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
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 group
20% yeast suspension* 0.005% methylene blue solution+ (prepared 1~2 days ahead) 10 cm3 1 % glucose solution distilled water 10 cm3
test-tube rack and 4 test-tubes Bunsen burner
3 labels or spirit marker graduated pipette or syringe 5-10cm3 test-tube holder beaker or jar, for water to rinse pipette or syringe
Result The methylene blue: in tubes 2 and 3 should be decolourized in a few minutes with tube 3 changing first.
* Add 40 g dried yeast and 0.4 g potassium dihydrogen phosphate (KH2PO4) to 200 cm3 distilled water in a tall 600 cm3 (or larger) beaker (a large jam jar will do). Cover the mouth of the container with aluminium foil and bubble air through the yeast suspension for one or two days using an aquarium aerator. Observe the suspension from time to time during the first two hours and control the air flow to prevent the yeast suspension frothing out of the jar.
+Dissolve 0.05 g in 1 litre of distilled water. Methylene blue stains skin and clothing. Lab coats should be worn
Experiment 14. Discussion – answers
1 The addition of distilled water to tubes 1 and 2 keeps the concentration of yeast and methylene blue the same in all three tubes.
2 The methylene blue accepts hydrogen atoms removed from glucose molecules during respiration. The reduced form of methylene blue is colourless.
3 Boiling will have killed the yeast. Dead yeast is therefore incapable of carrying out one or more stages in the transfer of hydrogen from glucose to methylene blue. (A similar answer may be given in terms of enzymes.)
4 Tube 3 will probably lose its blue colour first. Presumably if the hydrogen atoms for reducing methylene blue come from glucose, additional glucose will mean that more hydrogen atoms are available and decolourization will be more rapid.
5 Respiration will continue in yeast cells, using their own carbohydrate reserves such as glycogen.
6 It might be expected that increasing the glucose concentration would increase the rate of decolourization up to the point where all the available enzyme or enzymes were being used, or where the concentration of glucose was sufficient to plasmolyse the yeast cells.
7 If enzymes (dehydrogenases) are involved, it should be possible to extract them from yeast by grinding some dried yeast with sand and distilled water, and filtering. This could be the subject of further experiment, particularly if little or none of the carbohydrate reserve in yeast comes through in the filtrate.
8 Shaking the tubes introduces more oxygen which re-oxidises the methylene blue