How does substrate concentration affect the rate of reaction in an enzyme-controlled reaction?
The aim is to investigate the effects of increasing substrate concentration on enzyme activity on two different reactions involving enzymes from the same living being. This is by using sucrose (in table sugar) and hydrogen peroxide as the substrate, while using catalase and invertase (in yeast) as the enzyme respectfully. Therefore, to measure the rate of reaction by recording the volume of gas (carbon dioxide and oxygen) produced after 15 minutes.
The hypothesis is that as the substrate concentration increases the rate of reaction will also increase.
30 Vol hydrogen peroxide is highly concentrated and is a hazard as it is harmful. So can be harmful if swallowed and has a risk of damaging the eyes (CLEAPSS 2013). If any of the solution is swallowed or gets into the eye (wash the eye) and immediately seek medical attention.
The conical flasks, beaker and gas syringe are made of glass so are fragile. Therefore, there is a risk of injury if the equipment is handled carelessly.
Consumption of active dried yeast can be poisonous as yeast is a fungus that can harm the body (stomach) of the victim. Therefore, to avoid this risk it is best to wear protective gloves when handling the yeast. If consumed, seek medical attention immediately.
The expected outcome is that as substrate concentration increases the rate of reaction increases; as substrate concentration increases there are more particles that can collide with each other, so there are more frequent and successful collisions between the substrate molecules and enzymes. Therefore, there are more enzyme-substrate complexes formed leading to more products being formed in less amount of time (than with less substrate concentration), thus increasing the rate of reaction. Therefore, it is expected that as substrate concentration increases the volume of gas produced will also increase, which can be quantitatively measured and qualitatively observed.
· Dried yeast (56g)
· Table sugar (1kg)
· Distil water (1L)
· 30 Vol Hydrogen Peroxide
· 100cm^3 Gas syringe
· Clamp with stand
· 250cm^3 beaker
· 250cm^3 conical flask
· 10cm^3 syringe
· 25cm^3 Volumetric pipette with bulb
· Bung with delivery and flexible tube
· Thermostatic water bath (or kettle with a tub)
· Digital Stopwatch
· Digital mass scale
· Plastic mass boat
· Spatula (for calculating mass)
· Stirring rod
1. Preparing yeast suspension;
a) Measure 21 grams of dried yeast into a mass boat and measure accurately on the digital mass scale.
b) Pour 100ml of gently heated distil water into a beaker and mix in the yeast, ensuring the distil water is at 24 degrees Celsius.
c) Gently mix with stirring rod to distribute evenly.
2. Preparing sucrose solutions with different concentrations;
a) Measure 4g of table sugar using mass boat and digital scale.
b) Measure 100ml distil water into a beaker and then mix the measured sugar into beaker.
c) Stir with sirring rod until the solid sugar completely dissolves.
d) This creates a sucrose (substrate) solution with concentration of 0.4g/L . Therefore, label this solution as 0.4g/L. Therefore, repeat steps a-d with different masses of table sugar to create the other required concentrations (0.8, 1.2, 1.6, 2.0 g/L).
3. Preparing hydrogen peroxide solutions with different concentrations;
a) Measure out 10ml of the 30 Vol hydrogen peroxide into a beaker
b) Measure 40 ml of distil water and pour into the beaker and stir the beaker lightly with the stirring rod.
c) This creates a 20% concentrated solution of hydrogen peroxide. Label this beaker as 20%.
d) Repeat steps a-c with different volumes of hydrogen peroxide and distil water to create the other solutions with different concentrations (40, 60, 80, 100 %).
4. Setting up apparatus;
a) Attach the gas syringe horizontally to the clamp of the clamp stand. Ensure the gas syringe has the least amount of gas possible by making sure there is no empty space within the syringe.
b) Attach the delivery tube to gas syringe and to the bung of the conical flask. (Do not need to cover conical flask yet)
Yeast experiment with invertase:
1. Transfer 10cm^3 of the yeast suspension using the syringe into a conical flask.
2. Using a volumetric pipette transfer 25cm^3 of the 1.4 g/L sucrose solution into a graduated cylinder.
3. Pour the solution into the conical flask and place the bung on the conical flask straightaway without delay. Gradually rotate the flask in circles.
4. Begin the stopwatch and then record the volume of gas (carbon dioxide) produced after 15 minutes.
5. Then empty and clean the flask with distil water.
6. After preparation again, repeat steps 1-4 with the additional different concentrations.