enzymes lols

8/12/2019 Enzymes Lols

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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


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What are enzymes?

Enzymes areproteinsthat 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 Fe2+

, Mg2+

, Mn2+

, or Zn2+

, or consist of organic or metalloorganic moleculesknowns as co-enzymes.

Enzyme Classifications

Enzymes are classified according to the reactions they catalyze. The six classes

are:

1.

Oxidoreductases

2.

Transferases3.

Hydrolases

4.

Lyases

5.

Isomerases

6.

Ligases
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FACTORS AFFECTING ENZYMATIC ACTIVITY

A. EFFECT OF TEMPERATURE


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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.


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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.


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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.


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Effect

Of

pH


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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)


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Study Questions:

Enzymesare 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


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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 lesseffective. The low pH of the juices in the stomach can cause ulcers if they eat through the walls

of the small intestine or stomach.


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Effect of Enzyme

Concentration


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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.


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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.


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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.


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A.4 Effect of the

Substrate

Concentration


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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.


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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.


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Effect of

Metal-ion poisoning

on enzyme activity


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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)


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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


Yellow Green


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


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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 solutionretained 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.


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PANCREATIC AMYLASE


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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 1st

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 2nd

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


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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.


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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.


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Pancreatic Protease


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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.


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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


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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 amylasebreaks large polysaccharides like starches and glycogen

into smaller sugars such as maltose, maltotriose, and glucose. Maltase

secreted by thesmall intestinethen breaks maltose into the

monosaccharide glucose, which the intestines can directly absorb.

Trypsin, chymotrypsin, and carboxypeptidaseare protein-digesting enzymes

that break proteins down into their amino acid subunits. These amino acids

can then be absorbed by the intestines.

Pancreatic lipaseis a lipid-digesting enzyme that breaks large triglyceride

molecules into fatty acids and monoglycerides. Bile released by

thegallbladderemulsifies 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.

Ribonucleaseand deoxyribonucleaseare nucleases, or enzymes that digestnucleic 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.
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Pancreatic Lipase and

Bile


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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 dropwise.

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.


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Results:

pH 1 2 3

InitialpH

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 calledemulsification).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.


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