Factors effecting enzyme activity

good source as many chemical reactions happen in liver cells. The liver is

liquidised as it releases more enzymes. I have merely chosen temperature because this is what I was most interested in. I predict that as the temperature increases, the rate of reaction will also increase rapidly until at the optimum temperature of 37oC all the enzymes will be denatured and the rate of reaction will decrease as rapidly as it increased. This denaturing of the enzymes is due to the collision theory, the more energy the particles have, the more they move and there is more chance that they will collide, resulting in a faster the rate of reaction. However, as the temperature increases to its optimum, the substrate can no longer fit into the enzyme’s active site, and the reaction is therefore not catalysed. The graph below shows the effect of temperature on the rate of reaction

 

Preliminary Work:

I have carried out a few short experiments to aid me with the planning of my main method. These experiments were undertaken to see what volumes, concentrations, apparatus etc. that I could use during my coursework. I carried out my experiments to see which catalase gave off the most oxygen, the one which gave off the most oxygen would be the one which I use in my real experiments because this would then give me the best results. By doing my preliminary experiments, I worked out how often I should take my readings because I looked at how quickly oxygen was produced and whether I would be able to measure it accurately, I decided that I could measure the amount of oxygen produced every 10 seconds for a minute, because after one minute most of the oxygen had been produced and it would be pointless to carry on after that. I carried out my first set of experiments and produced this table of results:

Firstly, I made sure that the amount of potato, liver extract and apple was the same, by using 3cm of each one. This table tells me that the catalase liver produced the most oxygen after 2 minutes of reacting. Apple produced the least which means that the catalase in apple is not very concentrate and so it is pointless using this for my real experiment. I carried out another set of experiments to make sure that my results were accurate and this is what I got:

        

This table of results also tells me that the catalase liver produced the most oxygen so I am going to use liver during my real experiment. I could also use potatoes, however, this takes a very long time to crush up using the pestle and mortar so I am not going to use potatoes as it would just be a waste of time. Another factor that I am going to investigate throughout my preliminary work is what ratio of liver extract to hydrogen peroxide works best. I am looking for one that can last 1 minute. This is because I need to take a minute worth of readings to measure the rate of reaction. So from my results whichever ratio of hydrogen peroxide to liver extract lasts the longest, will be the one that I use. Here were my results:

From these results I found that the ratio 0.5:4 worked best and I will use this ratio for my actual experiment. Also, to make sure that this last results was not just anomalous I did the experiment again and got 122 seconds, so I know for definite that this will be the best ratio to use. I did not do the ratio 0.5:1 because this would just be a half and I have already done the ratio 1:2 so there would be little point in doing it again.

Fair Testing:

There are certain factors that could affect my experiments in different ways if I don’t keep them the same.

• I need to make sure that I keep the amount of liver and hydrogen peroxide the same throughout my experiments.
• Also, as well as keeping the amount the same, I need to keep the concentration of the hydrogen peroxide and liver extract the same all the way through.  I already know that concentration is one of the key variables, but in this case I want the key variable to be temperature.
• Another factor that could affect my experiment would be the level of pH, so to ensure that the pH stays the same at all times, I need to make sure that I use water from the same source throughout my experiment. To make sure that the pH is constant I will use some litmus paper, I am expecting the colour of the litmus paper to be green throughout my experiment.
• Apparatus can also affect the experiment in different ways,
• I need to make sure that when the apparatus is all set up, that it is airtight. Otherwise, oxygen from the atmosphere could also get collected in the upturned measuring cylinder, this would not be a fair test as the amount of oxygen would be inaccurate. To keep my apparatus airtight, I will insert the hydrogen peroxide through a syringe that is connected to a bung, with no air gaps. Also I will fill the upturned cylinder up with water completely to the top, so there are no air bubbles in it.

Method:

Firstly, you need to collect all the apparatus on the list below,

Apparatus:

• Trough
• Measuring cylinder
• Test tube
• Rubber bung with hole in the top
• Syringe
• Hydrogen peroxide
• Liver extract
• Rubber tubing
• Clamp
• Clamp stand
• Safety goggles
• Test tube rack
• Stop clock
• Blank table of results
• Thermometer
• Hot water basin

Then set the apparatus up like the diagram below:

I need to make sure that the temperature is right after being in the warm water basin, by measuring it with a thermometer. I am going to investigate how much oxygen is produced from 20oC to 70oC and I am going to increase the temperature by 10oC at a time. To  increase the temperature, place the test tube with the liver extract in it into a water bath which is at the correct temperature, whether it is 20, 30, 40, 50, 60 or 70oC. The test tube must be left in the water bath for approximately 5 minutes. This way, the liver extract will have acclimatised to the temperature the surrounding water.  I am going to take 3 readings each time to make sure that the test is accurate.   There should be 0.5cm3 of hydrogen peroxide and 4cm3 of liver concentrate in the test tube, pour the liver concentrate into the test tube first and put the bung on top. The upside down measuring cylinder should be full of water, so hold it under the tap so that it fills up with water, put your finger over the top and carefully place it upside down in the water so that no gas from the atmosphere will be able to get through. This way it will be an accurate way to measure how much gas has got into the measuring cylinder. Place the end of the syringe (with the 0.5cm3 of hydrogen peroxide) into the top of the rubber bung with the hole in it, carefully push the hydrogen peroxide out and start the stop clock. After every 10 seconds record the amount of oxygen that has been produced. Record your results in a table like the one below and repeat three times for accurate results. Below is a blank results table to show how I am going to record my results:

Obtaining Evidence:

During the biology coursework day, I carried out a series of experiments to investigate how temperature affects the rate of catalase activity.  Using my method I got the results below:

Firstly, I am going to measure the rate of reaction at 20oC. However, the closest that I could get to 20oC was 21oC as the temperature fluctuated quite a bit. Here is the table of results that I obtained from doing that experiment:

I then increased the temperature from 21oC to 29oC, to see whether increasing the temperature affected the rate of reaction, recorded my results in this table:

I then increased the temperature again to 38oC to see how increasing the temperature affected the rate of reaction, here is a table to show my results:

I then increased the temperature again to 50oC to see how increasing the temperature affected the rate of reaction, here is a table to show my results:

I then increased the temperature again to 59oC to see how increasing the temperature affected the rate of reaction, here is a table to show my results:

I then increased the temperature again to 70oC to see how increasing the temperature affected the rate of reaction, here is a table to show my results:

However, at 70oC all the enzymes had denatured and so I did not manage to obtain any results for this temperature.

Analysis:

        I managed with a series of experiments to obtain many results which I can now put into graphs and scientifically explain them. I now have six tables of results, therefore, I need to plot them onto a graph in excel and see whether there is any pattern or clear relationship between the increasing temperature and the amount of oxygen produced. From my results at 20°C, when I plotted time/secs against the average amount of oxygen produced/cm³, I found a very clear pattern. At the beginning the gradient of the slope was very high, this means that there was a very rapid amount of oxygen produced. As the line began to ease off and the gradient became less this meant that less oxygen was being produced and the rate of reaction began to slow down. Then, right at the end, the gradient became 0, meaning that no more oxygen was being produced. This is because all the oxygen had been produced and there was no more to be reacted. If the experiment was left then the gradient would still stay at 0 because all the oxygen had already been produced. Here is a graph to show my results at 21oC:

         I then increased the temperature to 30°C and saw how this affected the rate of reaction. These results behaved in the same way, in the sense that at first the rate of reaction Is very high and then begins to ease off until the gradient becomes 0. However, the average amount of oxygen produced was slightly higher, this shows that as the temperature increases the rate of reaction also increases. This means that there have been many more successful collisions and due to the activation energy being high at the moment of impact, they can break the bonds and produce new bonds, resulting in new products of the reaction, in this case oxygen. Below is a graph to show my results at 29°C:

        I then increased the temperature again, this time to 40°C, to see how this affected the temperature. I found that overall the average amount of oxygen produced was slightly less, this was because some of the enzymes had been denatured, the theory is that the optimum temperature for enzymes to work in is 37°C. When enzymes are denatured meaning that they are destroyed and cannot work any longer. Temperatures above 37°C/damage (denature) the intricate structure of enzymes, inactivating them and causing reactions to stop. This would be the reason why these results were slightly less. The graph, however, again showed the same pattern that at first the rate of reaction was very high and then began to ease off until no more oxygen could be produced and it was a flat line. Here is a graph to show my resultsat 38oC:

        I then increased the temperature again this time to 50°C and I found that most of the results were the same, although I expected the rate of reaction to have been less, due to enzymes being denatured. This graph also behaved in the same way, that the rate of reaction was very fast at first and began to ease off towards the end until the gradient was 0. Although at the beginning there was a much larger burst of oxygen produced at 50°C than in some of my other graphs, this may suggest an irregularity or anomaly in my results, either the results for 50oC are anomalously high or the results for 40oC were anomalously low, this needs to be looked at further . Here is a graph to show my results at 50oC:

        I then increased the temperature again to 60°C, to see how this affected the rate of reaction and to see whether the enzymes were denatured or not. This graph also behaved in the same way, that the rate of reaction was  faster at first and began to ease off towards the end until the gradient was 0. Although, the amount of oxygen produced was much less over the course of one minute. Normally the amount of oxygen produced is around 35cm3 on average, but in this case the average amount of oxygen produced is about 2.9cm3, which is much less than usual, so this shows very clearly that the enzymes are now definitely denaturing. Here is a graph to show my results at 59oC,

I then increased the temperature again to the highest temperature I am going to investigate, which is 70°C, however, the temperature was so high and I did not manage to obtain any results. This means that all over the enzymes had denatured.

To summarise, the graphs at 20, 30, 40, 50oC show a similar pattern with a fast rate of reaction at the beginning, then the rate tails off until it approaches a gradient of zero at around 60secs.  The same pattern is seen at 60oC but much less oxygen is produced, showing that the enzyme is denaturing and not working as efficiently. At 70oC no oxygen was produced so the enzyme was not working, it had been completely denatured by the high temperature.

I then decided to find which temperature generates the fastest rate of reaction to do this I took the average amount of oxygen produced at 30 seconds for each different temperature ranging from 20oC to 70oC, and divided it by 30, to get the amount of oxygen produced per second. The reason that I choose 30 was because it was the mid way point between 10 seconds and 60 seconds. If I choose to use 10 seconds then this would have been biased, as it would be too soon and the rate of reaction would have been very high, also if I choose to use the average amount of oxygen at 60 seconds, by this time the rate of reaction would have completely slowed down and this would also have been biased. The graph came out quite well and as was predicted, as it turned out to be bell-shaped. It showed that the optimum was somewhere between 30 and 40oC, and you could tell that the enzymes began to denature between 50 and 60oC. However, the enzymes did not begin denature until just after 50oC, which is not as predicted, so 50oC will be regarded as anomalous as the enzymes theoretically should have denatured by then. Here is a graph to show my final results:

As you can see from this graph, at 50oC the amount of oxygen produced/cm3 looks a little bit out of place, so I will be investigating further into why this could be in my evaluation. As the optimum temperature was somewhere between 30 and 40oC I have investigated further to find the initial optimum temperature between these two points, I have recorded my results in this table:

        

From this table, it is not very clear as to which temperature gives the highest amount of oxygen produced, so I have decided to make it clearer and put these results into a graph:

To conclude, from this graph it shows me that the optimum temperature for the catalase enzyme to work in is 37oC. I made sure that this was accurate as I did it twice and it worked out both times, so I know that this was not anomalous. It also agrees with my initial prediction that the optimum for enzyme activity is 37oC above which enzymes start to denature.

Evaluation:

During my evaluation I am going to be describing and evaluating ways in which I could improve my coursework if it was to be done again. I will also be commenting on any mistakes and any anomalous readings I have noticed.

Overall, I believe that most of my results went as predicted, and agreed with my hypothesis, which then helped me to verify my prediction. I believe that my results were accurate enough as shown by my graphs. You can tell this from my lines of best fit, as they are as I had predicted. However, one of the graphs which did not seem to agree with my prediction was at 50oC, here the results seemed anomalously high, alternatively the readings for 38oC may have been anomalously low. I think that the further analysis of optimum temperature between 35o C and 40o C resolves this issue. In this more detailed experiment,  at the temperature of 38o C , 38.8cm3 of oxygen was produced at 60secs, In my initial recordings at 38o C only 33.1cm3 of oxygen was produced at 60secs, so it seems more likely that this initial set of readings at 38oC  were anomalously low and should have been higher.

There are several explanations for this, maybe the liver extract did not acclimatise to the right temperature, if it only reached a lower temperature this would have given the lower readings. Also that particular suspension of liver extract may have had a lower concentration of catalase within it, meaning that less oxygen would have been produced than anticipated.  Also in all the experiments I only measured the temperature in the surrounding water bath and not the temperature in the catalase, so I would not have known the exact temperature of the catalase.

        

        If I were to do the experiment again, then there would be several ways in which I could make the experiment more accurate. To make sure that the temperature was exactly right, I would make sure that I had a thermometer that was very precise and I would measure the temperature of the catalase instead of the temperature of the surrounding water. I would have left the catalase to acclimatise in the surrounding water bath for much longer, probably for a duration of 15 minutes instead of just 5 minutes, which was most probably not long enough.  Also given more time I would have repeated any set of readings that after evaluation appeared to be anomalous and a poor fit.

        Despite the anomalous readings at 38oC, I consider my results were good enough to show clearly how rate of reaction of catalase changes with temperature, the more detailed investigation between 35 and 40oC also showed clearly that the optimum temperature for enzymes to work in is 37oC.

        I now know how temperature affects the rate of catalase activity. That the rate of reaction is speeded up as the temperature increases until its optimum at 37oC and then it rapidly drops again as fast as it increased, resulting in a bell shaped graph. However, there are several other factors that affect the rate of reaction, such as varying the concentration of catalase. To do this I would set up the apparatus like in the diagram I drew earlier in my coursework. I would need to ensure that the temperature stayed constant at 30 degrees, to ensure a fair test. First of all, take the 2% concentration of catalase. Test the pH of the solution using universal indicator. Then write down these two variables into a table, to ensure that the two variables are the same throughout the experiments. Once you have ensured that these two variables are right then add 0.2cm³ of catalase into the side arm boiling tube with the 1cm³ syringe. Then seal the side arm boiling tube with a bung. Through the hole in the syringe-adapted bung inject 5cm³ of hydrogen peroxide (10vols) with the 5cm³, into the side arm boiling tube, and then start the stop clock as soon as the hydrogen peroxide has been dropped in. Record how much oxygen is collected in the gas tube cylinder every 10 seconds for the next 60 seconds into your table of results. Repeat this method 3 times for each concentration, 2%, 4%, 6%, 8% and 10%, of catalase and a control with 0% catalase, distilled water.