An Investigation Into the Effect of Substrate Concentration On the Rate of Enzyme Activity.
AN INVESTIGATION INTO THE EFFECT OF SUBSTRATE CONCENTRATION ON THE RATE OF ENZYME ACTIVITY
INTRODUCTION
Hydrogen peroxide is a toxic waste product of respiration. It's conversion to water and oxygen is catalysed by the enzyme CATALASE, which is present in most cells.
2H202 2H20 + 02
hydrogen CATALASE ENZYME water oxygen
peroxide gas
Hypothesis
Increasing the substrate concentration will increase the rate at which hydrogen peroxide is broken down to water and oxygen by the catalase enzyme.
Null Hypothesis
Increasing the substrate concentration will not affect the rate at which hydrogen peroxide is broken down to water and oxygen by the catalase enzyme.
Possible Methods
There are a number of possible methods for carrying out this investigation. The easiest way to monitor the reaction is to measure the production of oxygen because it is the gaseous waste product:
) In this method the reaction takes pace in a test tube. The test tube is connected by a thin glass tube to a gas syringe. As the oxygen is given off by the reaction, it travels through the glass tube and into the syringe, this pushes the syringe outwards so there is an increased volume of oxygen in the syringe. As the volume of the syringe increases the amount can be measured on the scale of the syringe:
Gas syringe
H202 and catalase enzyme
The problem with this method is that the reaction starts as soon as the catalase enzyme is added to the hydrogen peroxide. If the bung is not put on top of the test tube very fast some of the waste oxygen might escape and so it would not be measured in the gas syringe. Also, the volume of gas changes with temperature so if the room temperature changes then the results would be affected. The syringe could become more slippery or stiff after each use, this could effect how far the end of the syringe is pushed out. This method is easy to use.
2) In this method, the catalase enzyme is immobilised in sodium alginate jelly beads. The beads are then dropped into a known volume of hydrogen peroxide. Oxygen gas is produced from the reaction causes the beads to rise in the hydrogen peroxide. The beads are then timed for how long it takes them to reach the surface from the bottom of the test tube:
This method provides more control over the start of the reaction so it is more accurate. Although technically it is more difficult and it is hard to make all the enzyme beads the same size. However the start and end of the reaction are easy to determine so this method will be used.
Apparatus and Chemicals
Test tube rack, test tubes, syringes (10cm3), filter paper, filter, beakers, stopwatch, glass stirrer, calcium chloride (1M), sodium alginate jelly (6cm3), yeast suspension (4cm3), hydrogen peroxide (1M), distilled water, catalase enzyme, heat block, ice and safety goggles,
Risk Assessment
There are many safety risks to consider whilst carrying out this experiment. Firstly the chemicals being used should be considered. Hydrogen peroxide is dangerous because it is corrosive. It causes burns and when it comes into contact with combustible material it may cause fire. In high concentrations it can have toxic effects where it is irritant and caustic to mucous membranes, skin and eyes. If hydrogen peroxide is swallowed that sudden evolution of oxygen may cause injury by acute distension of the stomach and may cause nausea, vomiting and internal bleeding. To take precautions with the chemical safety goggles will be worn to protect the eyes, handle all chemicals with care and label any containers carefully. If any of the hydrogen peroxide splashes into eyes, the eye will be flooded thoroughly with running water for at least 10 minutes. To ensure that the water bathes the eyeball correctly, it will be exposed by gently opening the eyelids and keeping them apart until the 10 minutes has finished. Any eye injury involving chemicals requires immediate medical advice. In the event of hydrogen peroxide splashing into the eye transport to a hospital will be arranged and information about the chemical and the treatment already given will be supplied. If any hydrogen peroxide comes into contact with skin the area will be washed under running, cold water for at least 10 minutes, not using soap. Any contaminated clothes will be removed and in a very serious incident then transport to hospital will be arranged where information about the chemical and prior treatment will be passed on. If any of the hydrogen peroxide is swallowed, then large amounts of water will be used as a mouth wash, the mouth wash will not be swallowed. Then the casualty will be given large amounts of water or milk to dilute the hydrogen peroxide in the stomach. Vomiting will not be induced and transport to a hospital will be arranged and information about the relevant chemicals will be given. If any of the hydrogen peroxide is spilt then goggles and gloves will be worn to mop the excess up and then the rest will be dilutes with running water.
Another safety risk is the glass test tubes that will be used. If broken, then sharp pieces of broken glass could result. This could cause cuts to the skin if not dealt with in a safe way. Open wounds could then get chemicals in which would also be hazardous. Shards of glass could cause blindness if they got into the eyes. To prevent this safety goggles will be worn and care will be taken with test tubes. If a test tube was broken and a wound resulted then the wound would be run under cold water and would then be bandaged up.
Another safety risk is the heat block which can get quite hot. If skin came into contact with the heat block then a burn could result. Care should be taken when placing the test tubes into the heat block. If a burn does occur then the effect area should be put under cold, running water for as long as possible to prevent blistering. In the case of a serious burn transport to a hospital would be arranged and information about previous treatment explained.
Another safety precaution to take is to place all bags and coats at the side of the lab so that no one could trip over them. The yeast being used has little safety danger as it has a low safety risk.
Ethical Considerations
The catalase enzyme can be obtained from many different sources. For example liver is a good source of catalase. However, because some people may object to the use of animal products in experiments, and there are alternative non-animal sources of catalase available, Yeast was used.
Plan
First some sodium alginate beads containing the catalase enzyme will be made. To do this, 4cm3 of yeast suspension and 6cm3 of sodium alginate will be drawn up into a syringe. To mix the solution thoroughly, the syringe will be turned upside down several times. To make the solution into beads, the syringe will be used to drop small amounts of the solution into a beaker containing calcium chloride, this will harden the beads and allow the experiment to be carried out. The calcium chloride will be drained off the beads using a filter and filter paper, then the beads will be rinsed to remove any excess calcium chloride. The beads will then be placed into a clean beaker to avoid contamination.
To determine which concentration of hydrogen peroxide will be appropriate for the experiment a number of tests on four different concentrations of hydrogen peroxide at a standard temperature will be carried out. The concentration that is found to be most appropriate (the one that did not take too long or too quickly) will be used to carry out the final experiment. To do this, four different concentrations of hydrogen peroxide will be made.
The different concentrations of hydrogen peroxide will be placed into four different test tubes, the test tubes will then be labelled by their concentrations. These test tubes will then be placed into a heat block at a temperature of 20oC, this will be done to keep the temperature constant. The test tubes containing the hydrogen peroxide will be left in the heat block for five minutes; this will allow the hydrogen peroxide solutions to rise to the temperature of the heat block. After the five minutes a bead will be placed into the 0.25M hydrogen peroxide. When the bead reaches the bottom of the test tube the stopwatch will be started, when the bead reaches the top of the hydrogen peroxide the stopwatch will be stopped. The time taken for the bead to travel from the bottom of the test tube to the top will be recorded. This will be repeated using the different concentrations of hydrogen peroxide.
INTRODUCTION
Hydrogen peroxide is a toxic waste product of respiration. It's conversion to water and oxygen is catalysed by the enzyme CATALASE, which is present in most cells.
2H202 2H20 + 02
hydrogen CATALASE ENZYME water oxygen
peroxide gas
Hypothesis
Increasing the substrate concentration will increase the rate at which hydrogen peroxide is broken down to water and oxygen by the catalase enzyme.
Null Hypothesis
Increasing the substrate concentration will not affect the rate at which hydrogen peroxide is broken down to water and oxygen by the catalase enzyme.
Possible Methods
There are a number of possible methods for carrying out this investigation. The easiest way to monitor the reaction is to measure the production of oxygen because it is the gaseous waste product:
) In this method the reaction takes pace in a test tube. The test tube is connected by a thin glass tube to a gas syringe. As the oxygen is given off by the reaction, it travels through the glass tube and into the syringe, this pushes the syringe outwards so there is an increased volume of oxygen in the syringe. As the volume of the syringe increases the amount can be measured on the scale of the syringe:
Gas syringe
H202 and catalase enzyme
The problem with this method is that the reaction starts as soon as the catalase enzyme is added to the hydrogen peroxide. If the bung is not put on top of the test tube very fast some of the waste oxygen might escape and so it would not be measured in the gas syringe. Also, the volume of gas changes with temperature so if the room temperature changes then the results would be affected. The syringe could become more slippery or stiff after each use, this could effect how far the end of the syringe is pushed out. This method is easy to use.
2) In this method, the catalase enzyme is immobilised in sodium alginate jelly beads. The beads are then dropped into a known volume of hydrogen peroxide. Oxygen gas is produced from the reaction causes the beads to rise in the hydrogen peroxide. The beads are then timed for how long it takes them to reach the surface from the bottom of the test tube:
This method provides more control over the start of the reaction so it is more accurate. Although technically it is more difficult and it is hard to make all the enzyme beads the same size. However the start and end of the reaction are easy to determine so this method will be used.
Apparatus and Chemicals
Test tube rack, test tubes, syringes (10cm3), filter paper, filter, beakers, stopwatch, glass stirrer, calcium chloride (1M), sodium alginate jelly (6cm3), yeast suspension (4cm3), hydrogen peroxide (1M), distilled water, catalase enzyme, heat block, ice and safety goggles,
Risk Assessment
There are many safety risks to consider whilst carrying out this experiment. Firstly the chemicals being used should be considered. Hydrogen peroxide is dangerous because it is corrosive. It causes burns and when it comes into contact with combustible material it may cause fire. In high concentrations it can have toxic effects where it is irritant and caustic to mucous membranes, skin and eyes. If hydrogen peroxide is swallowed that sudden evolution of oxygen may cause injury by acute distension of the stomach and may cause nausea, vomiting and internal bleeding. To take precautions with the chemical safety goggles will be worn to protect the eyes, handle all chemicals with care and label any containers carefully. If any of the hydrogen peroxide splashes into eyes, the eye will be flooded thoroughly with running water for at least 10 minutes. To ensure that the water bathes the eyeball correctly, it will be exposed by gently opening the eyelids and keeping them apart until the 10 minutes has finished. Any eye injury involving chemicals requires immediate medical advice. In the event of hydrogen peroxide splashing into the eye transport to a hospital will be arranged and information about the chemical and the treatment already given will be supplied. If any hydrogen peroxide comes into contact with skin the area will be washed under running, cold water for at least 10 minutes, not using soap. Any contaminated clothes will be removed and in a very serious incident then transport to hospital will be arranged where information about the chemical and prior treatment will be passed on. If any of the hydrogen peroxide is swallowed, then large amounts of water will be used as a mouth wash, the mouth wash will not be swallowed. Then the casualty will be given large amounts of water or milk to dilute the hydrogen peroxide in the stomach. Vomiting will not be induced and transport to a hospital will be arranged and information about the relevant chemicals will be given. If any of the hydrogen peroxide is spilt then goggles and gloves will be worn to mop the excess up and then the rest will be dilutes with running water.
Another safety risk is the glass test tubes that will be used. If broken, then sharp pieces of broken glass could result. This could cause cuts to the skin if not dealt with in a safe way. Open wounds could then get chemicals in which would also be hazardous. Shards of glass could cause blindness if they got into the eyes. To prevent this safety goggles will be worn and care will be taken with test tubes. If a test tube was broken and a wound resulted then the wound would be run under cold water and would then be bandaged up.
Another safety risk is the heat block which can get quite hot. If skin came into contact with the heat block then a burn could result. Care should be taken when placing the test tubes into the heat block. If a burn does occur then the effect area should be put under cold, running water for as long as possible to prevent blistering. In the case of a serious burn transport to a hospital would be arranged and information about previous treatment explained.
Another safety precaution to take is to place all bags and coats at the side of the lab so that no one could trip over them. The yeast being used has little safety danger as it has a low safety risk.
Ethical Considerations
The catalase enzyme can be obtained from many different sources. For example liver is a good source of catalase. However, because some people may object to the use of animal products in experiments, and there are alternative non-animal sources of catalase available, Yeast was used.
Plan
First some sodium alginate beads containing the catalase enzyme will be made. To do this, 4cm3 of yeast suspension and 6cm3 of sodium alginate will be drawn up into a syringe. To mix the solution thoroughly, the syringe will be turned upside down several times. To make the solution into beads, the syringe will be used to drop small amounts of the solution into a beaker containing calcium chloride, this will harden the beads and allow the experiment to be carried out. The calcium chloride will be drained off the beads using a filter and filter paper, then the beads will be rinsed to remove any excess calcium chloride. The beads will then be placed into a clean beaker to avoid contamination.
To determine which concentration of hydrogen peroxide will be appropriate for the experiment a number of tests on four different concentrations of hydrogen peroxide at a standard temperature will be carried out. The concentration that is found to be most appropriate (the one that did not take too long or too quickly) will be used to carry out the final experiment. To do this, four different concentrations of hydrogen peroxide will be made.
The different concentrations of hydrogen peroxide will be placed into four different test tubes, the test tubes will then be labelled by their concentrations. These test tubes will then be placed into a heat block at a temperature of 20oC, this will be done to keep the temperature constant. The test tubes containing the hydrogen peroxide will be left in the heat block for five minutes; this will allow the hydrogen peroxide solutions to rise to the temperature of the heat block. After the five minutes a bead will be placed into the 0.25M hydrogen peroxide. When the bead reaches the bottom of the test tube the stopwatch will be started, when the bead reaches the top of the hydrogen peroxide the stopwatch will be stopped. The time taken for the bead to travel from the bottom of the test tube to the top will be recorded. This will be repeated using the different concentrations of hydrogen peroxide.
