Use of a Redox Indicator to show Dehydrogenase Activity

Triphenyl tetrazolium chloride (also known as T.T.C) is an example of an artificial hydrogen acceptor. It is a redox indicator which is colourless when oxidised, however when reduced, it produces a red, insoluble precipitate called ‘formazans'. T.T.C can therefore be used to investigate the enzyme activity of dehyrogenase enzymes by showing a colour change when they are present. The purpose of this experiment is to see what effect temperature has on the activity of dehydrogenase enzymes within yeast cells. Materials/Apparatus: * Actively respiring yeast suspension. This is prepared by adding 10g of dried yeast to 1dm3 of distilled water, followed by mixing in 50g of glucose. This mixture should be allowed to stand for 24 hours before the experiment takes place. * Tiphenyl tetrazolium chloride is used as a redox indicator to investigate the activity of dehydrogenase enzymes when yeast suspension is exposed to different temperatures. * Distilled water for the preparation of the yeast suspension. * Test tubes to place the mixture of yeast and T.T.C. * Test tube rack to allow the test tubes to stand upright in the water baths. * Incubator to allow enzyme activity to occur at different temperatures * Syringes to accurately measure the right amount of yeast and T.T.C needed for each solution. * A Glass rod to evenly distribute the cells in the solution after the T.T.C has been added. * Crushed ice to allow the dehyrogenase activity to take place at 10degrees. * Beakers for the yeast suspension to be prepared in. * Thermometer to measure the temperature of the water bath containing the ice cubes. * Stopwatch to measure the time taken for the solution to change colour. NOTE: The colour change is completed once the solution has turned a ‘salmon pink' colour. Allow all solutions to reach the same colour before removing them from the water baths. Method: Prepare a solution of yeast cells by adding 10g of dried yeast to 1dm3 of distilled water, followed by mixing in 50g of glucose. This mixture should be allowed to stand for 24 hours before the experiment takes place. Once the yeast suspension has been allowed to stand for 24 hours, the froth should be removed and discarded. Set up a water bath by adding ice cubes to cold water, until the water has reached 10degrees. Continue to measure the temperature with a thermometer ensuring that the temperature is maintained. Set up separate incubators at 30, 40, 50 and 60 degrees. Using a syringe, place 5cm of yeast suspension into three separate test tubes and place in the incubator. Leave for several minutes and then add 0.5cm of T.T.C into each solution and place them back into the incubator set at 30degrees. Start the stopwatch immediately. Observe carefully for any colour changes that have developed. When the colour change has taken place, take the test tubes out of the incubator and note down the time taken for the colour change to take place. Repeat this procedure at 20, 40, 50 and 60 degrees. To measure the dehydrogenase activity at 20 degrees, carry out this procedure at room temperature. Table of results: Temperature (degrees) Time taken for colour change to occur (minutes) 10 No change 20 52.11 30 26.12 40 10.08 50 4.22 60 4.43 A bar graph has been produced to portray these results so that a comparison can clearly be seen. The graph has been drawn on graph paper. Conclusion: The results from this experiment indicate that temperature has a definite affect on the activity of dehydrogenase enzymes. The graph shows that as the temperature increases, the time taken for the solution to change colour decreases. This shows that dehyrogenase enzymes work faster at a higher temperature as there was no colour change when the T.T.C was added to the yeast suspension at 10 degrees. The temperature at which the dehydrogenase enzymes worked at their quickest was 50 degrees. This indicates that 50 degrees is the optimum temperature for the enzyme activity to take place as the colour change took slightly longer when placed in a water bath set at 60 degrees. This may be due to the fact that some of the dehydrogenase enzymes could have been denatured due to the high temperature. However, it is not quite clear whether 50 degrees is the optimum temperature for the enzyme activity to take place because this experiment took place using a limited amount of temperature ranges. If this investigation was to be repeated, a wider range of temperatures could be used so that an optimum temperature could be established. Overall, the results from this experiment support the hypothesis and therefore have provided successful and sufficient data which have confirmed the predictions that were made prior to the investigation taking place.