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a) To show the action of amylase on starch

b) To study the effect of temperature on enzyme action
c) To show the action of pepsin on egg white (protein)
d) (Pepsin)To show emulsification of fats

 Digestion is basically a process of breaking down big food particles

into individual molecules, tiny enough to squeeze through the intestinal lining into the
bloodstream. Your body uses mechanical and chemical means to do this. By
understanding the way the digestive process works - and how you can make it work
better, you can improve your own "gut feeling."

 !" The salivary amylase in the saliva hydrolyses the starch into a reducing
sugar

#$$%

a) Manipulated: Contents in the test tube

b) Responding: The presence of starch or glucose in the test tube
c) Fixed: Amount of contents in the test tube 

$&

The food we consume needs to be digested and transported by the circulatory

system to other parts of the body before it is utilised. The nutrients in the food we eat
are complex organic molecules which are too large to pass through plasma
membranes and enter body cells. In order for food substances to be used by the
human body, they have to be converted into a form that can be readily absorbed by
the body cells. The process that breaks down complex food substances into simpler,
soluble molecule that are small enough for the body to absorb is called digestion.
Carbohydrates, proteins and lipids are broken down into their c omponent monomers
or units through hydrolysis by digestive enzymes. Digestion breaks down
carbohydrates into glucose molecules, proteins into amino acids and lipids into
glycerol and fatty acids. These essential substances are required by the body cells to
carry out metabolic processes.

$$%$$!!$$ 

Beaker, blue and red litmus paper, measuring cylinder, thermometer, test tube, white
tile, dropper, stopwatch, 1% starch solution, dilute hydrochloric acid, dilute natrium
hydroxide, Benedict¶s solution, Iodine solution, egg, pepsin, rennin, milk, coconut oil,
bile from guinea pig gall bladder

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$' "&"$($) %$$" 

1. The mouth is rinsed

2. The chewing movement is performed to stimulate the flow of saliva and it is
collected in a test tube.
3. The saliva is tested with litmus paper to find out whether it is acidic or alkaline.
4. Dilute with about an equal volume of distilled water.
5. The saliva preparation is divided into 3 equal amounts in three test tubes and
they are labelled with B,C and D. Each tubes is filled as follows:

A ± 3 cm3 of distilled water (control experiment)

B ± 3 cm3 of the saliva preparation

C ± 3 cm3 of the saliva preparation and 3 cm 3 of dilute hydrochloric acid

D ± 3 cm3 of the saliva preparation and 3 cm 3 dilute natrium hydroxide

6. To each of the test tubes and 5 cm3 of starch solution and stirred thoroughly
7. After 30 minutes, one half of the contents of each test tube is tested with dilute
iodine solution and the other half is boiled with an equal amount of Benedict
solution.
8. The result is tabulated as shown in the table.

%

   *+ (

A Starch + Food sample Food sample in test This is
distilled water in test tube A tube A remains because food
turns blue- blue in colour. sample in A
black contains starch
B Starch + saliva Food sample Food sample in test This is
in test tube B tube B formed a because food
remains brick-red sample in B
brownish precipitate. contains
yellow. glucose
C Starch + saliva Food sample Food sample in test This is
+ dilute HCl in test tube C tube C remains because food
turns blue- blue in colour. sample in C
black contains starch
D Starch + saliva Food sample Food sample in test This is
+ dilute NaOH in test tube D tube D formed a because food
remains brick-red sample in D
brownish precipitate contains
yellow. glucose


 1. Test tube A act as controls in this experiment.
2. All the test tubes are maintained at 37oC because this is the optimum body
temperature for the action of salivary amylase.
3. Test tube B and D show the presence of a reducing sugar ( brick -red
precipitate is formed when tested with Benedict¶s solution).
4. The salivary amylase in the test tube B and D hydrolyses the starch into a
reducing sugar.
5. Enzyme salivary amylase can acts in dilute NaOH because it is alkaline
medium. This is because in our mouth, the ph is 6.5 -7.5 which is quite same
with ph dilute NaOH that is used.

From this experiment, what general conclusion can you draw regarding the nature of
amylase activity?

In conclusion, amylase work best in alkaline solution

'  "((()!$, )$ 

 !" The higher the temperature, the higher the rate of reaction of enzyme
until 37 oC and it will decrease after 37 oC and eventually the enzyme is denatured at
60oC.

#$$%

d) Manipulated: Temperature of the mixture in the test tube

e) Responding: Rate of reaction of enzyme
f) Fixed: The amount of saliva in each test tube


c

1. A solution of saliva is prepared in experiment (a)

2. 5 cm3 of this saliva solution is placed in a test tube and 5 cm3 of 1% starch
solution in another.
3. Both test tubes are allowed to stand at temperature.
4. A white tile is taken and a series of drops of dilute iodine solution is placed on it.
5. The two solutions are mixed in the tubes and the time of mixing them is noted.
6. By means of a clean glass rod, a drop of the properly stirred mixture is removed
and a drop of iodine is tested on the white tile. A deep blue colour appeared.
7. This test at intervals of one minutes is repeated, the glass rod is washing
between each test until the mixture fails to give a blue colour with iodine.
8. The total time taken between the mi xing of the saliva, the starch solution and the
end of the test is recorded. This is the time for all the starch to be converted to
maltose by amylase at room temperature.
9. The experiment at different temperatures examples at 5 o, 15 o, 25 o, 45 o, 55 o,
65o and 75 o is repeated.
10. It is important that for each of these experiments the saliva and the starch
solutions be warmed or cooled to the required temperature before they are
allowed to mix. For temperatures higher than room temperature use a water
bath, for temperatures lower than room temperature use ice cubes to bring the
temperature down.
11. For the above experiments, it may be necessary to work in groups, each group
working at a particular temperature.
12. The results are tabulated.

%

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 -./'

Room temperature 5 0.200
5o 18 0.056
15o 12 0.083
25o 8 0.125
45o 6 0.167
55o 9 0.111
65o No reaction -
75o No reaction -

Since the shorter the time taken the more active the enzyme will be, the activity (rate
of reaction) is denoted by 1/t (the reciprocal of the time taken).



1. At very low temperatures, the kinetic energy of the substance and enzyme
molecules are low. It takes a very long time for the substrate and enzyme to
bind. Hence, the rate if reaction is very low.
2. As the temperature rises, the kinetic energy of the substr ate and enzyme is
also increases. The number of collisions between the substrate and enzyme
increases and the number of enzyme -substrate complexes formed also
increases.
3. For every 10oC rise in temperature, the rate of enzyme -catalysed reaction
increase twice.
4. At the optimum temperature, the kinetic energy in the substrate and enzyme
increases. The number of collisions between substrate and enzymes also
increases but the formation of enzyme -substrate complex decreases. This is
because the increased kinetic energy causes the amino acid molecules in the
enzyme to vibrate violently.
5. Some bonds like hydrogen and ionic bonds which help hold the configuration
of enzyme break. The active site shape changes and is not able to bind with
the substrate. The enzyme is d enatured. The rate of reaction declines.
6. At 60oC, the enzyme denatured.


1. Plot a graph of 1/t against temperature. What is the optimum temperature?

A graph is plotted at the graph paper. The optimum temperature has been
denoted in the graph.

2. Describe how temperature affects enzyme catalysed biochemical reactions.

When the temperature is increase, the substrate molecules move faster. The
collisions between the substrate and enzyme molecules occur more
frequently. The random movement of molecule s and the more frequent
collisions between the substrate and enzyme molecules increase the chances
of the substrate molecules coming into contact with the active sites of an
enzyme. Thus, at higher temperature the rate of reaction between the
substrate and enzyme increase.

' "&"$(!!00&"-!' 

$ & Pepsin enzyme produced in the mucosal lining of the stomach
that acts to degrade protein. Pepsin is one of three principal protein -degrading, or
proteolytic, enzymes in the digestive system, the other two being chymotrypsin and
trypsin. The three enzymes were among the first to be isolated in crystalline form.
During the process of digestion, these enzymes, each of which is particularly
effective in severing links between particular types of amino acids, collaborate to
break down dietary proteins to their components, i.e., peptides and amino acids ,
which can be readily absorbed by the intestinal lining. In the laboratory studies
pepsin is most efficient in cleaving bonds involving the aromatic amino acids,
phenylalanine, tryptophan, and tyrosine. Pepsin is synthesized in an inactive form by
the stomach lining; hydrochloric acid, also produced by the gastric mucosa, is
necessary to convert the inactive enzyme and to maintain the optimum acidity ( ð H
1-3) for pepsin function. Pepsin and other proteolytic enz ymes are used in the
laboratory analysis of various proteins; pepsin is also used in the preparation of
cheese and other protein -containing foods.
 !" Enzyme pepsin hydrolyses protein into amino acid

c

1. A suspension of egg white is prepare d by beating up a little raw egg white

with boiling water in a small beaker and the suspension is allowed to cool.
2. To each of the three test tubes A, B and C , a few cm3 of the cooled egg white
suspension is added.
3. The tubes are placed in a water bath at about body temperature (about 37 oC).
Each tubes is filled as follows:

A ± 5 cm3 of 0.2% hydrochloric acid

B ± 5 cm3 of µartificial gastric juice¶ (prepared by dissolving 3.5 g of pepsin in
 100 cm3 of 0.2 % hydrochloric acid
C ± 5 cm3 of µartificial gastric juice¶ and 1 cm 3 of dilute natrium hydroxide
4. The mouth of each tube is plugged with a piece of cotton wool after adding a
little thymol to prevent decay.
5. The tubes are left to stand for about one hour and they are shake d
periodically. What happens to the egg white suspension in tube B? Does it
turn yellow, transparent and then gradually dissolve? Is there any change in
the other tubes?
6. The buiret test is carried out on the contents of each test tube at the end of
one hour.
7. The results are tabulated.

%

  $ * (


A Egg white + dilute HCl Food sample in Positive This is
test tube A turn because food
purple. sample in test
tube A
contains
protein
B Egg white + artificial Food sample in Negative This is
gastric juice test tube B turn because food
blue in colour sample in B
contains amino
acid.
C Egg white + artificial Food sample in Positive This is
gastric juice + dilute test tube C turn because food
NaOH purple. sample in test
tube C
contains
protein



1. Buiret test is used to detect the presence of protein.

2. Test tube A acts as control experiment.
12 In this experiment, test tube A and C have protein. This is because in test
tube A, dilute HCl does not hydrolysed egg white (protein) into amino acid.
Dilute HCl although in acidic medium, but it is not an enzyme. For test tube C,
the adding od dilute NaOH in mixture of egg white and artificial gastric juice
cause the protein is not be hydrolysed into amino acid. This is because dilute
NaOH is in alkaline medium, but the hydrolysed process to be carried out
must be in acidic medium. Hence, it cannot undergo hydrolysed process due
to alkaline medium. 

 ' "&)%($ 

$ & 

Bile or gall is a bitter-tasting, dark green to yellowish brown fluid, produced by the
liver of most vertebrates, that aids the process of digestion of lipids in the small
intestine. In many species, bile is stored in the gallbladder between meals and upon
eating is discharged into the duodenum.

 !" Bile acts as emulsifier to mix up water and coconut oil.

#$$%

$' $!%$ Contents in the test tube 

' !0 The condition of the contents after experiment is done 
' 34 The amount of solution in the test tube

(

1. A little coconut oil is shaken vigorously with some water in a test tube. What
do you observe after the mixture has been left to stand for a while?
2. A little bile from the gall bladder of a guinea pig is obtained and is added to
the mixture in (1).It has to be shaken vigorously; the mixture is then left to
stand for some time. Do you notice any difference before adding and after
adding the bile?

%

Contents Observations Inferences

Coconut oil + water Coconut oil and water does
not mix. Coconut oil is placed
in the top level while water in
the bottom level.
Coconut oil + water + Coconut oil, water and bile
bile mix each others.
This is because coconut
oil is a non-polar
molecules where as water
is a polar-molecules.
This is because bile acts
as emulsifier to mix up the
water and coconut oil.



1. Water is held together by hydrogen bonds and can interact efficiently with
anything that has well developed permanent charges; that's why it can
dissolve electrolytes so well.

2. Oils on the other hand are non polar, simply because the difference between
the electronegativities of H and C are so small. These molecules interact by
London (of van der Waals) dispersion forces and are induced dipole - induced
dipole interactions. These interactions become stronger as more molecules
 are bound together, at least up to some critical size. These interactions are
also strongest between molecules with similar polarizabilities.

3. The interactions between the oil an d water molecules are not strong, the oil
can't hydrogen bond as it is non -polar and water can't form strong London
forces to the oil; it has a different polarizability.

4. Now, a second part to this is that water molecules are much smaller than
most other molecules, so to accommodate oil molecules, many water
molecules have to have their hydrogen bonds broken.

5. When water has to surround non -polar parts of molecules it seems to form
cage like structures, where the water molecules on the surface of the cavity
form 4 hydrogen bonds to neighboring water molecules. These structures can
be seen in the crystal structures of certain hydrated electrolytes, called
"clathrates". In solution this is called hydrophobic hydration and is still a
subject of research.

6. The most interesting place where this difference between non -polar
"hydrophobic" and polar "hydrophilic" molecules is in biology where nature
uses these two in a myriad of ways: fats, which should stay in place, are oily,
while sugars, that need to be mo ved quickly around the body, are very
hydrophilic; cell walls are formed by amphiphiles, one end hydrophilic and the
other oily (hydrophobic), trans membrane proteins are anchored in the cell
wall by having hydrophobic sidegroups, globular proteins have la rge amounts
of hydrophobic sidegroups that make them fold with these on the inside, away
from the water.

7. The water molecule is made up of two hydrogen atoms and one oxygen atom.

8. Due to the ability of the oxygen atom to pull the hydrogen electrons towards
itself, it carries a negative charge. As a result, the hydrogen atoms carry a
more positive charge. The charges of these two molecules allow them to
make weak bonds with certain particles, like NaCl.

9. NaCl dissociates into a Na+ which is attracte d to the negative oxygen atoms
of the water molecules and hangs out there, and the Cl - which is attracted to
the positive hydrogen atoms in the water molecules and hangs out with them.

10. Likewise, molecules which have lots of charges on them are termed
hydrophillic, or "water loving." They interact well with the charges on water
molecules.

11. Unlike the water molecule, oil is made up of long chains of carbon atoms
(usually 8 Carbon atoms) which do not carry an electric charge. These long
chains without a charge are called hydrophobic or "afraid of water." Because
 they do not have a charge, there is no attraction between the charges on the
water molecule and the long carbon chains and can't hang out with the water
molecules. That is why oil does not mix with water. It is because it cannot
form any bonds with water.

12. Water is charged positively on the oxygen end, and negatively on the
hydrogen end. Oil does not have a charge (it's neutral), so the oil does not mix
with the water.

13. Bile has various components, some of which are produced by hepatocytes in
the liver. The main components include:

 Water
 Cholesterol
 Bile pigments
 Anions of the Bile acids
 Phospholipids (mainly lecithin)
 Bicarbonate and other ions

14. The biliary system has two main functions. One is to produce and deliver a
digestive juice, and the ot her is to provide a route through which certain kinds
of things are eliminated from the body. 

15. Bile is a lot like soap. Soap functions to help you lift oily stuff into water.
Without soap, oil floats as droplets on top of water and the two do not mix.
Soap is composed of molecules that have oil-like properties at one end and
water-soluble properties at the other. In a mixture of water and oil, soap will
form tiny bubbles with oil inside, and the droplets will freely mix into the water.
This is called "emulsification". Bile does the same thing. Bile is a chemical
mixture with molecules that have oil-compatible properties on one end and
water-compatible properties on the other. Bile makes tiny soap bubbles with
oily food on the inside and watery stuff all arou nd. Bile emulsifies oily food into
the otherwise watery juice flowing in the intestine. 

16. The reason that it's important to emulsify the oily food droplets is that there
are tremendously important nutrients and vitamins that are only available
inside the oily stuff. If the oil floats as giant droplets through the intestine
without mixing into the water, then all the digestive enzymes needed to break
it down would not function correctly. The digestive enzymes that have to act
on the oily food substances are floating in the watery juices. They can only act
on the oil at the oil -water interface. 

17. When chemistry depends on the conditions found at an interface such as the
surface where water touches oil, we describe it as "surface area dependent".
In this case, the job is to digest all the good stuff in the oil, so the best way to
do this is to make the oil droplets tiny. That way there's plenty of surface on
the droplets for the enzymes to do their digestion, and the droplets are
processed very quickly. 
 18. Without bile, the oil droplets would be very large and the process of enzyme
digestion would be incomplete by the time that the oil went all the way through
the small intestine. Once oil gets to the colon, the window of opportunity is
closed. The colon doesn't do a lot of nutrient absorption. Also, the rich nutrient
supply of oil, if it gets to the colon, causes a bacterial bloom, and it draws
water into the colon as well. The result is diarrhea. It's actually a very stinky
kind of diarrhea that floats (lik e oil) and it's called "steatorrhea". So that's the
first main function of bile. It's a juice that assists with the digestion and
absorption of fatty/oily nutrients that don't mix well with water. It works a lot
like soap. 

19. Another function of bile is to be a route of elimination.Not everything in the

body's tissues that needs to float out through the blood and then be filtered
out of the system can exit in the urine. The urine is a great way to get rid of
things that dissolve in water. However, some chemi cals that tbe body needs
to eliminate do not dissolve in water. These are often filtered out of the blood
stream by the liver, and are either modified so that they DO dissolve in water
(so that they can exit in the urine) or else they get put into the bile system and
are excreted into the intestine where they flow out with the stools. 

20. In particular, bile is rich in a chemical called "bilirubin" (notice the similar root
words?). Bilirubin is a breakdown product that is made out of used -up
hemoglobin. Hemoglobin is the oxygen carrying molecule in red blood cells
that makes them red. Bilirubin is why bile is a dark green, or even brown/black
color. 

21. If something happens and the bile system gets blocked (there are a number of
ways that this could happen), then the color of bile is absent from the poop.
The normal brown color that we're all used to is replaced by having no color at
all. The stools are actually silvery white, without bile.In the mean time, all that
stuff backs up in the blood stream, and people t urn bananna yellow as they
get sicker. 

22. Many substances are eliminated in bile, but bilirubin is the most obvious one
because of the striking color changes associated with blockage.So that's the
second function of bile. It's a route of elimination from the body, for things that
don't dissolve well in the water and which are cleared by the liver. 
% The hypothesis is accepted. Bile acts as emulsifier to mix up water and
coconut oil.


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. (n.d.). Retrieved February 21, 2010, from http://en.wikipedia.org/wiki/Bile

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http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

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p pp. (n.d.). Retrieved February 22, 2010, from

http://www.occc.edu/bbdiscovery/documen ts/Modules/Packing%20peanuts.ht
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cð . (n.d.). Retrieved February 24, 2010, from http://en.wikipedia.org/wiki/Pepsin

c p  pppp p . (n.d.). Retrieved February 23, 2010, from

http://library.thinkquest.org/11226/main/c14txt.htm

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ppp . (n.d.). Retrieved February 20, 2010, from

http://www.chemistryquestion.com/English/Questions/SpecialistChemistry/15_

water_oil.html