PRACTICAL 5: ENZYMES AND DIGESTION

Objectives:

a) To show the action of amylase on starchb) To study the effect of temperature on enzyme actionc) To show the action of pepsin on egg white (protein)d) (Pepsin)To show emulsification of fats

Introduction: 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."

Hypothesis: The salivary amylase in the saliva hydrolyses the starch into a reducing sugar

Variables:

a) Manipulated: Contents in the test tubeb) Responding: The presence of starch or glucose in the test tube c) Fixed: Amount of contents in the test tube

Literature reviews:

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 component 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.

Materials and apparatus:

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

Procedure:

a) To show the action of amylase on starch

1. The mouth is rinsed2. 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 cm3 of dilute hydrochloric acid

D – 3 cm3 of the saliva preparation and 3 cm3 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.

Results:

Test tube Contents Iodine test Benedict’s test Inferences A Starch +

distilled waterFood sample in test tube A turns blue-black

Food sample in test tube A remains blue in colour.

This is because food sample in A contains starch

B Starch + saliva Food sample in test tube B remains brownish yellow.

Food sample in test tube B formed a brick-red precipitate.

This is because food sample in B contains glucose

C Starch + saliva + dilute HCl

Food sample in test tube C turns blue-black

Food sample in test tube C remains blue in colour.

This is because food sample in C contains starch

D Starch + saliva + dilute NaOH

Food sample in test tube D remains brownish yellow.

Food sample in test tube D formed a brick-red precipitate

This is because food sample in D contains glucose

Discussion:

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

b) To study the effect of temperature on enzyme action

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

Variables:

d) Manipulated: Temperature of the mixture in the test tubee) Responding: Rate of reaction of enzyme f) Fixed: The amount of saliva in each test tube

Procedures:

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 mixing 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 5o, 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.

Results:

Temperature Time in minutes (t) Activity (1 / t)Room temperature 5 0.200

5o 18 0.05615o 12 0.08325o 8 0.12545o 6 0.16755o 9 0.11165o 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).

Discussion:

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 substrate 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 denatured. The rate of reaction declines.

6. At 60oC, the enzyme denatured.

Questions:

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 molecules 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.

c) To show the action of pepsin on egg white (protein)

Literature Review: 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 ( p H 1-3) for pepsin function. Pepsin and other proteolytic enzymes are used in the laboratory analysis of various proteins; pepsin is also used in the preparation of cheese and other protein-containing foods.

Hypothesis: Enzyme pepsin hydrolyses protein into amino acid

Procudures:

1. A suspension of egg white is prepared 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 37oC). Each tubes is filled as follows:

A – 5 cm3 of 0.2% hydrochloric acid

B – 5 cm3 of ‘artificial gastric juice’ (prepared by dissolving 3.5 g of pepsin in

100 cm3 of 0.2 % hydrochloric acid

C – 5 cm3 of ‘artificial gastric juice’ and 1 cm3 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 shaked 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.

Results:

Test tube

Contents Observation Buiret test Inferences

A Egg white + dilute HCl Food sample in test tube A turn

purple.

Positive This is because food sample in test

tube A contains protein

B Egg white + artificial gastric juice

Food sample in test tube B turn blue in colour

Negative This is because food sample in B

contains amino acid.

C Egg white + artificial gastric juice + dilute NaOH

Food sample in test tube C turn

purple.

Positive This is because food sample in test

tube C contains protein

Discussion:

1. Buiret test is used to detect the presence of protein. 2. Test tube A acts as control experiment.3. 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.

d) To show emulsification

Literature Review:

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.

Hypothesis: Bile acts as emulsifier to mix up water and coconut oil.

Variables:

a) Manipulated: Contents in the test tubeb) Responding: The condition of the contents after experiment is donec) Fixed: The amount of solution in the test tube

Inferences:

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?

Result:

Contents Observations InferencesCoconut oil + water Coconut oil and water does

not mix. Coconut oil is placed in the top level while water in the bottom level.

This is because coconut oil is a non-polar molecules where as water is a polar-molecules.

Coconut oil + water + bile

Coconut oil, water and bile mix each others.

This is because bile acts as emulsifier to mix up the water and coconut oil.

Discussion:

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 and 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 moved 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 large 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 attracted 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 other 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 around. 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 (like 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 chemicals 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 turn 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.

Conclusion: The hypothesis is accepted. Bile acts as emulsifier to mix up water and coconut oil.

References:

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

Effect the temperature on enzyme activity. (n.d.). Retrieved February 21, 2010, from

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

Enzyme amylase action on starch. (n.d.). Retrieved February 22, 2010, from

http://www.occc.edu/bbdiscovery/documents/Modules/Packing%20peanuts.htm

Pepsin. (n.d.). Retrieved February 24, 2010, from http://en.wikipedia.org/wiki/Pepsin

Protein digestion - a way through gut. (n.d.). Retrieved February 23, 2010, from

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

Why water and oil does not mix. (n.d.). Retrieved February 20, 2010, from

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

water_oil.html