Hypothesis- As the concentration of the enzyme protease is increased so will the rate of protein breakdown. However after a bit the enzymes will stop working (denature) as all the active sights will be full and so increasing the concentration will have no effect on the reaction rate.
Apparatus & materials- The following will be needed in order to carry out the experiment
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Laboratory coat, disposable gloves and goggles to ensure safety. Goggles will ensure that nothing gets in to the eyes and that they are protected from the enzyme. Laboratory coat and gloves will make sure that clothing and the hands are protected.
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Five different test tubes for the different concentrations of solution. The testubes will be labelled one- six to ensure that there is no mix up between the tubes. A testube rack will be used to place the tubes to ensure that they are not handled and are on a stable surface.
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2.55mm syringe to measure the enzyme protease solution. A syringe is more appropriate to use than a beaker or measuring cylinder as only small measurements are needed therefore the syringe will make the measurements more accurate and the results valid.
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A 10cm-measuring cylinder to transfer 5cm of protein to the testubes and the different amounts of distilled water.
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A stirring rod to mix the solutions to ensure that the enzyme is spread all across the tube.
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A stopclock will be used to record the time for the reactions to take place. A stopclock will be much more accurate to use that just timing the reaction from a watch or normal clock as milli seconds can also be recorded.
Expected results- After finding out about the concentration and how this affects the rate of reaction the graph should look as follows:
The concentration
should be directly proportional
to the rate of reaction.
Fair testing- To make sure that the results are reliable the following precautions will be taken:
In the experiment the enzyme concentration is the independent variable. This is because the concentration is being changed to see the effect of different concentration of enzymes on the rate of reaction. Thus the dependant variable will be the rate of reaction. The control in the experiment will be the protein and it will be kept the same in all the tubes. This is because the rate at which the proteins react will be dependant on the enzymes. By controlling the amount of proteins present it will prove if the hypothesis that the greater the concentration of enzyme the faster the reaction is correct.
All the testubes need to be clean before the experiment is conducted as any substance left over in the testubes could affect the validity of the results. This is because it could cause the solution to appear less clear. The tubes should also be dry and not contain any water as this could cause the proportion of water to be used unequal. Therefore all equipment to be used and the workbench will need to be fully clean before the start of the experiment
To ensure random errors are not made a standardized procedure must be followed. Also care and concentration should be kept in mind when conducting the experiment to ensure a valid and reliable set of results. If the experiment is to be repeated the same procedure must be followed.
The testubes for all the different concentrations must be equal in size. This is because a bigger testube compared to a small one will have a bigger surface area for the enzymes to work. Using different sizes could affect the results because the solution may not be as much concentrated if using as small tube compared to a big one.
The temperature of the water baths must be kept at 20c to ensure that this variable is controlled and that the results are accurate as much as possible
Safety-
- The first and foremost thing before the start of the experiment is to wash hands with warm water and soap. This will ensure that no unwanted substances are transferred during the experiment.
- The workbench needs to be tidy and all stools tucked underneath the table so that there is no risk of tripping over or a spillage. If equipment is broken or if there is a spillage it should be cleaned up immediately as it could cause a hazard for someone else.
- Loose pieces of clothing and hair will need to be tied back so that they do not come in the way whilst conducting the experiment. A laboratory coat, gloves and goggles must be worn as all times during the experiment to protect clothing and the body. The enzymes could irritate the body so by wearing gloves it will ensure that they do not come into direct contact with skin
- Care must be taken that liquid does not come into contact with electrical equipment
- Food and drink must not be consumed in the science laboratory especially when conducting the experiment
Ethical issues-. Many people such as animal rights activist would be against this experiment as the enzyme Pepsin is derived form animals. The is enzyme produced in the mucosal lining of the stomach that acts to degrade protein.²
Method- Gather all the apparatus before the start of the experiment so that it does not affect the concentration and care taken during the experiment
Make sure that all the testubes are clean so that no substance is left behind which could affect the validity if the results. If a testube is rinsed make sure that it is thoroughly dry before using it. Place the tubes in a testube rack so that there is a stable surface to work with and that the tubes are not just left on the table. By placing the tubes in a rack it will ensure that they are not handled as this could have an affect on the validity of the results. The testubes should be marked one-six so that there is no mix up between them.
Use a 10cm-measuring cylinder to measure the different amounts of distilled water and add it to each tube. By using a measuring cylinder with small measurements will ensure that the amount will be more accurate. Next add the enzymes into the same testubes using a syringe to measure to the required amounts. A syringe will be much more suitable to use compared to a beaker or measuring cylinder as the measurements are quite small so it will ensure that the measurements are accurate. The following amounts of enzyme, distilled water and protein will be used.
Net use a measuring cylinder to add 5cm of Albumen to each tube. As soon as the protein is in the tubes start timing using a stopclock. A stopclock should be used as the figures obtained will be much more accurate than a normal clock as mille seconds can also be recorded. Before deciding if the solutions are clear give each tube a shake so that it is absolutely certain that it is not only one part that has gone clear but the entire tube. To make sure that the results are accurate and valid use the same procedure for the rest of the tubes. Repeat the experiment at least twice so that there is a reliable set of results.
Results- The results obtained are shown in the following table
Conclusion- The results show that as the concentration of the enzyme was increased the time taken for the reaction decreased. Thus it took less time for the solution to go clear. So it is write to conclude that the prediction that the rate of reaction would become faster is correct.
From drawing up the results as a lined graph a clear correlation between enzyme concentration and rate of reaction can be seen. There is a positive correlation. As the enzyme concentration increases the rate of reaction also increases linearly. This shows that as there are more enzymes present the reaction is faster as the enzyme are able to catalyse more of the proteins. With a lower concentration of enzymes there are not enough active sights for the reaction to take place quicker. The graph supports the fact that higher concentration causes more collisions between the molecules as, as the concentration increased the reaction time decreased. The initial prediction of how the graph would look is therefore correct.
The
After studying the graph it is clear that they support the hypothesis and although the results are not perfect they support a valid conclusion. The line of a best fit shows a clear trend. Although there is one anomalous result it is not very far away from the line of best fir therefore the results are still valid.
Evaluation- Although an acceptable set of results was acquired there are some limitations with the results. Improvements could be made to make sure that the experiment is conducted in a better way and that no mistakes are made with the procedure.
The results would have been more valid and accurate if the experiment had been repeated at least twice. Because there was not enough time the experiment was only conducted once. To make sure that there is a reliable and valid set of results next time the results from the rest of the class will be acquired. Also form this average can be worked out which will be much more precise. This will ensure that there is a much more reliable set of results.
Eyesight could have contributed to some inaccuracy. Because an automatic stop clock was not used it could be possible that the stopclock was stopped or started earlier/ later. This could have affected the results as the stopclock could have been stopped at a wrong time. This could be a systematic error. To avoid this next time an automatic stopclock should be used. Another problem of eyesight is the interpretation of when the solution goes clear. Because no one persons eyesight is the same the results cannot be deemed accurate and valid however they would still be reliable. However the results cannot be guaranteed absolutely reliable, as the interpretation of when the solution goes clear could be different for all the tubes. To overcome this problem a colorimeter could be used to measure the light intensity. A colorimeter would ensure that no random errors are met. Also by making sure that the colorimeter is calibrated before each use would ensure that systematic errors are not made.
Because only small concentrations were used in the experiment the time when the enzymes stopped working could not really be identified. Larger concentrations for example 10, 20, 30 could be used. This way the point at which the enzymes started to denature could have been identified.
Care was not taken to ensure that other variables such as PH and temperatures were controlled. This could have affected the results and although the anomalous result is not far off it could be that the anomalous result is because one of the two variables was not controlled
Further work- Only one type of enzyme was used therefore it cannot really be concluded if this is the general trend for all enzymes. Another investigation with different enzymes but using the same protein could be conducted to see if the general trend is the same. To ensure that the results would be accurate the same methods, apparatus and precautions would need to be taken. However the improvements and modifications mentioned would need to be taken into account
