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Experiment 14. Dehydrogenase in yeast

During respiration, hydrogen atoms are removed from glucose molecules by enzymes called dehydrogenases and passed to various chemicals called hydrogen acceptors. As the hydrogen atoms pass from one hydrogen acceptor to another, energy is made available for chemical reactions in the cell. In this way, substances such as glucose provide energy for vital reactions in living organisms. In this experiment, a dye called methylene blue acts as an artificial hydrogen acceptor. When this dye is reduced by accepting hydrogen atoms it goes colourless.

(a) Place about 30 mm of yeast suspension in a test-tube and, using a test-tube holder, heat this suspension over a small Bunsen flame until the liquid boils for about half a minute. Then cool the tube under the tap.

(b) Label three test-tubes 1-3.

(c) Using a graduated pipette or syringe, place 2 cm3 of the boiled yeast suspension in tube 1.

(d) Using the graduated pipette or syringe, draw up 4 cm3 unboiled yeast suspension and place 2 cm3 in tube 2 and 2 cm3 in tube 3.

(e) Rinse the pipette or syringe and use it to place 2 cm3 distilled water in tubes 1 and 2.

(f) With the pipette or syringe, place 2 cm3 1 % glucose solution in tube 3.

(g) Prepare a water bath by mixing hot and cold water from the tap to obtain a temperature between 35 and 45 °C. Place all three tubes in this water bath. Rinse the pipette or syringe.

(h) Copy the table given below into your notebook.

(i) After 5 minutes draw up 6 cm3 methylene blue solution in the pipette or syringe and place 2 cm3 in each tube. Shake all three tubes thoroughly and return them to the water bath, noting the time as you do so. Do not shake the tubes again.

(j) Watch the tubes to see how long it takes for the blue colour to disappear, leaving the creamy colour of the yeast. A thin film of blue colour at the surface of the tube may be ignored but the tubes should not be moved. Record the times in your table.

(k) The experiment may be repeated by simply shaking all the tubes again until the blue colour returns.

|Tube |Contents |Time for methylene blue to go colourless | |1 |Boiled yeast | | |2 |Unboiled yeast | | |3 |Unboiled yeast + 1% glucose | |

Experiment 14. Discussion

1 Why was distilled water added to tubes 1 and 2?

2 What causes the methylene blue solution to go colourless (according to the introduction on p. 14.01)?

3 How do you explain the results with tube 1?

4 In which of tubes 2 and 3 was the methylene blue decolourized more rapidly? How can this result be explained?

5 If the hydrogen atoms for the reduction of methylene blue come from glucose, why should the methylene blue in tube 2 become decolourized at all?

6 What do you think would be the effect of increasing the glucose concentration in tube 3? Explain your answer.

7 How could you extend the experiment to see if enzymes in yeast are capable of reducing methylene blue?

8 Why, do you think, the colour retuned on shaking the tubes?

Experiment 14. Dehydrogenase in yeast - preparation

Outline Methylene blue, acting as a hydrogen acceptor, is decolourized during the respiration of yeast. Addition of small amounts of substrate increases the rate of decolourization.

Prior knowledge An elementary idea of respiration as a process which releases energy during the breaking down of carbohydrates; yeast is a microscopic living organism.

Advance preparation and materials-per...
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