Volume of enzyme
(Keeping the volume of sucrose and concentration of both the same) Increasing the volume of sucrase will increase the rate as there will be more enzymes therefore more enzymes will react per unit time and the rate will be faster. I believe that the volume of enzyme is directly proportional to the rate, but this is just a prediction. This could be a factor to investigate.
Volume of substrate
(Keeping the volume of sucrase the same and the concentration of both solutions the same). Increasing the volume of sucrose will decrease the rate as there will be more sucrose to be broken down and this will take longer so the rate in slower. I believe that the volume of substrate is inversely proportional to the rate, but this is just a prediction. This could be a factor to investigate.
Rate of stirring
The rate of stirring will not affect the rate much, but in increasing the speed of stirring the more the two substances will be mixed, so there is a greater chance a free enzyme will meet a sucrose molecule, so the rate would be slightly faster.
Effect of pH
The pH also affects the enzymes and substrate in different ways.
Changes in the pH affect the attraction between the substrate and enzyme, and thus the efficiency of the conversion process, therefore pH different from the optimum mean a slower rate.
Enzymes are affected by changes in pH. The most favourable pH value - the point where the enzyme is most active - is known as the optimum pH. This is pH6.2
PH can have an effect of the state of ionisation of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine functional groups in their side chains. If the state of ionisation of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or an enzyme might become inactive, this process is called denaturisation. This dramatically reduces the rate as the substrate no longer fits the active site.
Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate con not bind to the active site or it cannot undergo catalysis. (Taken from http://academic.brooklyn.cuny.edu/biology)
Temperature
The temperature affects the enzymes and substrate in many ways.
Firstly it affects both by the kinetic energy theory. The higher the temperature the more kinetic energy the enzyme/substrate will have therefore they are both moving at a faster rate. As a result there are more frequent collisions so enzymes are more likely to breakdown the substrate, and a faster rate is achieved. It is often said that an enzyme's rate of reaction doubles for every 10° C rise in temperature.
Another effect on enzymes (taken from www.enzymes.co.uk)
Enzymes are proteins and proteins are constructed from chains of amino acids (20 possible different ones). The analogy of the amino acids being like links in a chain is a good one, although it should be visualised as having 20 different types of links all that have different shapes and properties. As an unfolded chain, the enzyme has no catalytic activity. Only the folded structure forms the catalytic or active site. However this folded structure will generally be held together by non-covalent interactions unlike the covalent bonds that hold the amino acid links together. These consist of interactions such as ionic (salt) bridges, hydrogen bonds, hydrophobic and hydrophilic interactions and so on.
There is usually a doubling of the reaction rate for every 10°C rise in temperature.
Temperature coefficient Q10 = rate of reaction at T + 10°C = 2
Rate of reaction at T°C
At very high temperatures although chemically you are increasing the chances of sucrose breakdown, you are also increasing the chances of the breakdown of the three-dimensional structure of the enzyme. As the heat in the system increases, the vibrational energy of the entire alpha-sucrase molecule also increases. This puts a strain on the weak interactions that hold the enzyme together. At temperatures just above optima, there may be a situation where the enzyme is in a sort of equilibrium where it temporarily loses some of its structure and then regains it to work again this is why the graph does not suddenly drop after the optimum. At higher temperatures these bonds literally get shaken apart and the three-dimensional structure of the protein destabilises. This is called denaturisation and will dramatically slow down the rate as the substrate no longer fits the active site of the enzyme.
Optimum temperature for sucrase = 37°C
My Factor
I have chosen to make my variable factor Temperature. All other factors must be kept the same (how is stated later).
I will vary the Temperature by the use of a water bath. This will help maintain the temperature required at a constant during each experiment.
Both the enzyme and substrate will be heated in the water bath to the temperature required for about five minutes so that the breakdown occurs at the desired temp.
The rest of the experiment will take place inside the water bath to ensure a constant temperature is kept.
I know from research the optimum temperature for sucrase is 37°C. I will use this value to concentrate some values of my range.
(http://www.usdsu.org/faculty/madsens/powerpoints/Spring%202004/Lecture%20stuff/Lecture%2010%20Chapter%2021%203%2030%202004%20%20ALL%20sections.pdf) - Research page.
Temperature Range
I will be looking at a wide spread of temperatures to help make my results more reliable. My range will be:
10°C
20°C
30°C
34°C
37°C
40°C
50°C
60°C
I believe that this range is wide enough to find the temp at which sucrase denatures and find the optimum.
Rate
I will find out the rate buy using 1/T. Therefore I will need to know the time it takes for the total breakdown of sucrose. This will be achieved by timing how long it takes for a clinistix to change colour from pink to blue i.e. to show when the end point is and glucose is produced.
Maintenance of other factors
It is very important to keep ALL other factors the same to achieve more accurate and reliable results. This is because only the factor I have chosen will vary the results and nothing else.
Concentration of Enzyme
The concentration of enzyme will be kept the same as is will be taken from the same source. The concentration is 5% sucrase.
Concentration of Substrate
This will also be kept the same by taking the substrate from the same source. The concentration will be 10% sucrose.
Volume of enzyme
This will be kept the same by accurately measuring out the volume using measuring cylinders, syringes and teat pipettes to get exactly 5cm3 of sucrase solution.
Volume of substrate
This will also be kept the same by accurately measuring out the volume using measuring cylinders, syringes and teat pipettes to get exactly 5cm3 of sucrose solution.
Rate of stirring
This is not easy to keep the same but I will try my best to stir all experiments for the same amount of time at the same rate.
pH
I will not be adding any other foreign substances e.g. acids/alkalis so the pH should stay the same through out the experiment.
Hypothesis
My prediction is that as the temperature increases so will the rate (at a coefficient Q10 of 2). I believe this because as the temperature increases so will the kinetic energy of the molecules in the solution. Therefore they are moving faster, and there are more frequent collisions which increase the chance the enzyme will react with a sucrase molecule and increase the rate.
But this will only increase up to the optimum. This is because above the optimum the structure of the enzyme begins to weaken and fall apart and become denatured. At this point the rate will begin to decrease.
The rate will continue to decrease up to a point where no sucrose is broken down as all sucrase enzymes have been denatured where the rate is 0. This is at about 50-60°C.
(More detailed explanation given on page 6).
Plan
- All variables are to be kept the same except temperature. This will be done as stated before.
- Set water bath to desired temp, use thermometer to get it exact.
-
Measure out 5cm3 of sucrose solution into a test tube using a measuring cylinder, syringe and teat pipette. Repeat this 3 more times
-
Measure out 5cm3 of sucrase solution into a test tube using a measuring cylinder, syringe and teat pipette. Repeat this 3 more times
- Place all 8 test tubes in a test tube rack (on separate rows for sucrase and sucrose so you do not mix them up) then place the test tube rack into the water bath when it is at the desired temperature for about 5 minutes.
- After 5 minutes remove 1 of the sucrase solutions and 1 of the sucrose solutions. Place a clinistix in the sucrose, and then add the sucrase. Mix for a bit then start the stop clock
- Place test tube back into water bath and observe the clinistix for a colour change from pink to blue. Stop the stop watch at a definite change to blue.
- Re do experiment with remaining solutions to get 3 more results
- Re do whole experiment at a different temp according to range (given before).
Apparatus list
This is a list of all the apparatus I will be using during the experiment.
Safety precautions
Wear goggles to avoid solutions getting in eyes
Beware of high temperatures of water.
Table for results
Modifications
Different range used to get more results near optimum temp °C:
10, 20, 30, 34, 37, 40, 44, 54, 64
No buffer used
Sucrose concentration changed to 1M
Table of results
Analysis
As the graph shows the rate increases to a point of 37°C (being the optimum) then begins to decrease till there is no rate at 54°C (where all enzymes were denatured)., just as I had predicted.
Looking at the line of best fit the line is ‘bell shaped’ just as I had predicted.
I will use the Table to work out a couple Q10’s to see if they are close to what I had predicted.
Q10 = rate of reaction at T + 10°C
Rate of reaction at T°C
For 10°C - 20°C Q10 = 7.34 = 2.033
3.61
For 20°C to 30 °C Q10 = 14.59 = 1.963
7.43
I predicted from searching the net that the Q10 should be 2(see plan for more detail) and the Q10s for my experiment was very close to 2, 2.033 and 1.963.
Why this happened
Looking at the graph from 10°C to 37°C there is a steady increase of rate. This is because of the kinetic energy theory. The higher the temperature the more kinetic energy the enzyme/substrate will have therefore they are both moving at a faster rate. As a result there are more frequent collisions so enzymes are more likely to breakdown the substrate, and a faster rate is achieved.
The rate only increased to a point of 37°C then began to decrease. 37°C is the optimum and at temperatures above that week the ionic (salt) bridges, hydrogen bonds, hydrophobic and hydrophilic interactions.... get shaken apart since as the temperature increases the partials vibrate faster due to the kinetic energy theory stated above. This breaking of bonds will alter the 3D structure of the enzyme, and denature the active site so the substrate will fit no more, At temperatures just above optima, there may be a situation where the enzyme is in a sort of equilibrium where it temporarily loses some of its structure and then regains it to work again this is why the graph does not suddenly drop after the optimum.
On the graph the rate comes to absolute 0 at 54°C and this is when ALL enzymes are denatured.
Evaluation
I feel the experiment went quite well, as the practical went smoothly and looking at the analysis most of my results follow my prediction.
Looking at the graph there was only 1 apparent anomaly which is at 44°C. The rest of the results follow the line of best fit accurately.
From the table there is not a lot of deviation between replicates so I believe the results were quite accurate.
I recon my experiment was a fair test as all of the factors were maintained accurately
I believe 4 replicated were enough to get a reliable average and spot any anomalies.
The range could have been wider after the optimum to determine the point at which the enzymes denatured more accurately.
Problems
During the experiment there possibly was some unwanted mixing of solutions, which could explain why the 44°C result was consistently to fast in all the replicates.
The cleaning of test tubes was not very good; some residue of enzyme could have been left over in a test tube where sucrose was measured out into, starting breakdown.
It was hard to get the same colour on the clinistix for the end point
There were two different types of clinistix (one which reached end point much faster) so using the wrong clinistix could effect results drastically.
Improvements
Use a wider range of temperature at he optimum and after to accurately determine the denature point
Use a burette to accurately measure out solutions
Have the two solutions on different sides of the room with its own set of equipment to stop any contamination of solutions (the accidental addition of sucrase to the sucrose source could seriously effect the results as breakdown will already occur).
Use a magnetic stirrer to evenly mix solutions.
Use a bigger concentration to make the time to end point of clinistix more, so results are more accurate times.
Use a colorimeter to determine the colour of clinistix at the end point accurately.