate in the samples.
8

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

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 which10

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.
11

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.
12

Aldehyde oxidase
H2C=O + H2O + FAD
FADH2

HCOOH +

Aldehyde oxidase

FADH2 + O2

FAD

+ H 2O2

MB as a model hydrogen acceptor, on its


addition to the system studied, is converted to
a reduced form (leucoform),
Aldehyde oxidase MBH2:
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
13

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

14

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.

15

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