There are many factors which affect the function and efficiency of an enzyme, it is important to control these in the experiment so as to obtain accurate results and vary others to investigate its effect. The factors are as following;
pH - Certain enzymes work best in a specific Ph e.g. pepsin found in the stomach works best at a pH of 1-2 while trypsin found in the small intestine is highly active at a pH of around 8. An incorrect pH can render an enzyme useless as the bonds present in its structure are destroyed by the acidity or alkalinity of its surroundings. A perfect pH for a certain enzyme will mean that the rate of the reaction taking place will increase while a pH far deviated from the ideal will cause a slower less efficient rate of reaction. pH is not the desired variable in this experiment and will have to be controlled using a pH7 buffer which will control the change of pH during the experiment.
Temperature - Temperature also affects the efficiency of an enzyme. When an enzyme is present in conditions with an optimum temperature, the reaction in which it is involved is sped up. The molecules in the reaction move faster and with more kinetic energy resulting in a greater chance of more frequent and successful collisions. However, if the temperature is too high, the hydrogen bonds in the enzyme are disrupted and broken changing the shape of the enzyme and the active site. This change in shape means that the lock and key system is no longer possible and the enzyme is useless and ‘denatured.’ Low temperatures decrease enzyme activity as the molecules vibrate to a lesser extent and with less energy, the chance of successful collisions is decreased and the reaction is slowed. Temperature is not the desired variable for this experiment and so will have to be controlled by making sure that all of the reactions occur at room temperature, 21°C.
Concentration – Substrate concentration – A high substrate concentration would obviously mean that collisions are more frequent and therefore the reaction would occur at a greater rate. A low substrate concentration would mean the opposite with lower chance of collisions between enzyme and substrate and therefore a slower reaction. At the point when enzymes reach their maximum rate, there would be no effect in the addition of extra substrate. This is enzyme saturation (V-max) and the reaction will continue to operate at saturation level. Substrate concentration is the desired variable for this experiment so different concentrations of substrate will be used in separate tests to investigate their effect.
- Enzyme Concentration – A high enzyme concentration would be expected to give a greater rate of reaction. The chance of a successful collision between an enzyme and substrate would be greater and so the reaction would proceed at a greater rate while a lower concentration of enzymes would obviously give a lower rate of reaction due to the smaller likelihood of collisions between enzyme and substrate. However, this is not the desired variable and so will have to be controlled. I will have to make sure that I take my enzyme sample from the same radish each time and that the filter paper discs used are completely saturated with catalase before use in the tests so as to make sure the same concentration of enzyme is used each time.
Enzyme Inhibitors - Competitive inhibitors - are named so because they compete with the relevant substrate for the active site and slow the reaction. In a high enough concentration, competitive inhibitors can cause the reaction to stop completely.
- Non-competitive inhibitors - these latch onto the enzyme outside the active site and change the enzyme shape so that it is unable to lower activation energy and thus slows the rate of the reaction.
To control enzyme inhibitors, I will have to make sure that there is none present in the sample of catalase or substrate used in the test.
Substrate concentration will be used as the variable in this experiment. The concentration will be varied by changing the proportions of buffer and hydrogen peroxide in the solution. Different proportions will give different concentrations of Hydrogen Peroxide and therefore will give different rates which can then be investigated. To ensure accuracy, it would be best to measure the solutions with burettes as they are much more precise than syringes
Apparatus
Analysis
Table of results for final experiment
N.B. as you can see, the top two rows of volume proportions are not corresponding to those below them. The concentration of the Hydrogen peroxide was tripled for these particular tests so that the reaction could be observed with a much higher concentration thus expanding the scope of results obtained.
The graph of the results following the analysis shows that as the concentration of hydrogen peroxide increases, so does the rate of reaction thus proving my initial prediction correct. However, when the substrate concentration begins to increase further, it has a lesser effect on the rate itself. This is enzyme saturation (V-max) where the enzymes cannot work any faster and rate of reaction peaks. This is why we see the line in the graph levelling off.
According to the collision theory, as substrate concentration increases, the number of particles present in the solution obviously increases. The likelihood of frequent and successful collisions between enzyme and substrate particles is greater, rate increases and so the reaction is carried out in a smaller time. A lower substrate concentration means less particles present and so the frequency and likelihood of a successful collision is decreased and thus the rate also. If the concentration and therefore rate increase, more oxygen is produced and in a quicker amount of time so the paper disc will float to the top in a shorter period of time. The figure below is a representation of different substrate concentrations.
The disc floated because the catalase which was embedded in the fibres of the filter paper produced oxygen during the reaction. The oxygen which was also trapped between the fibres in the paper was less dense than water and so caused the paper disc to float. Obviously the faster the reaction was, the faster the oxygen was produced and the faster the disc floated to the top of the liquid.
The effect of concentration on an enzyme controlled reaction
Substrate concentration – A high substrate concentration would obviously mean that collisions are more frequent and therefore the reaction would occur at a greater rate. A low substrate concentration would mean the opposite with lower chance of collisions between enzyme and substrate and therefore a slower reaction. At the point when enzymes reach their maximum rate, there would be no effect in the addition of extra substrate. This is enzyme saturation (V-max) and the reaction will continue to operate at saturation level. Substrate concentration is the desired variable for this experiment so different concentrations of substrate will be used in separate tests to investigate their effect.
Enzyme Concentration – A high enzyme concentration would be expected to give a greater rate of reaction. The chance of a successful collision between an enzyme and substrate would be greater and so the reaction would proceed at a greater rate while a lower concentration of enzymes would obviously give a lower rate of reaction due to the smaller likelihood of collisions between enzyme and substrate. However, this is not the desired variable and so will have to be controlled. I will have to make sure that I take my enzyme sample from the same radish each time and that the filter paper discs used are completely saturated with catalase before use in the tests so as to make sure the same concentration of enzyme is used each time.
Higher substrate concentrations gave a quicker time as the reaction occurred much quicker. If either enzyme or substrate concentration is high, the chance of an enzyme colliding with the substrate is higher; it is these collisions which are part of the reaction itself and the more these collisions occur and the more frequently they occur the quicker the whole reaction is.
To clarify, with a higher substrate concentration, more oxygen is produced and quicker and so the disc floats to the top faster. High substrate concentration also means there is more for the enzyme present to ‘work on’ and so the rate is initially higher.
Analysis Continued.
There was an anomalous result which can be seen in the graph and there are a number of reasons why it could have occurred. The filter paper disc may not have been completely saturated with catalase from the sliced radish thus changing the time taken for the reaction. It is most likely that the solution used for that particular test was improperly prepared so that all three times were incorrect and not corresponding to the other results. So as to avoid affecting the results of the whole experiment, the anomaly was left out of the graph. I used error bars on some points in the graph so that it was possible to see how far off the actual results were from the line of best fit. All the error bars were quite small showing that the experiment was indeed performed with a high degree of accuracy.
The buffer stopped the pH from fluctuating and affecting the experiment. An incorrect pH can render an enzyme useless as the bonds present in its structure are destroyed by the acidity or alkalinity of its surroundings. A perfect pH for a certain enzyme will mean that the rate of the reaction taking place will increase while a pH far deviated from the ideal will cause a slower less efficient rate of reaction. It was necessary to use buffer as pH was not the desired variable.
All the tests were performed at room temperature. Temperature affects the efficiency of an enzyme. When an enzyme is present in conditions with an optimum temperature, the reaction in which it is involved is sped up. The molecules in the reaction move faster and with more kinetic energy resulting in a greater chance of more frequent and successful collisions. Low temperatures decrease enzyme activity as the molecules vibrate to a lesser extent and with less energy, the chance of successful collisions is decreased and the reaction is slowed. Temperature was regulated to the best of my ability with room temperature (21 degrees Celsius) being the standard temperature for each individual test.
Spearman Rank Correlation
Formula for the spearman rank correlation.
Rs = 1 - (6∑D2)/ n(n2 – 1)
Where n is the number of different concentrations used during this experiment, in this case n = 7.
The worked out formula gives my results an Rs value of 1. This means that the correlation of my results and graphs is perfect and the fact that Rs is a positive number shows that the correlation is a positive one. I predicted that rate would increase with the increase of substrate concentration and the formula shows that my prediction was correct. All results are corresponding and relevant and I am confident that the experiment was carried out correctly.
Evaluation
The method used was suitable to obtain the information that was required. There were a few problems with the experiment itself however the prediction made was corresponding to the results obtained. The main hindrance was the source of catalase. By soaking it up with a filter paper disc, there was no definite way of telling when the disc was saturated with the enzyme therefore affecting the amount of enzyme used for each individual test. The slight variation in the amount of enzyme used for each test would obviously affect the time taken for the disc to return to the surface. I would suggest a beaker full of catalase solution for future experiments of this sort so as to be sure that the filter paper discs are saturated with enzyme. The PONY vials in which the tests were carried out were also too small to be able to accurately tell how quickly the disc returned to the surface. I believe taller vials would be easier to use as longer times would be easier to accurately monitor. Of course more hydrogen peroxide and buffer would have to be used so as to increase solution height but the improvement in results would give a much more accurate experiment overall. I would also suggest the use of burettes for the measuring of solutions so as to improve accuracy. Any inaccuracies in the measuring of buffer or hydrogen peroxide will affect the pH or rate of the reaction thus giving inaccurate results. The most important error I felt was the actual motion of the filter paper disc which varied uncontrollably. Sometimes the disc would float to the surface in a vertical position and at other times it would be diagonal or horizontal. The resistance of the motion due to its position meant that times would vary thus hindering or promoting the rate. Usually the rate was promoted when the disc would float to the top in a vertical position giving least resistance and thus increasing the rate of reaction. This would account for the one anomalous result noted in the experiment.
One set of anomalous results was obtained and I believe this was due to inaccurate measuring of solutions for one particular concentration of hydrogen peroxide. Their presence was not crucial to the experiment and the particular results were therefore left out of the graph and trend line. Other than for the set of anomalous results the rest of the measuring would appear to have been done accurately using marked syringes, one for each solution so as not to mix them and affect results.
The results obtained were reliable, even the error bars which were small were very close to the trend line. Larger error bars would suggest a less accurate and reliable experiment with very variable results where as smaller ones would suggest a much more accurate experiment. I don’t believe my trend line would be greatly affected if my error bars were smaller. A changed trend line would probably change the possible conclusions drawn but this does not apply to my experiment.
In conclusion I can say that my initial prediction was right and that the rate of reaction increases with a greater substrate concentration. It is clear in the results table and graph that this is true. It was also proved right that enzyme saturation (V-Max) would occur when a significantly large amount of substrate was used in the test and this is shown in the graph where the line levels out towards the higher concentration of Hydrogen Peroxide. I am confident that my results are correct because the Rs value worked out in the analysis was 1 which signifies a perfect correlation of results. The fact that it is a positive number also shows that there is a positive correlation as was predicted.
