Investigation to study how temperature affects the activity of an enzyme.

Temperature

An increase in temperature generally results in an increase in enzyme activity (i.e. Q10 theory: a 10 degree temperature increase doubles the rate of reaction). As the temperature rises, the movement of enzyme molecules and substrate molecules increases. This causes more collisions between enzyme and substrate and therefore more reactions. If the temperature rises above approximately 40-50 degrees Celsius, the enzyme activity eventually slows down because the enzyme is denatured by heat. The shape of the enzyme’s active site was changed when it was denatured and it will not combine with the substrate anymore.                                                                                                 Using the above information I can predict that as the temperature of the enzyme reaction increases by ten degrees it will double the rate of reaction up until around 45 degrees when the catalase will denature. Beyond this temperature the catalase will not work and the rate of reaction will decrease to nil.

Substrate Concentration

When the concentration of substrate is low collisions between the enzyme and substrate molecules are infrequent and reaction proceeds slowly. As the substrate concentration increases, the rate of reaction increases proportionately as there are more collisions between the two reactants. When the enzymes begin to approach the maximum rate at which they can combine with reactants and release products, the effects of increasing substrate concentration lessen. When enzymes reach the point where they are reacting as quickly as possible an increase in substrate concentration will have no effect. At this point the enzyme is saturated and the reaction remains at the saturation level.                                                         Bearing in mind the above information I predict that as the level of catalase is increased (i.e. the volume of potato) the rate of reaction will increase up to the enzymes saturation point, which can only be found by performing the experiment.

Factor chosen to Investigate:

        Looking at the selection of hypotheses shown above and considering other factors to investigate I have chosen to investigate the affect that temperature has on the rate of enzyme reactivity. I have chosen this factor, as it will be easy to control, which is needed to enable a fair test.

If I did not control temperature I would have to use room temperature and assume that it was always the same (which is very unlikely) or use a water bath, which is a lot of hassle if you are also changing another factor.

Prediction for this Factor:

        As stated under the heading Optimum Conditions and Hypotheses, I predict that as the temperature increases by ten degrees the rate of reaction will double (Q10 theory). This trend will be manifested until the optimum temperature of around 40o to 50 o is reached, after which the enzyme will denature and the reactions will not take place.

Preliminary Work and Resulting Changes to the Method:

        Preliminary work and experiments were carried out to determine whether or not the prospective method was suitable and to establish what range of results to expect.

Preliminary Method:

• The apparatus was set up as shown in the diagram
• The water bath was set up at the lowest and highest temperatures (10 and 60 degrees)
• At both of these temperatures the enzyme (contained in one 3cm cylindrical section of potato) and substrate (catalase and Hydrogen peroxide) were added together in the test tube.
• The amount gas collected after two minutes was measured and recorded.

Preliminary Results:

Preliminary Conclusion Notes and Changes to the Method:

We were briefed that it was a good idea to devise method to yield lots of O2 easily and accurately and were told that the supply of hydrogen peroxide was limited

• Firstly, measurements were collected after two minutes, which produced little oxygen to measure; therefore the reaction time was doubled to four minutes.
• The potato slice used was in one 3cm cylinder and this yielded poor results; therefore a 4cm cylinder cut into 1cm pieces was used to create a larger surface area to enable more of the enzymes to used in order to produce more oxygen.
• As tap water was at 22oC Ice was added to reach 10oC.
• In the preliminary experiment the H2O2 was poured onto the potatoes which was time consuming and inaccurate; therefore the H2O2 was placed into the test tube and placed in the water bath to get to the water bath temperature and then the potato was added to reduce the amount of oxygen from escaping as the bung was fitted.
• Measurements far above the recognized denaturation temperature (of about forty to fifty degrees) i.e. sixty degrees were used to determine whether or not the Q10 theory continued past the denaturation point. It did not and so higher temperatures were not needed to identify another trend.
• It was discovered that there was a time lag for the H2O2 to reach the temperature of the water bath, of about 20-30 seconds; therefore before the potato is added the H2O2 will be placed in the water bath for 30 seconds.
• 10ml of H2O2 was used in the preliminary experiment but produced little oxygen; therefore it was doubled which doubled the substrate volume in order to increase the rate of reaction.
• At first a gas syringe was to be used to measure the oxygen produced but the gas produced wouldn’t exert enough pressure to move the syringe and so the gas displacing water method (as shown in the diagram) was used

Safety:

• Hot water will be used and so the experiment will have to be performed standing up with hair ties and shirts tucked in.
• Hydrogen Peroxide is HARMFUL and must be handled with care; safety glasses must be worn at all times.
• Knives will be used and therefore it is imperative that a sensible nature is adopted to ensure a safe practical.

Fair Test/Controlled Variables:

• PH level: - the same amount of hydrogen peroxide will be used 20ml for each experiment and the pH level will be the same as it will all be from the same bottle
• Substrate Concentration: -The same amount of H2O2 will be used, as is all of the same concentration as it came from the same bottle.
•  Enzyme concentration; - assuming that the enzymes are spread evenly throughout the potato, gain a compromise between making sure there are no inaccuracies due to the catalase leeching out of the potato into the H2O2 and keeping the same amount of enzyme/potato constant by changing the potato pieces for every new temperature.

Diagram:

        See Attached.

Apparatus

• 250ml plastic beaker
• Water bath at 60 degrees C
• Boiling tube
• Thermometer
• Hydrogen Peroxide
• Potato (which contains catalase)
• Bung with delivery tube hole
• Delivery tube
• Basin of water (room temp)
• 25ml measuring cylinder
• Retort stand with boss and clamp
• Stop clock
• White tile
• Knives
• Potato borer
• Digital top-pan balance

Method:

• The apparatus was set up as shown in the diagram but without mixing the H2O2 with the potato.
• It was made sure that there were no air bubbles in the measuring cylinder and the test tubes was filled with 20 ml of H2O2 and placed in the water bath at ten degrees C (using ice in the water bath if necessary).
• The H2O2 was left in the water bath for 30 seconds and then the four 1cm potato cylinders were added and the bung was immediately closed over the test tube.
• The stop clock was started immediately.
• After four minutes the delivery tube was removed from below the measuring cylinder and the amount of oxygen collected was read.
• The glowing splint test was then carried out to make sure that the gas collected was indeed, oxygen.
• This experiment was repeated and an average of the two results was calculated.
• This method was also applied the other variables of 10, 20, 30, 40, 50, and 60 degrees C.

Extension work (see also evaluation)

As the results in the next section show the highest area of enzyme reactivity was recorded between 40oC and 50oC. Therefore some extension work as carried out to show what temperature yielded the highest amount of Oxygen or the optimum temperature. This was simply achieved by performing a few strategic experiments:

• A temperature of 45 o (directly between 40 o & 50 o) was used and this gave a reading of 26.25cm3.
• Then a lower temp of 43 o was used to find out if the optimum temp was higher or lower. This gave a reading of 24.75cm3, which implied that the optimum temp was higher than 45 o.
• Seeing that the optimum temperature was above 45o and below 50o a temperature of 46o was used which produced 28.00cm3.
• To determine whether or not the optimum temperature was above 46o a temperature of 47o was used which yielded only 10.5 cm3.

This proved that the optimum temperature for the catalase enzyme working to break down H2O2 in this experiment was 46o to the nearest oC.

Results:

The values in red should be nil, as the enzymes should have denatured after the optimum temperature. However even though the H2O2 was heated to the water bath temperature the enzymes in the potatoes were not, and could not have been heated to the water bath temperature, and in these cases there was a time lag for the enzymes to reach the high temperatures and denature which allowed them to react for a short while and therefore produce some oxygen.

Manipulating Data:

        For this section I am going to find the rate of reaction and see if the Q10 theory is evident in these results.

If we assume that the volume of oxygen produced is a form of the rate of reaction then the amount of oxygen produced in four minutes is also a form of rate of reaction.

        We can see from this graph that the temperature is increased by 10o C the rate of reaction increases by approximately 200% (or in other words doubles).

        i.e. 15 o gives off 3.2cm3 of oxygen and 25 o gives off 5.8cm3 of oxygen;

              35 o gives off 13.5 cm3 of oxygen and 45 o gives off 27 cm3 of oxygen (which is exactly double)

        This shows that the Q10 theory that every 10o C increase in temperature doubles the rate of reaction is followed closely by this set of results.

 Conclusion:

         These results and manipulation of data us proves that our quantative prediction that as the temperature of the enzyme reaction was increased by 10o the rate of reaction would double. The results also showed us that this trend was apparent up until the denaturation point where the enzymes active site was altered and therefore the reactions couldn’t take place. The investigation also shows that the optimum temperature for catalase activity is 46oC (under the general conditions of the experiment).

        The reason for the increase in temperature is due to the collision theory; when you increase the temperature this gives the H2O2 and catalase molecules more energy; this makes them move around faster and so there are more forceful effective collisions, between the two molecules, with enough force, to create a reaction.

        Above the denaturation temperatures of above 46o the physical form of the active site is altered. This means that the H2O2 compound cannot physically fit into the active site and so the two molecules cannot combine to react and break down the H2O2 

Evaluation:

        The results were quite accurate as shown by the fact that they complied with the Q10 theory and that the repeat readings were all within three units of eachother.  The procedure used was sufficiently capable of fulfilling our needs but if we had better, more accurate equipment our results could have certainly been more accurate. The data that we collected was not exact correct, as there was some sources of error that rendered the results 100% accurate. However these sources of error were applied to all the experiments and so were in a way ‘controlled’ thus implying that although not full accurate the results are relative to eachother and still adopt the scientific trends associated with this experiment, therefore allowing us to draw and support firm conclusions. The essence of the procedure followed was accurate and could have produced fully accurate results if more accurate equipment was available.

        There were no very apparent anomalous results but the values of oxygen given off for thirty degrees C were a little low when the results were converted to oxygen given off per second. However this may have been due to the rounding down and up of figures. Otherwise, the results could have been affected/reduced in accuracy, by one or more of the sources of error listed below:

• At the end of the experiment oxygen may have been trapped in the delivery tube and some may not have even entered the delivery tube from the test tube. As a result not all the oxygen that was produced was collected.
• The time lag of thirty seconds was obviously not enough to warm the H2O2 to the water bath temperature and the fact that we couldn’t warm the actual enzymes in the potato to the water bath temperature before the potato was added allowed the enzymes to react at high temperatures before they actually reached the water bath temperature themselves and were denatured. This was shown in the last three measurements in red on the results table (which should have been nil) where temperatures above the denaturation point should have caused the enzymes to denature. However there was a time lag before the enzymes could reach that temperature and therefore they reacted with the H2O2 during that time lag.
• As stated in the ‘Preliminary Conclusion Notes and Changes to the Method’ we were not able to use gas syringes and measuring cylinders were used. The scale on these was poor and readings had to be approximated to the nearest half a cm3 which caused inaccuracies in the results.

However as stated before these sources of error occurred in all the experiments and therefore the results are at least relative to eachother if not fully accurate.

        As shown in the extension to the methodology further work was proposed and carried out to find the optimum temperature for catalase, which was 46o. However if I wanted to further my investigation even more I could investigate more temperatures and conclude whether catalase stops reacting or slows down at a certain temperature, perhaps below freezing when it is harder for the molecules to move around.