BIOLOGY Volume 1 - A Guide to General Biology - 2004

4. ENZYMES

4.3. Factors Affecting Enzyme Reaction Rates

4.3.5. Laboratory Practical Work

Experiment 4.1. Investigating The Effect of Enzyme Concentration on the Hydrolysis of sucrose catalysed by sucrase (invertase)

Materials and Equipment

2% sucrose solution

1%, 0.75%, and 0.5% sucrase (invertase) solutions

Benedict's reagent

12 test tubes with a rack

Water baths maintained at 38 °C and 100 °C

Glass stirring rods

Timer

Distilled water

Labels

Bunsen burner

Procedure

1. Add 2 ml of clear blue Benedict's reagent to 2 ml of clear, colourless 1% sucrase solution. Heat the mixture in a water bath at 100 °C for 5 min (Benedict's test).

2. Repeat step 1 using 2 ml of clear, colourless 2% sucrose solution, and then with 2 ml of distilled water.

3. Bring 5 ml of 1% sucrase solution to the boil.

4. Pipette 1 ml of Benedict's reagent into each of eight clean, dry, labelled test tubes (1–8).

5. Place 5 ml of 2% sucrose solution into a test tube labelled S and put it into a water bath maintained at 38 °C throughout the experiment.

6. Place 5 ml of 1% sucrase solution into a test tube labelled E and put it into the 38 °C water bath.

7. Leave both test tubes and their contents in the water bath for 5 min to allow them to reach the required Temperature.

8. Add the enzyme solution to the sucrose solution and invert the test tube to mix the two solutions thoroughly.

9. Immediately start the timer and return the test tube containing the reaction mixture to the water bath.

10. Continuously stir the reaction mixture throughout the entire experiment.

11. After 30 s of incubation, transfer 1 ml of the mixture into tube 1.

12. At 30 s intervals, take similar samples and transfer them one by one into tubes 2—8.

13. Heat tubes 1—8 in a boiling water bath at 100 °С for 5 min. Note the time when a brick-red precipitate first appears, indicating a positive reaction for reducing sugars.

14. Repeat the same experiment, this time using the boiled enzyme solution (see step 3).

15. Repeat the entire procedure twice using 0.75% and 0.5% sucrase solutions.

16. Record your observations and explain the results obtained.

Experiment 4.2. Investigation of catalase distribution in soaked pea seeds and the Effect of temperature on enzyme activity

Catalase is an enzyme that catalyzes the decomposition of hydrogen peroxide to form molecular oxygen, which is released as gas bubbles:

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Hydrogen peroxide is produced in certain PLANT AND ANIMAL Cells as a metabolic byproduct. This compound is toxic to cells, and catalase ensures its efficient removal. Catalase is one of the fastest-acting Enzymes known.

Materials and Equipment

A handful of soaked peas

Hydrogen peroxide solution

Test tubes and rack

Water baths at 40, 50, 60, 70, 80, and 100 °С

Clock or stopwatch

Thermometer

Scalpels, scissors, and forceps

Test tube holder

Glass stirring rod

White ceramic tile

Procedure

1. Test for the presence of catalase. To do this, mash a single pea seed and apply a few drops of hydrogen peroxide to it.

2. Peel the seed coats off three peas and test the seed coats and cotyledons for catalase activity separately.

3. Place two test tubes containing distilled water into a water bath set at 40 °С.

4. Boil three whole peas in a separate test tube, and then place them into one of the test tubes in the water bath.

5. Place three unboiled peas into the other test tube in the water bath.

6. Leave the test tubes in the water bath for a sufficient time to reach its temperature (about 10 min).

7. Test each of the peas for catalase activity.

8. Repeat the same experiment at 50, 60, 70, 80, and 100 °С.

9. Record your observations and explain the obtained results.

Experiment 4.3. Investigating the effect of various pH levels on enzyme activity

Materials and Equipment

Benedict's reagent

Buffer solutions with pH 3, 5, 7, 9, and 11

1% starch solution

Water bath at 38 °С

Bunsen burner

Asbestos sheet

Test tube holder, test tube rack

5 ml graduated pipettes

Thermometer

Timer

Distilled water

Stock amylase solution (such as that contained in saliva)

Procedure

1. Rinse your Mouth with 5 ml of distilled water and spit it out.

2. Take 10 ml of distilled water into your mouth, rinse for 1 min, and collect the liquid.

3. Bring the volume of this salivary amylase solution up to 40 mL with distilled water.

4. Test the amylase, starch, and buffer solutions for the presence of reducing sugars using Benedict's reagent.

5. Label one of the test tubes "pH 3" and add 2 mL of the starch solution to it.

6. Add 2 mL of the pH 3 buffer solution to the same test tube and mix both solutions thoroughly.

7. Boil at least 4 mL of the enzyme solution and transfer 4 mL of it into the appropriately labeled test tube.

8. In another labeled test tube, add 4 mL of the unboiled enzyme solution; place all three test tubes in a water bath and wait briefly (about 1 min) for them to warm up to 38 «С.

9. Pour a small amount of Benedict's reagent into each of the 11 test tubes and number them 1–11. The next three steps must be performed very quickly.

10. Once the solutions in the water bath have reached its temperature, add the buffered starch solution to the unboiled enzyme solution.

11. Mix both solutions thoroughly by inverting the test tube, and then return the test tube to the water bath.

12. Start a timer and immediately transfer a small amount of the reaction mixture (approximately equal in volume to the Benedict's reagent used) into test tube 1.

13. Shake the mixture vigorously throughout the entire experiment.

14. After 1 min, transfer a second portion of the reaction mixture (roughly the same volume as the first) into test tube 2.

15. Repeat this procedure at 1-min intervals for another 9 min (i.e., fill test tubes 3–11 with the collected samples).

16. For test tubes 1–11, record the incubation time required for the first signs of a positive Benedict's reaction to appear (formation of a brick-red precipitate).

17. Repeat the same experiment using the boiled enzyme solution, starting from step 7.

18. Repeat the entire experiment from scratch with each of the remaining buffer solutions.

19. Plot a graph showing the dependence of hydrolysis time on pH and explain the results obtained.