Use of a Redox Indicator to show Dehydrogenase Activity

Triphenyl tetrazolium chloride (also known as T.T.C) is an pillow slip of an artificial hydrogen acceptor. It is a oxidation-reduction indicator which is colourationationless when oxidised, however when reduced, it produces a red, insoluble precipitate called formazans. T.T.C can whence be used to study the enzyme occupation of dehyrogenase enzymes by showing a colour reposition when they are present. The purpose of this try is to see what effect temperature has on the application of dehydrogenase enzymes within barm cells.Materials/Apparatus* actively respiring barm suspension. This is prepared by adding 10g of modify barm to 1dm3 of distilled pissing, followed by mixing in 50g of glucose. This mixture should be brooked to wrack for 24 hours before the sieve engenders pop come forth.* Tiphenyl tetrazolium chloride is used as a redox indicator to investigate the drill of dehydrogenase enzymes when yeast suspension is uncovered to different temperatures.* Distil led water for the preparation of the yeast suspension.* Test tubes to slip the mixture of yeast and T.T.C.* Test tube rack to allow the test tubes to stand upright in the water tubs.* Incubator to allow enzyme action to occur at different temperatures* Syringes to accurately measure the right amount of yeast and T.T.C needed for each effect.* A glass rod to evenly distribute the cells in the solution after the T.T.C has been added.* Crushed grouch 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 term taken for the solution to variety colour. bloodline The colour careen is completed at once the solution has turned a pink-orange pink colour. Allow all solutions to reach the uniform colour before removing them from the water baths. rulePrepare a solution of yeast cells by adding 10g of dried yeast to 1 dm3 of distilled water, followed by mixing in 50g of glucose. This mixture should be allowed to stand for 24 hours before the audition takes place. Once the yeast suspension has been allowed to stand for 24 hours, the froth should be upstage and discarded.Set up a water bath by adding ice cubes to ratty 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 common chord separate test tubes and place in the incubator. Leave for several minutes and accordingly add 0.5cm of T.T.C into each solution and place them back into the incubator inflexible at 30degrees. Start the stopwatch immediately. Observe guardedly for any colour turns that check developed. When the colour deviate has taken place, take the test tubes out of the incubator and note muckle the time taken for the colour c hange 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. control panel of resultsTemperature (degrees)Time taken for colour change to occur (minutes)10No change2052.113026.124010.08504.22604.43A bar represent has been produced to portray these results so that a comparison can clearly be seen. The represent has been drawn on graph paper. cultureThe results from this experiment indicate that temperature has a decisive 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 blistering 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 t he optimal 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 control 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 take the hypothesis and therefore have provided no-hit and sufficient data which have corroborate the predictions that were made prior to the investigation fetching place.