Investigate the effect of temperature on dehydrogenase activity in yeast. At which temperature is tetrazolium chloride (TTC) discoloured most rapidly?

Investigate the effect of temperature on dehydrogenase activity in yeast.

At which temperature is tetrazolium chloride (TTC) discoloured most rapidly?

Introduction

Enzymes are composed of many specialised organic substances, such as polymers of amino acids, which act as catalysts to regulate the speed of the many chemical reactions involved in the metabolism of living organisms. Individual enzymes are naming by adding –ase to the name of the substrate with which they react. There are many types of enzymes such as… hydrolytic, oxidising, and reducing.

Hydrolytic… speed up reactions in which a substance is broken into simpler compounds through reaction with water molecules.
Oxidases… these are oxidising enzymes that speed oxidation reactions.
Reductases… reducing enzymes that accelerate reduction reactions.

“Enzymes are highly specific in their actions. Each enzyme catalyses either a single chemical reaction or a group of closely related reactions”

Enzymes operate by attaching themselves to their substrate molecule at the active site. The theory that enzymes and substrates bind is called the ‘lock and key’ hypothesis. As the enzyme (A) and the substrate (B) form a complex (AB), the substrate is raised in energy to a transition state, and then breaks down to products (C) plus an unchanged enzyme (E)…

A + B → AB → C + E

In this experiment the only variable which can be changed is the temperature, to show that it is the only influence on the dehydrogenase activity. I will be observing the speed at which the colour changes with the redox indicator that I am using, tetrazolium chloride (TTC). This indicator is an artificial hydrogen acceptor; when oxidised is colourless, but when reduced it forms red, insoluble compounds called formazans. The compound …

NH2 – N=CH – N=NH

…Is called a formazans.

Enzymes of the dehydrogenase class can be revealed I their location because they convert tetrazolium salts in solution to coloured, insoluble formazans. TTC can be used to show the presence of active dehydrogenase enzymes by a change in colour. I will be aiming to find out the rate of the reaction and see the changes in temperature over time using this information.

Hypothesis

Enzymes are chemicals that increase in activity as the temperature increases. Once the enzymes reach a specific temperature, their active sites become denatured. I expect there to be a pattern and this pattern will be clearly shown by a graph. As the temperature increases initially, I expect the time to shorten, but then at a higher temperature I expect the time to plateau off and then decrease rapidly, as the active sites become denatured. I expect the optimum temperature to be around 60 °C. temperature should have a positive effect until a certain temperature, where it will decrease. There will be a stable rise in activity.

Aim

Once I reach the initial colour, I will place it in a beaker of ice to prevent any further colour development through the formation of formazans. This low temperature inhibits and completely stops the development of formazans. I will have seven data points to work with, ranging from 20 °C and 80 °C, and I will match these points to the colour in the test tube held in the beaker of ice. I want to see a distinct change in times due to the range of temperatures. I also expect to see a plateau on the ...

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Aim

Apparatus

Water baths (30 °C, 40 °C, 60 °C and 80 °C)
Kettle
Glass beakers x 2
Stopwatch
Thermometer
Test tube rack
Test tubes x 20
Graduated pipettes x 2
Syringe
Safety glasses
Lab coat
Protective tongs for water bath
Wool insulator for glass beaker

Materials needed

Triphenyl tetrazolium chloride (0.5%) – 20 cm3
Respiring baker’s yeast suspension (100g-dried baker’s yeast to 1 dm3 of water, mixed with 50g glucose). To be made up two hours before use in practical.
Water and ice.

Plan

I will wok systematically through the range of temperatures after doing my control test. The procedure will be the same for each temperature apart from those temperatures that cannot be performed with the use of the water baths. For these tests I will boil water in a kettle and pour it into a beaker. I will then balance it to the desired temperature with cold water. I will then insulate it with a wool cover to make sure that there is minimum heat loss from through the beaker. The temperature will be constantly observed, and if it drops, it will be immediately rectified by the addition of more water from the kettle. This procedure applies to the 50 °and the 70 °C tests. The 20 °C test will be performed at room temperature, so there will be no need for any water baths or beakers of water. I will then work through the following method.

Method

I will set up the apparatus and get the materials needed to conduct the experiment.
Using the graduated pipette I will measure out 10 cm3 of yeast suspension and transfer it to a test tube.
Using the syringe, I shall measure out 1 cm3 of TTC solution into a separate test tube.
I will stand these test tubes in the desired temperatures in the water bath/beaker of water, and wait until they reach the same temperature as the water surrounding it. This will be about 5 minutes.
I will transfer the TTC solution to the test tube of yeast suspension, all with the use of the tongs.
The stopwatch will now be started.
I will observe the discolouration until it reaches the same colour of the control test tube in the beaker of ice.
Once both test tubes match, I will remove them from the water bath/beaker of water and stop the stopwatch. I will then record the time taken for the discolouration in my results table.
I will clean the current apparatus up and repeat the temperature again.
I will proceed with the remaining temperatures with their repeats.
After all the experiments have been finished with, the apparatus will be put away in its relevant container for cleaning.

Fair test

I will be ensuring that my experiment is as fair as possible by limiting the variables to just one, temperature. The volume of TTC and respiring yeast solution will be a constant. I will be repeating each temperature to ensure that the original was a valid result, and subsequently I shall be making an average of the result. This will narrow down the margin of error and increase the efficiency of my results.

To make sure that I time up to the same colour change in each test I will make up on control test tube and keep it by me throughout the experiment as a point of reference. I will compare the control test tube’s colouration with the current test tube to make sure that my timing is accurate and I am timing the same colour change for each temperature.

To stop any more discolouration of the control, I will put it in a beaker of ice. This will end any reactions taking place in the test tube and will keep the colour of the solution as a record. I will set a certain colour for the TTC to reach, and time the reaction until the colour has been reached.

When making measurements of the solutions, I will always measure from the bottom of the meniscus to ensure that the amount that I am transferring to the test tube is always the same.

To ensure that the reaction takes place fairly in all reactions the yeast suspension and TTC solution are placed in the water bath/beaker for five minutes. This is because we need the contents of the test tubes to be at the same temperature as the water bath/beaker to say it is an experiment at that temperature. Also, when mixing the two after the five minutes they need to be the same temperature for the reaction to take place as efficiently as possible.

Safety Precautions

The yeast solution must always be covered up and the bottle of TTC must have the top on it, when not in use. This will limit spillage, and also prevent inhaling anything.
Lab coats and safety glasses must be worn at all times.
When using the water baths, tongs must be used to hold the test tubes.
All used solutions should be retained in a large beaker and not poured down the industrial drain, as the baker’s yeast is a living organism.
When making up the yeast solution, inhaling the yeast powder should be avoided if possible.
All used sharp apparatus should be put in a specified container.
All spillages will be cleaned up immediately.
Ensure that the TTC solution is not consumed or put on skin for prolonged time spans. Avoid spilling the liquid.

Analysing Evidence and Results

Discussion

You can see from my results that my prediction of what was going to happen seemed correct. This was based on biological knowledge of enzymes. I found that once the temperature rose to 80°C the discolouration of the solution came to a stop. This was because the active sites had been distorted and the high temperature of 80°C denatured the dehydrogenase. Enzymes cannot exist at these high temperatures, and the yeast simply died under these conditions. The negative results were when there was no distinct colour change or a colour match with the control test in the beaker of ice. These tests were deemed negative, as they would alter the accuracy of my results if they stood.

There seems to be no anomalies or data that do not fit into the pattern that I predicted.

From my results the optimum temperature is 70°C.

As you can see from the results obtained, in the initial stages, as the temperature increases by 10°C the time for discolouration almost decreased by half (the Q10 value). This is not unusual for this kind of reaction in baker’s yeast. This continued until the time for TTC to form formazans and discolouration began to level off to a plateau and then decreased to a negative result at 80°C. You can see this pattern from the line graph.

It seems the formation of formazans is increased with higher temperatures, leading to more rapid discolouration of the solution within the test tube. At extremely high temperatures, they are unable to form, as the enzymes cannot make their products.

Conclusion

In conclusion, the dehydrogenase activity is shown through the discolouration of TTC solution by development of formazans. At higher temperatures the enzyme activity becomes more efficient and faster. The time decreases approximately by half as the temperature goes up by 10°C. My results prove this theory. After the temperature becomes too high the yeast dies and the enzyme activity inside it becomes zero, as the active sites of the enzyme have been denatured. This leads to no discolouration of the TTC solution. The optimum temperature in my results was between 65°C and 70°C. This is the temperature at which TTC solution will discolour most rapidly.

Evaluation of evidence and procedures

There was quite a range of data points in my investigation, ranging from 20°C to 80°C, a total of 8 points of reference. This was included to ensure that my results were as accurate as possible. This range of temperature I feel was fairly wide, and enough to show a definite pattern of results to prove a biologically based prediction. There was not much difference between what I expected the optimum temperature to be, showing that my results were accurate.

The procedure in which the results were obtained seemed to be quite efficient, which is evident from the precision of my results. There were a few minor details that I feel could have been rectified if possible, but they would not make a big impact on the eventual results. This was the transferring of the TTC solution to the test suspension. I felt that sometimes not the full 1 cm3 was going into the solution of yeast. This could change the time. Maybe for future experiments, the bottle or container of TTC could be put into the water bath/beaker of water to reach its temperature with the test tube of yeast. Then the measurement of 1 cm3 could be taken from the bottle or container directly. This would ensure that the full 1 cm3 went into the mixture.

A wider range of data points would increase the accuracy of the results and lead to a much firmer conclusion. Also, if there could be water baths at all the required temperatures hen it would be much fairer.

Maybe for a experiment, tests could be done on various types of yeast and what their optimum temperature for discolouration of TTC solution, and how the enzyme activity compares to that of other types of yeast with altering temperature. You could then compare all the data together to come to another conclusion about dehydrogenase activity. I am sure that there will be some differences in time between the different types of yeast. This could definitely be explored further with other yeast; for example, you could to it with brewer’s yeast.

Teacher Reviews

Here's what a teacher thought of this essay

Adam Roberts

5th of Sep 2013

**** A clearly written account of the investigation. The author has a good knowledge and understanding of the background theory. The discussion of trends and results could have been more thorough, however.

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