Effect of Concentration of Substrate on Enzymes
EFFECT OF CONCENTRATION OF SUBSTRATE ON ENZYMES
AIM - To find the effect of 'concentration of substrate' on an enzyme controlled reaction and
HYPOTHESIS - To prove the hypothesis: as the concentration doubles so does the rate of reaction, A point is reached however, where all sites are being used and by raising the substrate concentration the rate won't increase as the limiting factor is the amount of enzymes.
RESEARCH - A test that could be done is timing the speed of a reaction with obvious end results. In most instances it is hard to record the exact finish.
The reactants being used are hydrogen peroxide and the Enzyme - Catalase. This reaction produces O2 and H2O. These are not easy to see being made and a way is needed to facilitate this. pH is hard to measure to see the H2O2 change to neutral. A colorimeter could be used for accurate colour tests, but not for an accurate time scale as there would be time lapses between testing and getting the colour change. It is hard to see the change at exactly pH7. When pH changes from 4 to 6 it's on a very steep part of the graph showing pH change...
If phenaphalin (an indicator that changes at pH 8) is used (or another indicator that changes at pH 7 of after) it's on the flatter part of the graph where there is little difference between each pH. It is therefore harder to see when the change occurs because it is so gradual. Each indicator has it's flaws and is better to use the most accurate and easiest to measure (screened methyl orange). This though would show a pH change at pH6 and would not show the end of the reaction.
The end or rate of reaction can be measured in many ways.
* It can be tested by pH changing to 7 - finds end of reaction.
* Reacting the end products with something (i.e. washing up liquid and measuring the height of the foam. it with something - finds rate of reaction.
* Measuring the end products - finds rate of reaction.
The experiment where the oxygen pushes the water up the tube is the most accurate
a gas syringe is easier to read the results of but because the volume is much larger the change is almost unnoticeable in reactions that do not produce much oxygen. This experiment could be adapted to suit this investigation.
The easiest way to do the experiment is to have a certain time and record either the H2O or O2 given off by the reaction in that time. Oxygen can either be contained in a gas syringe or used with the pressure to push the water up the tube. Then measurements could be taken of this distance. The higher the substrate concentration therefore, the further the water should rise in a set time until the V max where the graph and the results should level off.
PREDICTION - Enzymes are biological catalysts that speed up reactions without changing. Each enzyme has an active site. Every enzyme catalyses its own specific reaction. Many things change the rate at which the reaction goes.
When the substrate joins with an enzyme an enzyme-substrate complex is formed.
An enzyme is a globular protein made up of many amino acids. These have R groups that interact with the substrate, the substrate is broken down and leaves the active site. The enzyme is unchanged and ready to form more enzyme-substrate complexes with available substrate. An enzyme can also bind together two products.
Usually energy is needed to start a reaction, enzymes reduce the amount needed and are therefore very useful in industry. Also, because they are reusable they are very cheap.
There ...
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When the substrate joins with an enzyme an enzyme-substrate complex is formed.
An enzyme is a globular protein made up of many amino acids. These have R groups that interact with the substrate, the substrate is broken down and leaves the active site. The enzyme is unchanged and ready to form more enzyme-substrate complexes with available substrate. An enzyme can also bind together two products.
Usually energy is needed to start a reaction, enzymes reduce the amount needed and are therefore very useful in industry. Also, because they are reusable they are very cheap.
There are many variables to an enzyme controlled reaction. The variable in this experiment is concentration of substrate. As the concentration rises the rate also does. When the concentration is too high a point occurs that all the active sites are working non-stop and no matter how many more substrate molecules are added the reaction will not speed up.
INITIAL EXPERIMENT - A yeast solution was made, dissolving the yeast in water so that the surface area of the enzyme was the largest it could be. Yeast in granules has a small surface area to react with the substrate, and crushed it is still very large. When added to the hydrogen peroxide it sank to the bottom and only started to react when shaken. When added to hydrogen peroxide as a liquid it is dissolved, reacted and mixed much quicker. Shaking the solution changes the results so this is the best way to mix the enzyme and substrate.
A 25% yeast solution was made, 200 ml and 5g of water. This was chosen because it was the highest amount of yeast to dissolve in 200ml of water without leaving a residue/precipitate.
Solutions were made up of
H2O2
H20
Percentage concentration (%)
2 ml
8ml
20
4ml
6ml
40
6ml
4ml
60
8ml
2ml
80
0ml
0ml
00
Because the experiment is on substrate concentration these are made up to 10ml each and then 10ml of the yeast solution was added at the last minute to the experiment.
The apparatus was set up using clamp stands and boss heads to hold everything straight. The hydrogen peroxide is syringed into the conical flask, the height of the water in the tube is marked when the bung is in the flask and at 3 cm above that.
When the bung is put into the flask the water rises due to the pressure.
The yeast is added and the bung (covered in Vaseline to create better seal) replaced.
The reaction is timed until the water has risen 3 cm. For it to rise 3cm there was enough time to measure in the highest concentration and did not take too long for the lowest to rise.
This is repeated three times for each concentration of substrate. The times are converted into rate and plotted on a graph.
FAIR TESTING AND VARIABLES - To ensure that the experiment is as fair and accurate as possible, these factors vary the rate of reactions
Each enzyme works at a different pH, enzymes in the stomach work at a lower pH that those in the mouth. The molecular shape in the enzyme is vital to the reaction. If the 3D structure shape changes the substrate will not be able to fit the active site and the reaction will not occur.
The more H+ bonds in the substance the more hydrogen bonds in the enzyme are broken. The H+ ions break the positive-negative bonds in the enzyme molecule and change its shape. The active site does then not fit the substrate and the reaction can't take place. The pH can be monitored using universal indicator. Using a buffer could control the pH, a buffer solution contains either a weak acid and its salt, or a weak base and its salt. The acid provides H+ ions when the pH rises (goes alkali) and the salt (mainly - the acid does provide some) provides - ions depending on the chemical structure .
E.g..
H2CO3 (aq) = H+ (aq) + CO3- (aq)
The - ions would be the CO3 from the salt.
The + ions would be the Hydrogen from the acid.
Then H2CO3 would be formed again.
Certain buffers are used in different situations so reactions can be kept at a certain pH.
The pH of this reaction gets lower due to the H2O2 being acid and the end product, water, being neutral. The enzyme Catalase can work in these conditions so its not going to denature. Therefore the pH would be seen (if indicator was added) to rise during the experiment the end pH if the experiment was left to complete being pH7.
The temperature may rise due to the energy being produced in the reaction. All of the experiments would rise in temperature by different amounts - the smallest concentration will give off less heat energy because it is not reacting as fast.
As the temperature increases the energy of the molecules both substrate and enzyme increases. They collide more often and faster, because they collide more frequently as the reaction goes faster. If the temperature gets too high, the bonds break and the 3D shape changes, again the site will not fit the substrate and the reaction will not work. The temperature will have to be kept low. A water bath will be used in the main experiment to sit the conical flask in and keep the temperature constant.
Inhibitors will change the rate of the reaction. All inhibitors, reversible inhibitors, both non-competitive and competitive, non-reversible inhibitors change the active site of the enzyme meaning it can now not react with the substrate. No inhibitors will be used and the solution mixture is the same so there will be no difference.
Enzyme concentration as with substrate concentration affects the reaction. The more particles to react the more collisions. The reaction rate increases, there is a maximum point when either. When there is an increase of enzyme concentration there are no more substrate molecules to react with. With substrate concentration all the active sites are full and adding more substrate will not change the reaction speed. The enzyme concentration is kept constant at a 2.5% solution. The substrate concentration is varied, the solutions are at 20%, 40%, 60%, 80% and 100%. The measurements are written in the initial experiment.
The pressure in the experiment is another key factor. If the readings on the tube are marked before the bung is in place when it is put on the conical flask the pressure will rise and so will the water. The pressure is the main factor that will ensure the experiment will work. Any leak that will let the oxygen out will ruin the results. Vaseline is used around the bung and any other joining of materials will be covered in it. If the pressure is lowered during the experiment the water level will drop. This will be very noticeable, the leak will be found by testing each piece of equipment separately and the experiment would be rerun.
RISK ASSESSMENT -
* Hydrogen peroxide is an irritant and harmful. Safety precautions like safety goggles and lab coats must be worn so to prevent burning.
* If hydrogen peroxide is swallowed wash mouth out with glasses of water, seek medical attention quickly.
* If the liquid is spilt on the skin or clothes flood the area with water, remove clothing and seek medical attention.
* If hydrogen peroxide gets in eyes, rinse for 4-10 minutes with water and seek medical attention.
* If hydrogen peroxide is spilt in the lab make sure the proper safety coat, gloves and protective eye glasses are adorned. Cover with a mineral absorbant and wash into a bucket. Rinse the surfaces into the bucket add water to dilute in and washing liquid. There may be different regulations in different places/areas about disposal these must be followed.
* All beakers and test tubes containing H2O2 have to be labelled.
* The bottle is held with the label under your so that the liquid does not disfigure the cautions.
* Hydrogen peroxide is dangerous with many other chemicals. Avoid mixing it with any substances where the out come is unknown or may be a potential health risk.
* When using a glass pipette do not hold at bulb. High pressure will make the glass shatter. Hold at top to apply pipette filler.
APPARATUS -
Glass pipette - 50ml
Distilled water - 260ml
Glass beaker - 250ml
Yeast granules - 5g
Hydrogen peroxide 20vol - 90ml
Syringe - 10ml x 2
Semi-permanent pen
Boiling tubes x 10
Boss head x 2
Clamp stand
Plastic tubing
Bung
Vaseline
Conical flask
Syringe - 10ml
Stop clock
Test tube holder
Ruler
METHOD -
. The yeast solution is made up;
Using a glass pipette 200ml of distilled water is put into a 250ml glass beaker. Bring the water above the measure line on the pipette, create a vacuum by putting a finger over the top and spin the pipette around. This will release the water slowly, stop when the bottom of the meniscus rests on the line. This will be repeated four times to get the desired amount. 5g of yeast is measured on a 3dp balance. The accurate number read off (as close to 5.000 as possible) is written down and an accurate percentage yeast concentration can be found.
Eg.
5g Yeast - 200ml water
Divide both by two to get 100ml of water
2.5g Yeast - 100ml water - 2.5% solution
The mixture must be stirred until all the yeast is dissolved. The mixture cannot be heated to prompt this as Catalase denatures at.......
2. Hydrogen peroxide solutions are made using 10ml syringes the hydrogen peroxide is drawn up (again measured to the bottom of the meniscus) and put into separate boiling tubes. The corresponding amount of distilled water (see table on next page) is added by the same method (using a different syringe) to the boiling tubes. The syringes should be labelled with semi-permanent pen to prevent contamination.
H2O2 (ml)
H2O (ml)
2
8
4
6
6
4
8
2
0
0
3. The apparatus has to be set up as in diagram. The clamp stands and boss heads are used to hold the apparatus steady and upright. To fill the tubing with water run a bath/sink of water and submerse the whole tube until it is 1/2 full. This may take some time as if it is too full all of the water must be poured out otherwise air bubbles will form.
4. The hydrogen peroxide solution being used is poured into the conical flask. The bung (covered with Vaseline) is placed in the conical flask and the water line is marked with
semi-permanent pen. A line 3 cm above it is also marked.
5. 10ml of yeast is added to the chonical flask using a 10ml syringe (line on the bottom of the meniscus) . The bung is put in (covered with Vaseline) and the stop clock started. Help is needed for a fairer/more accurate test.
6. The reaction is timed until the water has risen by 3 cm (when the bottom of the meniscus in the tubing is resting on the second line.)
7. All concentrations are repeated three times. By looking at the results it is possible to see anomilies and these will then be repeated again to give better averages.
8. A rate ( against concentration graph is plotted.
