enzymes, this is it!

8/3/2019 ENZYMES, This is It!

1/47

Experiment on Enzymes

Prepared by:

GROUP 3


2/47

DEMONSTRATION OF

TYPICAL ENZYMES

Experiment 4


3/47

Objectives

1. To be able to demonstrate the catalytic property ofproteins.

2. To be able to learn the different enzymatic activitythrough the use of various enzymes.


4/47

Discussion

J.J Berzelius

` Recognized the existence of catalyst in biological

materials in 1835` Coined the term catalysis

` He noted that potatoes contained something thatcatalyzed the breakdown of starch and suggested that

all natural products are formed under the influenceof such catalysis.


5/47

Louis Pasteur

` Introduced the chemical nature of biological catalyst.

` He demonstrated the fermentation(anaerobicbreakdown of sugar to CO2 and ethanol), occurredin the presence of living cells that it contained hadbeen killed by heat.

Discussion


6/47

Edward Buchner

` discovered by accident that fermentation wascatalyzed by clear juice that he had prepared bygrinding yeast with the sand and filtering out theunbroken cells.

` Looking for a way to preserve the juice, he addedsugar but the sugar break down rapidly and the

mixture frothed with CO2 .` This discovery made it possible to explore metabolic

processes such as fermentation in a greatly simplifiedsystem.

Discussion


7/47

Arthur Halden and William Young,

` two scientist discovered that yeast extracts containedtwo different types of molecules, both were

necessary to occur.

` small dialyzable, heat-stable molecules (inorganicphosphates)

`

large, non-dialyzable molecules (enzymes) weredestroyed easily by heat

Discussion


8/47

James Sumner` Proved that enzymes are proteins

` succeeded in purifying the crystallizing the enzyme ureasefrom beans.

John Northop` generalized Sumners conclusion by isolating and

characterizing a series of digestive enzymes and later ongave birth to thousands of different enzymes beingpurified which provide and immediate answers to atomicresolutions

` almost all of these molecules have proved to be proteins.

Discussion


9/47

Enzymes

` synthesized in a living cell

` These speeds up reactions more efficiently thaninorganic catalyst since these will decrease the energyof activation more than organic catalyst do.

` The effectiveness of enzymes as catalyst is

demonstrated by the high reaction rates atphysiological temperatures

Discussion


10/47

4.1 Enzymes in the Cells

MATERIALS:

` Fehling's reagent

` 0.2 Iodine solution` Hydrogen Peroxide

` Filter paper

` beaker

` test tubes

` 0.3% KH2PO4

` 0.03 M urea` Potato

` 5 grams soybeans

` Cheese cloth


11/47

Procedure

I.ExtractionofEnzymeA

1.Place 5ml freshly prepared starch solution in 2 separatetest tubes.

2. Heat one test tube to boiling, add 1ml saliva, continue boilfor 1 minute. Set aside to cool.

3. To the other test tube, add another 1ml saliva.

4. Place the two tubes in a beaker of water at 40C. Heat it

for30 minutes.5. Test both solution with Fehlings reagent and 0.2% Iodine

solution.

6. Note any evidence of color reactions.


12/47

Procedure

II.ExtractionofEnzyme B

1. Wash, peel and grate a potato.

2. Transfer the gratings and adhering liquid to clean cheesecloth,which has been folded into a bag.

3.Move the bag up and down through 100ml water contained ina beaker. Do this for 5 minutes. Notice the substances thatsettle out.

4. Decant and filter the supernatant liquid. Set aside.

5.Add 5 ml of the filtrate to each of two test tubes.

6. Place one tube in boiling water bath for 10 minutes.Removeand cool.

7. To each test tube, add 2ml of hydrogen peroxide. Shake thetest tubes well. Set them aside for 30 minutes.

8. Observes the results in each test tube.


13/47

Procedure

III.ExtractionofEnzyme C

1. Soak 5g of soybeans in H20 for at least 1 hour.

2. Blend the soaked soybeans in a blender. 10ml H20 per gram of dry soya

The H20 from the soaked bean can be used. Blend until mixture is smooth(for about 15 mins.)

3. Filter the soya puree through filter paper in a funnel

4. Collect the filtrate

5.Add 5ml of the filtrate each to 2 test tubes

6. Place 1 test tube in boiling H20 for 30 seconds.Remove andcool

7. To each test tube, add 2ml of 0.30M urea

8. Observe the results.


14/47

Demonstration of Typical Enzymes

Color Reaction with

Fehlings Reagent

Color Reaction with

Iodine Solution

Tube 1 (pre-

boiled with

saliva)

Light blue (lighter thantube 2) with blue lining

on top and white

precipitate at the

bottom

Black

Tube 2 (unboiled

with saliva)

Dark blue with blue

violet lining on top

Very dark violet

with violet bubbles

(like ink)

` EnzymeA


15/47

Enzyme A

TUBE 1 TUBE 2


16/47

Action with Hydrogen

Peroxide

Tube 1 (boiled for 10

minutes)

Brownish Green

Tube 2 (unboiled) Orange

` Enzyme B



17/47

Enzyme B


18/47

` Enzyme C

Action with 0.3 urea

Tube 1 (boiled) Precipitate at the bottom

Tube 2 (unbo

iled) No change at all



19/47

Post Laboratory Questions

What are the enzymes demonstrated in thisexperiment?

` EnzymeA Amylase

` Enzyme B Peroxidase

` Enzyme C - Urease


20/47

What are the actions of these enzymes

` Amylase - an enzyme that breaks starch down

into sugar.Amylase is present in human saliva, where it

begins the chemical process of digestion.` Peroxidase catalyzes hydrogen peroxide, breaks down

hydrogen peroxide to water and a single molecule of

oxygen

` UreaseAn enzyme that cataylzes the hydrolysis of urea

to form ammonium carbonate.



21/47

What are the substrates used in this experiment?

` EnzymeA Saliva

` Enzyme B Hydrogen Peroxide

` Enzyme C - Urea



22/47

What are the clinical significances ofenzymes?` Enzymes are substances that catalyze biochemical

reactions.Although they actively participate in thereaction, they are not changed in the process. They are

almost always found in cells so any increase in their bloodlevels would indicate cellular destruction. These are

important clinical enzymes which are measured in clinicalchemistry to determine the presence or absence of a

pathologic conditions.



23/47

CLINICAL SIGNIFICANCE OF ENZYMES

1. Peroxidase breaks down hydrogen peroxide in the body toavoid its build up in the body which can be harmful in the body2.Acid phosphatase (ACP) is primarily present in redblood cells (RBC), breast milk, and semen. It is tested to

determine the occurrence of prostate cancer, breastcancer and some red blood cell pathologic conditions.3. Alkaline phosphatase (ALP) is found in bones,kidneys, liver, placenta, intestines and platelets.Elevated ALP levels would indicate bone and liverdiseases like osteomalacia, liver cirrhosis, and the like.

4. Creatine phosphokinase (CPK) is found in thebrain, heart, and muscles. The significance of elevatedresults therefore are diseases related to the heart, brainand muscles.

What are the clinical significances of

enzymes?


24/47

What are the clinical significances of

enzymes?

4. Lactate dehydrogenase (LDH) is nicknamed the"ubiquitous enzyme" because it is almost present in all cells inthe body. It is found in red blood cells, cardiac muscles, liver,lungs, spleen and kidneys. It is elevated in liver diseases andheart conditions like myocardial infarction (MI).5. Gamma Glutamyl Transferase (GGT) is an alcoholicmarker. Its elevation indicates liver disease and chronicalcoholism. It is only elevated after prolonged usage ofalcohol.6. Alanine amino transferase (ALT) is a liver enzyme. It ismarkedly elevated in liver disease.

7. Aspartate amino transferase (AST) is a cardiac enzyme.It is significantly elevated in myocardial infarction, and slightlyelevated in liver disease. CK, LDH, and AST are the threecardiac enzymes.


25/47

What are the characteristics ofenzymes?` Enzymes are proteins that speed up chemical reactions.

` Without enzymes, they would be too slow to sustain life.

` Body is essentially a chemical processing plant, so enzymes

are crucial in every aspect of physiology.Characteristics of enzymes which enable them to be effective:

` Enzymes are unaffected by the reactions that they

speed up.

` Enzymes are highly specific.

` Enzymes can speed up the same chemical reaction

going inopposite directions.



26/47

Factors Affecting Enzyme Activity



27/47

Objectives

1. To be able to isolate the enzyme from a potato

extract.2. To be able to learn the different factors that will

affect the activity of enzymes.


28/47

Discussion

An enzyme is` considered a catalyst, a substance that speeds up

chemical reactions without itself undergoing anychange.

` There are factors that will affect its activity namely:pH, substrate concentration and temperature.


29/47

Enzymes` Stable only over a limited range ofpH.

` The effects of pH can reflect the pKa of ionizinggroups, on either enzyme or the substrate

` May also undergo changes in conformation when pHis varied.

` Depending upon the severity of these changes,

activity may or may not be restored when theenzyme is returned to its optimal pH.

Discussion


30/47

Enzymes` Substrate concentration influence the rate of an enzyme

catalyzed reaction. Initially, the rate is responsive toincrease in substrate concentration.

` The maximum rate(Vmax) occurs because theenzyme is saturated with substrate and cannot workany faster under conditions imposed.

Discussion


31/47

Enzyme` Like all chemical reactions, enzyme catalyzed reaction

rate increase with temperature.

` However, because enzymes are proteins, there is atemperature limit beyond which enzyme becomesvulnerable to denaturation.

` Enzyme catalyzed reaction has an optimum

temperature, usually range 25 to 40 degrees celsius.Above or below these values the reaction rate will belower.

Discussion


32/47

5.1 Factors Affecting Enzymatic Activity

MATERIALS:` 2% NaF SOLUTION

` 0.01M HCL

`

Cheesecloth` pH buffer

` Potato

` 0.1M Na2Co3` Hydrogen peroxide

` Thermometer

` ice

` pH 7 buffer

`

CuSO4` Test tubes

` Beaker


33/47

Procedure:

A. Preparationofenzyme extract1. Obtain 10g peeled potato

2. Cut the potato into small pieces and place in themortar.

3. Add 5ml distilled water.

4. Grind the mixture thoroughly. Place in a 100ml beakerand add 30ml 2% NaF solution. Continue grinding untilextract is produced.

5. Stand for a minute or two and then pour into a 100mlbeaker through cheesecloth held in a funnel.


34/47

Procedure

B.Effect ofpH1. Prepare four-labeled test tubes that contain the following

solutions:

A. 2ml 0.01M HCl

B. 2ml ofpH 4 buffer

C. 2ml ofpH 7 buffer

D. 2ml of0.1M Na2CO3

2. Add 1ml hydrogen peroxide to each test tubes followed by 1ml

potato extract.

3. Mix well and place all four-labeled test tubes in the 370C water

bath.

4. Wait forfew minutes and then examine each test tubes for

reactions.

5. Observe and record all the results (measure the foam height)


35/47

Procedure

C. Substrate Concentration1. Prepared four-labeled test tubes that contain the following

solutions:

A. 4ml hydrogen peroxide

B. 2ml hydrogen peroxideC. 1ml hydrogen peroxide

D. 0.50 hydrogen peroxide

2. Place all four-labeled test tubes in the 370C water bath.

3. Obtain 4 test tubes and put 2ml potato extract into each tubeand place in the 370C water bath for 2 minutes.

4. Observed and record the results (measure foam height)


36/47

D.Enzyme Concentration1. Prepared three-labeled test tubes that contains the following:

` 1.5ml potato extract

` .50ml potato extract + distilled water

`

.10ml potato extract + distilled water` Use a dropper to add distilled water making sure that the level of

tube B & C will be the same as the level of test tubeA.

2. Place all three-labeled test tubes in 37 oC water bath.

3. Obtain 3 test tubes and add 1ml hydrogen peroxide into each

tube and place in 37 oC water bath. Boil for 5 minutes.4. Observed and record the results (Measure the foam height)

Procedure


37/47

Procedure

TESTTUBE TEMPERATURE

1 Ice bath(approximately 0 oC)2 20-25 oC

3 37 oC

4 70 oC

E. Effect of temperature1. Prepared four labelled test tubes.

2. Place 1ml of potato extract in each test tube

3. Place each tube at a constant temperature for 10minutes

4. Remove the test tubes from the bath and add the same amount

of hydrogen peroxide to each test tube. Put the test tube

back into the bath.

5. Compare the rates of oxygen production by measuring the foam

height in each tube after 5minutes of putting it back at the water

bath.


38/47

TestTube H2O2 Potato

Extract

CuSO4

1 5ml 5ml 0

2 5ml 5ml 10 drops

Procedure

F. Effect of an inhibitorFill up 2 separate test tubes with substances

indicated below:

Observe and record the results


39/47

F. Effectofan Inhibitor


40/47


` I. Effects of pH

Test Tube Result

A.

2

m

l0.01M

HCL0

mm

B.2ml of pH 4 buffer 2mm

C.2

m

l of pH 7 buffer0

mm

D.2ml of0.1 Na2CO3 40mm


41/47

Effectsof pH


42/47


` II. Substrate Concentration

Test Tube Result

A. 4 ml H2O2

14 mm

B.2ml H2O2

19mm

C.1ml H2O2

18mm

D.0.5ml H2O2

19mm


43/47

Substrate Concentration

A B C


44/47

` III. Enzyme Concentration


Test Tube Result

A.1.5ml potato extract 24 mm

B.0.50ml potato

extract + distilled water

10mm

C.0.10ml potato

extract + distilled water

0mm


45/47

` IV. Effects of Temperature


Test Tube Result

A. Ice Bath

(approximately 0oC)

7 mm

B.20 25oC 17 mm

C. 37oC 18mm

D. 70oC 3 mm


46/47

Effectsof Temperature

A B C


47/47

Thank you

enzymes, this is it!

Documents