Experiment no.

8
ENZYMES

1-Nur-1
Group 2:
Ana Mariella A. De Castro
Louie Bryan P. Calimlim
Meg Andrea A. Cepe
Paolo T. Barrios



What are enzymes?
Enzymes are proteins that participate in cellular metabolic processes with the
ability to enhance the rate of reaction between biomolecules. Some enzymes can
even reverse a reaction from the direction it would normally take, by reducing the
activation energy (Ea) to the extent that the reaction favours the reverse
direction. Simlarly, enzymes can catalyze reactions that might not otherwise
occur, by lowering the Ea to a more "affordable" level for the cell.
Enzymes are proteins made by cells that act as catalysts to speed up
chemical reactions that occur in cells.
Enzyme Composition
Enzymes can have molecular weights ranging from about 10,000 to over 1 million.
A small number of enzymes are not proteins, but consist of small catalytic RNA
molecules. Often, enzymes are multiprotein complexes made up of a number of
individual protein subunits.
Many enzymes catalyze reactions without help, but some require an additional
non-protein component called a co-factor. Co-factors may be inorganic ions such
as Fe
2+
, Mg
2+
, Mn
2+
, or Zn
2+
, or consist of organic or metalloorganic molecules
knowns as co-enzymes.
Enzyme Classifications
Enzymes are classified according to the reactions they catalyze. The six classes
are:
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases








FACTORS AFFECTING ENZYMATIC ACTIVITY

A. EFFECT OF TEMPERATURE








Procedure:
Label 3 tubes as A, B and C. Put 2.0 mL of 1% starch solution into each of the tube.
Label another 3 tubes with A-1, B-1 and C-1. Add 1.0 mL SALIVA INTO EACH OF THE
TUBE.
Put tube A-1 in an ice bath, B-1 in a 37C water bath and C-1 in a boiling water bath for
about 5 minutes.
Transfer tube A into the ice bath and pour the contents of tube A-1 to tube A.
Mix and replace back to the ice bath.
After 10 minutes, add 2 drops of Iodine solution.
Note and record the color of the solution.
Repeat the same procedure for B-1 to B and C-1 to C, using instead 37C water bath for
B and the boiling water bath for C.
Note and record the color of the solution after the 10 minutes incubation and adition of
iodinr solution.
Rank the tubes from 1 to 3 (1-greatest digestion is colorless and 3-least or no digestion
is a dark shade of blue.









Study Questions:

WHAT IS OPTIMUM TEMPERATURE? WHAT IS THE OPTIMUM TEMPERATURE OF YOUR
ENZYME?
Its the temperature at which the reaction can take place at its most efficient. Usually the
'optimum' will be a temperature which results in the greatest (or purest) yield of product. The
optimum temperature of the enzyme is 37C.

IN THIS PROCEDURE, IDENTIFY WHICH IS THE ENZYME, THE SUBSTRATE AND THE
MANIPULATED VARIABLE.
enzyme- saliva
substrate- starch
manipulated variable- temperature


HOW WOULD YOU COMPARE THE DEGREE OF DIGESTION IN THE DIFFERENT
TEMPERATURES?
As the temperature increases, so will the rate of the
reaction. This is because when heat is applied, it excites the
particles more and as a result, they collide faster and so the
reaction rate increases. When a particular temperature is reached, the rate of reaction will
dramatically decrease because the
amylase is working outside its optimum temperature range. Usually a rise of about 10C will
double the rate of reaction. After the enzyme reaches its limit, which is 40C, I think the
reaction
rate will decrease rapidly and then stop. This is because the
enzyme has become denatured and the bonds holding the enzyme together started to break;
therefore denaturing the active site.


PROVIDE AN EXPLANATION FOR YOUR OBSERVATIONS IN THE DIFFERENT
TEMPERATURES.
TEMPERATURE OBSERVATION
ICE BATH SLIGHTLY COMPLETE
37C COMPLETE DIGESTION
BOILING WATER LEAST/ NO DIGESTION

GRAPH THAT SHOWS THE RELATIONSHIP BETWEEN ENZYME ACTIVITY AND
TEMPERATURE.


HOW CAN IODINE SOLUTION DETERMINE THE DEGREE OF DIGESTION IN THE GIVEN
TUBES? EXPLAIN THE PRINCIPLE BEHIND THE USE OF THIS REAGENT.
Iodine solution can determine the degree of digestion by the change in color of the solution.





Effect
Of
pH




Procedures:
1. Prepare 3 test tubes and label as 4, 7, 10. Add the following solutions as described
below
Contents Tube 4 Tube 7 Tube 10
Starch Solution 2.0 mL 2.0 mL 2.0 mL
Buffer Solution pH4 = 2.0mL pH4 = 2.0mL pH4 = 2.0mL
Saliva 1.0mL 1.0mL 1.0mL

2. Add the solutions in order: starch solution, buffer solutions and them the saliva
3. Put saliva in all test tubes at approximately the same time
4. Warm all test tubes in a 37degrees celsius water bath for 10mins
5. After heating, add 2 drops of iodine solution to each of the test tubes
6. Record the color of the solutions and rank the tubes from 1-3 (1-greatest digestion is
colorless and 3-least or no digestion is a dark shade of blue or violet)













Study Questions:

Enzymes are affected by changes in pH. The most favorable pH value - the point where the
enzyme is most active - is known as the optimum pH.
The optimum pH of the enzyme is pH7
Enzyme- Saliva
Substrate- Starch Solution
Manipulated Variable- pH level


Different enzymes are more effective at different pH levels. If the pH levels are too high or too
low for a particular enzyme, it might get denatured and will no longer perform its function.
Components in saliva help keep the pH in your mouth between 6.5 and 7 so that the enzyme
salivary amylase can start to break down carbohydrates. Those that function in the intestines,
including peptidases and maltase, work best at a pH around 7.5










Test Tube 4 which
has Buffer solution
of pH4
Test Tube 7 which
has Buffer solution
of pH7
Test Tube 10 which
has Buffer solution
of pH10
Slight Digestion Greatest/Complete
Digestion
Least/No Digestion







Iodine test is used to determine the presence of starch. Salivary amylase can hydrolyze or
breakdown starch into sugar. So If the iodine solution was added into the solution and it turned
dark violet solution, it means that there is still the presence of starch and there was no
digestion that occurred. On the other hand, if it turned into a colorless solution, it means that
starch was already digested. The principle involved was denaturation.
Iodine test determines the degree of digestion in the given tubes by the change of color in the
solution.
Antacids increase the pH in the stomach, which might make the enzymes in the stomach less
effective. The low pH of the juices in the stomach can cause ulcers if they eat through the walls
of the small intestine or stomach.









Effect of Enzyme
Concentration




Procedure:

Prepare 4 clean test tubes and label from 1 to 4.
Put 2.0 mL of 1% starch solution into each test tube.
Add a drop of iodine solution into all test tubes.
Note and record the resulting color of the solution.
In a separate tube, warm 2.0 mL saliva in a 37 C water bath for 5 minutes.
Tube 1 serves as the control of the experiment.
Add 3 drops of warm saliva to tube 2.
Mix quickly and note the time needed for complete digestion to take place.
Do the same for tubes 3 and 4 but instead add 6 drops saliva to tube 3 and 10 drops
saliva to tube 4.
Rank the tubes from 1 to 3 (1-fastest digestion and 3- slowest digestion)
Record all your observations.



Study Questions:

In this procedure, identify which is the enzyme, the substrate and the manipulated
variable.
a. Enzyme - saliva
b. Substrate - starch
c. Manipulated Variation - enzyme concentration

Account for the result obtained after addition of iodine in the tubes containing the
starch solution.
The result obtained was blue violet.



What is a control? What is its importance in the experiment?
Control is the portion that you are not performing experiments on. It is just there
to serve as the blank state that experiments compare to. The control is used to know
the degree of digestion by looking at the color.
How would you compare the degree of digestion in the different amount of saliva?
The degree of digestion is directly proportional to the amount of saliva used. The
more amount of saliva, the faster the degree of digestion there is. An increase in the
amount of saliva, the rate of the reaction will increase.
Provide an explanation for your observations in the different amounts of saliva.
The solution with 10 drops of saliva has the fastest digestion because it has the
greater amount of enzyme concentration. The solution with 3 drops of saliva has the
slowest digestion because it has the least amount of enzyme concentration.

Draw a graph that will show the relationship between enzyme activity and amount of
enzyme.



How can iodine solution determine the degree of digestion in the given tubes? Explain
the principle involved behind the use of this reagent.
The iodine solution is used to determine the degree of digestion by the change of
color from blue-violet to colorless.




A.4 Effect of the
Substrate
Concentration




Procedure:
Prepare 4 clean and dry test tubes and label from 1 to 4.
Put 1.0 mL of 1% starch solution into tube 1, 2.0 mL into tube 2, 3.0 mL into tube 3 and
5.0 mL into tube 4.
Add 1.0 mL distilled water to all of the tubes. Mix thoroughly.
Add 1.0 mL saliva to all the tubes (Put saliva at approximately the same time).
Thoroughly mix all the tubes and place in a water bath set at 37C for 5 minutes.
Remove all tubes from the water bath at the same time.
Prepare another set of 4 tubes, labeled as 1a to 4a.
Add 2.0 mL of Benedicts solution into each of the tubes.
Add 5 drops from your incubated mixture, i.e. Tube 1 to tube 1a, Tube 2 to tube 2a, and
so on. Mix thoroughly.
Warm all the tubes in a boiling water bath for 5 minutes.
Note the changes in color of the solution and resulting precipitation (if any).
Rank the tubes accordingly (1-greatest digestion and 3-least or no digestion).
Record all your observations.

















Study Questions:

IN THIS PROCEDURE, IDENTIFY WHICH IS THE ENZYME, THE SUBSTRATE AND THE
MANIPULATED VARIABLE.
enzyme- saliva
substrate- starch
manipulated variable- concentration of the substrate

HOW WOULD YOU COMPARE THE DEGREE OF DIGESTION IN THE DIFFERENT AMOUNTS
OF STARCH SOLUTION ADDED?
Increasing the substrate concentration increases the rate of reaction (enzyme activity).
However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites
of all the molecules are occupied most of the time. At the saturation point, the reaction will not
speed up, no matter how much additional substrate is added. The graph of the reaction rate
will plateau.


PROVIDE AN EXPLANATION FOR YOUR OBSERVATIONS IN THE DIFFERENT AMOUNTS OF
STARCH SOLUTION.
As the substrate concentration increases, enzymatic activity also increases but when it reach a
certain point called the saturation point the reaction will not speed up, no matter how much
additional substrate is added.





DRAW THE GRAPH THAT WILL SHOW THE RELATIONSHIP BETWEEN ENZYME ACTIVITY
AND AMOUNT OF SUBSTRATE.


HOW CAN BENEDICTS REAGENT DETERMINE THE DEGREE OF DIGESTION IN THE GIVEN
TUBES? EXPLAIN THE PRINCIPLE AND PROVIDE THE EQUATIONS BEHIND THE USE OF
THIS REAGENT.
Benedicts reagent is used to detect the presence of certain types of carbohydrate. That is why
by using this in this particular test we can identify which test tube has a greater digestion than
the rest.

IN WHICH TUBE DID YOU OBSERVE GREATEST DIGESTION? LEAST DIGESTION? PROVIDE
AN EXPLANATION FOR YOUR OBSERVATION.
greatest digestion- tube 1
least digestion- tube 4
test tube 1 had the greatest digestion because theres simply enough substrate concentration
for the enzyme to work. While that of test tube 4 which had the most substrate concentration
reached its saturation point.






Effect of
Metal-ion poisoning
on enzyme activity




Procedure:
1.Prepare 4 clean and dry test tubes and label it from 1-4
2. Put 1.0 mL of saliva into each test tube
3. To the first tube, add 3.0mL of distilled water ; 3.0mL of lead acetate solution to tube 2;
3.0mL of mercuric chloride solution to tube 3; and 3.0mL of 15 silver nitrate solution to tube 4
4. Place all test tubes in a water bath set at 37degrees celsius for 10 mins
5. Remove from the water bath at the same time
6. Prepare 4 clean dry vials and label 5-8
7. Transfer 3.0mL of 1% starch solution to each vials
8. Add 2 drops of iodine solution. Mix thoroughly
9. Transfer 10 drops of the solutions into the vials: tube 1 to vial 5, tube 2 to vial 6 and so on.
10. Incubate at room temperature for 10mins while mixing the contents of the vials
11. Record the color of the solutions and indicate if digestion took place (with complete
digestion-colorless, with partial digestion- violet or red and no digestion- dark shade of blue)



Test Tube 1/ vial 5 Test Tube 2/ vial 6 Test Tube 3/ vial 7 Test Tube 4/ vial8
Colorless
Greatest digestion
Yellow
Least or no digestion
Pink
Least or no digestion
Yellow Green
Least or no digestion

Enzyme- saliva
Substrate- starch
Manipulated variable- lead ,mercury, and silver (heavy metals)
We are probably aware that compounds containing heavy metals such as lead, mercury, copper
or silver are poisonous. This is because ions of these metals are non-competitive inhibitors for
several enzymes.
Enzyme inhibitors molecules that interact in some way with the enzyme to prevent it from
working in the normal manner





There are a variety of types of inhibitors including: nonspecific(pH and Temp),
irreversible(heavy metals), reversible - competitive and non-competitive. Poisons and drugs are
examples of enzyme inhibitors.
The 3 test tubes containing heavy metals obtained the same result because of the starch was
not broken down because of the enzyme inhibitor (heavy metals). Therefore, the solution
retained the purple or other color given by the iodine solution.
The test tube containing distilled water had a colorless solution because of the successful
enzyme activity. Distilled water doesnt have any enzyme inhibitor thats why starch was
successfully broken down by the amylase. The absence of starch in the solution resulted to the
disappearance of the color of the iodine solution.









PANCREATIC AMYLASE





Procedure:
B1.
1. In 2 clean testubes, put 1.0ml of 1% startch solution each.
2. To testube 1, add 1.0ml saliva and to testube 2, add 1.oml of 5% pancreatin and 1.0ml of
0.5% sodium carbonate solution.
3. Mix thoroughly and place in water bath set at 37 C for 10 mins.
4. Divide the solutions into to parts.
Benedicts Test
1. To the 1
st
portion, add 3.0ml Benedicts reagent.
2. Warm in a boiling water bath for 5 minutes.
3. Note the change in color.
4. Record all your observations.
Barfoeds Test
1. To the 2
nd
portion, add 3.0ml Barfoeds reagent.
2. Warm in a boiling water bath for 2 minutes.
3. Note the change in color.
4. Record all your observations









Study Questions:

In this procedure, identify which is/are the enzymes, the substrate and the manipulated
variable.
The enzyme is the salivary amylase and pancreatic amylase.
The substrate is the starch
The Manipulated variable is the 0.5% sodium carbonate solution.




What is the principle behind the use of the Benedicts and Barfoeds Test?
Redox Reaction.




What is the purpose of the 0.5% sodium carbonate solution in the experiment?
The purpose of the sodium carbonate is to activate the pancreatic amylase in the tube. This
resembles the process when the food stops digesting once it travels through the esophagus
going to intestine. The food starts to digest again once it reaches the duodenum which contains
bicarbonate which provide the proper environment for thr function of pancreatic amylase.

How does salivary amylase compare with pancreatic amylase in its ability to digest
carbohydrates?
Salivary amylase is the first enzyme to digest starch but pancreatic amylase is the one who
digest thoroughly starch into simple monosaccharide sugars.












Pancreatic Protease




Procedure:
B2.
1. Prepare 3 clean and dry test tubes and transfer the following solutions as follows:
Test tubes 1 2 3
Egg white 2.0 ml 2.0ml 2.0ml
CuSO4, 1% 5 drops 5 drops 5 drops
3N NaOH 2.0ml 2.0ml Add until basic to
litmus
3N Hcl - Add until acidic to
litmus
-
Pepsin - 2.0ml -
Pancreatin - - 2.0ml
2. Pepsin and pancreatin must be added simultaneously.
3. Quickly mix the contents of all tubes thoroughly.
4. Place all tubes in a water bath set at 37 C until change in color is observed.
5. Remove all the test tubes from the water bath at the same time.
6. Compare the intensity of the colored solutions produced.
7. Record all your observations.






Study Questions:
In the procedure, identify the function of every compenent used in the test.

components Function
Egg White Source of proteins which is Albumin
1% CuSO4 Reagent for Biuret Test
3N NaOH Reagent for Biuret Test
3N HCl Speeds up the pepsin
Pepsin Serves as an enzyme
Pancreatin Serves as an enzyme

What is the name of the test used? What is the principle behind its use?
The name of the test used is Biuret Test.
The principle involved in this test is

Draw the chemical equations involved in the tests and identify the substance
responsible for the visible results.


What is the purpose of adding 3N NaOH until basic to litmus? 3N HCl until its acidic to
litmus?
Pepsin is maximally active under the acidic conditions found in the gastric juice of the
stomach.
The purpose of adding NaOH is to make the solution alkaline so that complexation
reaction may happen and will provide proper environment for the functioning of
enzymes.

What is pepsin? What is pancreatin? Pancreatic juice? Specify the peptide bonds broken
by each type of proteose.
Pepsin, the powerful enzyme in gastric juice that digests proteins such as those in meat,
eggs, seeds, or dairy products.
Pancreatin is usually obtained from the pancreas of pigs or cows. The pancreas is an
organ in animals and people that makes chemicals amylase, lipase, and protease
that are needed for proper digestion. Pancreatin is used as medicine.






How does pepsin compare with pancreatic proteases in its ability to digest proteins?
Pepsin only break down proteins peptide bond but pancreatic juice can digest different
compounds other than protein.



Additional informations:

Pancreatic amylase breaks large polysaccharides like starches and glycogen
into smaller sugars such as maltose, maltotriose, and glucose. Maltase
secreted by thesmall intestine then breaks maltose into the
monosaccharide glucose, which the intestines can directly absorb.
Trypsin, chymotrypsin, and carboxypeptidase are protein-digesting enzymes
that break proteins down into their amino acid subunits. These amino acids
can then be absorbed by the intestines.
Pancreatic lipase is a lipid-digesting enzyme that breaks large triglyceride
molecules into fatty acids and monoglycerides. Bile released by
the gallbladder emulsifies fats to increase the surface area of triglycerides
that pancreatic lipase can react with. The fatty acids and monoglycerides
produced by pancreatic lipase can be absorbed by the intestines.
Ribonuclease and deoxyribonuclease are nucleases, or enzymes that digest
nucleic acids. Ribonuclease breaks down molecules of RNA into the sugar
ribose and the nitrogenous bases adenine, cytosine, guanine and uracil.
Deoxyribonuclease digests DNA molecules into the sugar deoxyribose and
the nitrogenous bases adenine, cytosine, guanine, and thymine.




















Pancreatic Lipase and
Bile





Procedure:
Prepare 3 clean and dry test tubes and transfer the contents as follows:
Test tube 1 2 3
Vegetable Oil 20 drops 20 drops 20 drops
Pancreatin 3.0 ml - 3.0 ml
Sodium
Choleate
- 3.0 ml 3.0 ml
Distilled Water 3.0 ml 3.0 ml -
2. Mix the contents of all tubes thoroughly.
3. Note and record the initial pH using pH paper.
4. Adjust the initial pH to 7 (or a little bit higher) by adding 0.1% NaOH drop
wise.
5. Immerse all the tubes in a water bath set at 37 C for 1 hour.
6. Check the pH of your solutions after every 15 minutes.
7. Remove all tubes from the water bath at the same time.
8. Record the final pH.








Results:
pH 1 2 3
Initial
pH
10 6 10
Final
pH
10 7 9

Lipase is a fat digesting enzyme that can break the bonds joining fatty acids
with glycerol. Maximum lipase activity is produced when fat is emulsified
by bile salts. The major digestion of fat occurs in the small intestine. The
digestion of fat in the small intestine is dependent upon the presence of
bile, which is produced by the liver, and the lipase enzyme produced by the
pancreas (the gallbladder serves only to store and concentrate the bile).
Since fat is insoluble in water, the fat entering the intestine from the
stomach is in the form of large droplets, which contain the fat-soluble
vitamins A, D, E and K. The detergent action of bile salts lowers the surface
tension of these large droplets allowing them to breakup into smaller
droplets and preventing large droplets from reforming (a process called
emulsification).In this way, more surface area is presented to the lipase
enzymes, permitting the digestion of fat and the release of the fat-soluble
vitamins. Lipase activity creates fatty acids which are acidic, thus lowering
the pH.

Vegtable oil served as the lipid sample.
Sodium Choleate is an emulsifying agent that transform large lipid droplet
into small lipid droplets. It aids in the suspension of triglycerides in water.
Bile salts are synthesized in the liver and stored in the gallbladder. When
the stomach and intestines digest food, the gallbladder releases bile.
Pancreatin is the enzyme used to breakdown lipids.



Emulsification -the breakdown of large fat globules into smaller, uniformly
distributed particles. It is accomplished mainly by bile acids in the small
intestine. Emulsification is the first preparation of fat for chemical digestion
by specific enzymes.
Hydrolysis pancreatic lipase hydrolyzes triglyceride into monoglyceride
and fatty acids.