Investigate the effect of enzyme concentration on the rate of reaction.

Biology Coursework- ENZYMES

AIM: To investigate the effect of enzyme concentration on the rate of reaction.

PREDICTION:

An enzyme is a biological catalyst. They speed up the rate of a reaction however they do not affected themselves whilst doing this, which is why they are catalysts. Enzymes are made to be specific, this means that they can have only one substrate that they will work on. Each enzyme has an active site that is where their own specific substrate’s molecule will fit into.

However, having equal amounts of enzyme and substrate concentration still I think would not react as fast as it would if there was a higher concentration of enzymes, as each and every substrate molecule must react with an active site. Being equal amounts of each, until the substrate does not react with enzyme the reaction would not be complete and there would be a lower chance of a successful collision with an active site that is available.

EQUIPMENT:

METHOD:

Set up the equipment/apparatus required.
Set the water bath on a temperature of about 35oC – 45oC
Measure out 1ml of Amylase concentration (use a measuring cylinder or pipette)
Pour the Amylase into a boiling tube
Measure out 5ml of Starch (use a measuring cylinder or pipette)
Add one or two drops of iodine indicator into the boiling tube containing Starch
Place the boiling tube containing Starch and Iodine indicator into the beaker containing water at a temperature between 35oC – 45oC
Add the 1ml of Amylase concentration to the boiling tube containing Starch and Iodine and start the stopwatch straight after.
When the mixture of Starch and Amylase containing Iodine indicator turns colourless, stop the stopwatch and record your results (time taken for reaction to complete).
Repeat the experiment using different concentrations in the same method as above and record these results on the table of results. The concentrations I am hoping to use are 0.2%, 0.4%, 0.5%, 0.6%, 0.8% and 1%.

SAFETY: To ensure safety during the experiment, safety precautions must be met which include:

Tying long hair backwards
Wearing safety goggles or glasses to protect chemicals such as iodine in this case from going in the eyes.
Iodine is an irritant.
Standing up and making sure nothing is in the way such as bags and coats/jackets.
Make sure the equipment require is only on the work surface to prevent accidents.
Keeping breakable equipment such as boiling tubes away from the edge of the table or work surface.

FAIR TEST / VARIABLES:

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SAFETY: To ensure safety during the experiment, safety precautions must be met which include:

Tying long hair backwards
Wearing safety goggles or glasses to protect chemicals such as iodine in this case from going in the eyes.
Iodine is an irritant.
Standing up and making sure nothing is in the way such as bags and coats/jackets.
Make sure the equipment require is only on the work surface to prevent accidents.
Keeping breakable equipment such as boiling tubes away from the edge of the table or work surface.

FAIR TEST / VARIABLES:

PRELIMINARY WORK:

Before doing the actual experiment a preliminary test was done, so that a prediction could be made and notes can be made on what problems may occur.

METHOD FOR PRELIMINARY TEST:

Set up all the equipment/apparatus as required in the experiment.
Using the pipette place one drop of iodine indicator into each well of the spotting tile.
Measure out 5cm3 of Starch using a pipette or measuring cylinder.
Measure out 1cm3 of Amylase using a pipette/measuring cylinder and put it into the test tube.
Add the 5cm3 of Starch to the test tube containing Amylase and start the stopwatch straight after.
Using the pipette, take out a little solution from the test tube and place in into one of the wells on the spotting tile containing iodine indicator every 30 seconds staring from point blank.
When there is no change in colour what so ever stop the stopwatch.
Record the results and the findings
Repeat this experiment using warm water and follow the method.

RESULTS:

This preliminary test (spotting tile method) is not very reliable because the solution could have become colourless within the 30 seconds, this is a good reason why a better way to conduct this experiment is by; using a test tube containing 5ml of Starch and one or two drops of iodine. Then add the 1ml of Amylase and starting the stopwatch straight after, timing how long it takes for the solution to go clear. In this way you can be more precise and tell more accurately the total time the experiment took for the reaction to complete.

While conducting the preliminary test I found that substances would be left on the inner wall of the test/boiling tube although they were rinsed, which would contaminate the substances in the experiments to follow (when changing the concentrations). Therefore I have decided to use clean test/boiling tubes to prevent contamination between solution and give me better results. Instead of using pipettes, syringes would give more accurate results as precise measurements can be taken this way making the test or experiment fair. A thermometer will also help to keep the temperature of the water constant and would tell you when the water needs to be changed. For the preliminary test I used a kettle to heat the water, but I found it difficult to keep it at 370C (temperature for best reaction rate), which lead into replacing or changing the water after a period of time and waiting for it to reach the right temperature before carrying out the experiment further. Therefore, I will use a water bath in the next experiment in order to keep it at a constant/optimum temperature providing me with more accurate and better results.

DILUTION TABLE:

OBTAINING EVIDENCE

RESULTS TABLE TO SHOW THE EFFECT OF THE CONCENTRATION OF AMYLASE ON THE RATE TO REACTION

(*) Results for time taken on 2nd attempt are taken from TOBIAS

I think that my method will be successful enough to provide me with quality/ reliable results. This because my method is through and can be followed step by step with ease. I have also used the correct equipment required for this experiment and have taken all the necessary precautions in order to make readings with good accuracy using precise techniques. Although I didn’t plot a graph as I was taking the readings, I recorded the results in a table precisely taking readings over a large enough range from 10% to 100% enzyme concentration.

ANALYSIS

In my plan I predicted that as the enzyme concentration increases, the rate of reaction increase but the time taken for the amylase to break down decreases. The graphs back up my theory. I can see the reaction is slowest when the enzyme concentration is the least, because there are not enough active sites for the substrate to fit into, therefore more enzymes need to be added to speed up the reaction. Also the reaction is faster when the enzyme concentration is greater, because there are enough active sites and substrate to have a collision with each other successfully, enabling fully saturated enzyme substrate complexes to be created. As I had predicted at 20% concentration of amylase the rate of reaction was is 0.001055966, but at 100% concentration of amylase the rate of reaction is 0.008810572. So this itself shows/proves that the higher the concentration of enzymes the faster the rate of reaction.

EVALUATION

Although I conducted my experiment/ practical and my results were quite accurate there are still quite a few sources of error. During the experiment I kept in mind the mistakes I made and which I would also like to impove on, if I was to conduct this experiment again. Even though I may have made a few mistakes during this experiment I think it was successful as I still proved that my prediction was correct, however I do not think that my results are very accurate due to the following in which I have also stated how they can be improved:

We noticed that you could not tell exactly when the experiment was complete or could not be precise when the solution went clear. Therefore using a colorimeter which picks up the change in colour you could be more precise and have accurate results.

Different amounts of iodine were added to the solution which could affect the reliability of my results, so measuring or have a set amount of iodine each time would give you more accurate results.

Using the kettle you could not exactly be precise on the temperature of the water and have to repeatedly change it when it cools down. Temperature will affect the reliability of your results, bearing in mind that high temperatures would also denature the enzyme. Therefore, a water bath should be used as it can be kept at a set temperature throughout the experiment again giving you accurate results.

After the experiment test tubes could be contaminated, even if rinsed out therefore effect they next experiments results. To prevent this fresh, clean glass ware should be provided or time given to wash/ rinse the test tubes toughly.

Looking back at point 4, I have found an anonymous result shown on the results table in red, which I think is because of contamination from the previous experiment. Contamination can easily speed up the process, slow it down, have an odd result or have nothing at all happen, but in this case it has been slowed down and not following the trend.

As an improvement changes I could make to my experimental method to carry out the same experiment are shown below:

Water bath instead of a kettle
Fresh, clean test tubes or extra time to have them cleaned
Syringes used instead off pipettes or measuring cylinder as they are more accurate.

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