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ACTIVITY

6

Enzymes

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INTRODUCTION

ACTIVITY 6

Enzymes

Enzymes are proteins. They are biological molecules that significantly speed up the rate of the chemical reactions that take place within cells. Unlike inorganic catalysts, enzymes show a high degree of specificity. This means that the enzymes catalyze only one reaction for a specific substrate. Some enzymes help break large molecules into smaller pieces that are more easily absorbed by the body. Other enzymes help bind two molecules together to produce a new molecule.

All enzymes have a protein component and are affected by a number of physical and chemical factors that denature proteins, e.g. extremes of pH and temperature, presence of metal and non-metal ions, presence of alkaloidal reagents to name a few .In addition, enzyme and substrate concentrations also affect enzyme activity.

The molecules that an enzyme works with are called substrates. The substrates bind to a region on the enzyme called the active site. Consequently, the reactions that occur accelerate greatly once the substrates bind to the active site of the enzyme. The chemical reactions result in a new product or molecule that then separates from the enzyme, which goes on to catalyze other reactions.

Enzymes are named by adding the suffix -ase to the name of the substrate that they modify (i.e., urease and tyrosinase), or the type of reaction they catalyze (dehydrogenase, decarboxylase). Some have arbitrary names (pepsin and trypsin).

Metabolic processes provide energy for the maintenance of life support systems of organisms. These include the degrading of chemical compounds of relatively high potential energy to products with low potential energy. Th energy evolved is collected, stored and utilized by the cell for growth, reproduction, synthesis, repair of cellular materials and other functions which keep life systems operational. These processes are made possible by catalytic substances called enzymes.

OBJECTIVES

1. To determine the factors that affect enzyme activity .

2. To observe reactions catalyzed by enzymes.

MATERIALS test tube , test tube rack, test tube holder, 5-mL pipet, dropper, 600-mL beaker, wire gauze, burner, tripod, thermometer, spot plate

REAGENTS distilled water, potato extract, 3M NaOH, solution, 0.1 MCuSO4, 0.1M NaCl, 1% AgNO3, tannic acid, 95% ethanol, 6% H2O2, iodine solution

PROCEDURE

I. Preparation of sample extract

Activity 6

1. Wash a potato and grate it, including its skin into a fine pulp.

2. Extract a juice into a beaker and re- extract the pulp after adding a little distilled water. Strain through cheesecloth and discard the pulp.

3. Use the extract for the following test:

Hazard warning: Hydrogen peroxide burns skin on contact.

1. Mix 5 ml of extract and 5ml of 6% H2O2 in a test tube.

2. Immediately insert a glowing wooden splint or a lighted matchstick into the test tube (do not drop inside the tube!). Observe what happens and identify what gas is present.

3. To 5ml of catalase extract, add 5ml of 3M NaOH. Mix the two solutions. Add 5 drops of 0.1M CuSO4 solution to the mixture. Observe and note the color formed.

Preparation of Enzyme

Factors that Affect Enzyme Activity

1. Collect 10-15 ml of your saliva and use it to test for the effect of pH and temperature.

2. Label the test tubes and mix the substances indicated below.

1 ml starch solution

10

1ml 0.1 M NaCl

1ml 0.1 M NaCl

2ml saliva

2 ml starch solution

10

1ml 0.1 M NaCl

1ml distilled water

2ml saliva

3 ml starch solution

10

1ml 0.1 M NaCl

1ml 0.05 M NaOH

2ml saliva

3. Mix well by shaking each test tube and place in water bath. Maintain at 37degree Celsius.

4. To test for complete hydrolyzation, while still in the water bath, take two drop of each solutions from the three test tubes and place them into separate depressions of a spot plate.

5. Add two drops of iodine solutions to each solution. If a dark blue color is observed, repeat this procedure after another three minutes. A dark blue color should appear on each spot.

6. After three minutes, take another two drops from each of the solutions and repeat the addition of iodine solution. If a dark blue color is still observed, repeat this procedure after another three minutes. Keep doing this until the blue color is no longer observed and only the iodine color is observed.

7. Take note of the time it takes for the blue color of the starch-iodine mixture to disappear in the samples taken from each tube, indicating that all the starch had been completely hydrolyzed into glucose. Record all observations.

Effect of Temperature

1. Label 3 test tubes 1 to 3. Add 10 ml of 1% starch solution and 1 ml of 0.1 N NaCl solution to each.

2. Place test tube number 1 in a beaker of water at room temperature.

3. Place test tube number 2 at 37 degree Celcius.

4. Place test tube number 3 in a boiling water bath.

5. Place 2ml of the saliva into each test tube and mix.

6. To test for complete hydrolyzation, take two drops of each solution from the three test tubes and place them into separate depressions of the spot plate.

7. Add two drops of iodine solution to each solution (in the spot plate) every three minutes. Observe the color change in each spot.

8. After three minutes, take another two drops from each of the solutions and repeat the addition of iodine solution. If a dark blue color is still observed, repeat this procedure after another three minutes. Keep doing this until the blue color is no longer observed and only the color of the iodine is seen.

9. Again note the time it takes before the starch is completely hydrolyzed, or when the blue color with iodine no longer appears. Record results and observations.