I will control the variable by carrying out the whole experiment in a water bath at 37◦c. Enzymes like it warm but not too hot. Enzymes are biological catalysts, which speed up chemical reactions. They work most efficiently at the optimum point (37◦C). If they are below 37◦C they will work slower. If the temperature goes over 45◦C they will be denatured due to the high heat.
2. The pH will also affect the rate of reaction. The optimum pH is the pH at which the maximum rate of reaction occurs. Changes in the pH alter the ionic charge of the basic groups that help to maintain the specific shape especially the active site. This can also cause the hydrogen bonds to break and will lead to denaturing.
If any change in the pH occurs then it will affect the ionic and hydrogen bonding in an enzyme, and subsequently the shape of the enzyme is altered. This causes the rate of enzyme activity to moderate as the active site changes so that the substrate can no longer be broken into products. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction.
In this experiment 5cm³ of buffer solution of pH 7.0 will be used to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (catalase). This will ensure no variation in pH that might result in an increase or decrease in rate of reaction. I’ve chosen pH 7 because at pH7 or neutral there is maximum rate, whereas for the majority of enzymes prefer extremely high pH (acidity) or low pH (alkalinity) results in complete loss of activity, and therefore no reaction. I will control this variable by adding buffer of pH7 as catalase works best and at which is the condition it works most efficiently at.
3. The enzyme concentration would affect the rate of reaction too. If the substrate concentration was high and the enzyme concentration was increased then the rate of reaction would also increase. However if the substrate concentration was low, after a certain point it wouldn't matter how much you increased the enzyme concentration, the rate of reaction would not increase because there are too many active sites to not enough substrate. Provided that there is an excess supply of substrate, an increase in enzyme concentration leads to a corresponding increase in the rate of reaction. Where the substrate is in short supply (i.e. it is limiting) an increase in enzyme concentration has no effect. This is due to the larger numbers of catalase molecules which increased the frequency of successful collisions between reacting particles (Hydrogen Peroxide) at the increased numbers of active sites. Lowering the enzyme concentration decreased the number of active sites available and this slowed down the reaction. Eventually, increasing the concentration of enzyme had no further effect on the rate of the reaction (point x) due to the limiting factor of the number of substrate molecules. The source of enzyme will be cut up into very small equal pieces (grounded) to ensure the enzyme concentration is not changed. 3.0 g of liver and peas will be used for each experiment.
4. If there is an unlimited amount of substrate available, then the initial rate of reaction of the experiment will increase proportionally with enzyme concentration. If the substrate concentration is restricted then after a certain point, an increase in enzyme concentration will have no effect. Reactions involving enzymes begin quickly, but gradually slow down; this is because when the enzyme and substrate are first mixed there's a large amount of substrate molecules so almost every enzyme active site is busy concerting the substrate into products, and the rate of reaction depends on how many enzyme molecules there are. Eventually however if the substrate concentration isn't continually increased then the reaction slows down as enzymes are left waiting for substrate molecules. If there are sufficient substrate molecules to occupy all of the enzymes' active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the great quantity of substrate, due to the 'saturation' of the enzyme. If there is a set amount then initially enzyme activity will increase as many enzyme-substrate complexes are formed. Substrate concentration is my independent variable, as I will be changing its concentration levels. I will measure and observe how the substrate concentration affects the reaction by using various concentrations with a set amount of enzyme concentration.
As the substrate concentration increases, there will be a point at when the rate of reaction is independent of the substrate concentration; this is shown by the graph.
The enzyme can only form a specific number of enzyme- substrate complexes in a period of time. So if more substrate is added, it won't work any faster. A maximum velocity has been reached.
5. Incubation time- 10 minutes.
6. Inhibition competes with the substrate for the active sites of the enzyme - these are known as competitive inhibitors, they can also attach themselves to the enzyme away from the active site - this action alters the shape of active site so that the substrate is unable to occupy it and the enzyme cannot function properly. The type of molecule which attaches itself away from the enzyme's active site - causing it to not function properly anymore is known as a non-competitive inhibitor. Inhibitors therefore slow the rate of reaction. As none are to be added to this investigation, there will be no effect from inhibition.
7. Enzyme Cofactors are non-protein substances, which influence the functioning of enzymes, as some enzymes only work in the presence of these chemical cofactors. They include activators that are essential for the activation of some enzymes. Co-factors also influence the functioning of enzymes, although they are not bonded to the enzyme. Unless enzyme cofactors are present in the peas or liver tissue containing the catalase, they will not be included in this investigation and therefore will not affect the rate of reaction and the results of this experiment.
8. A larger surface area would expose more particles for the hydrogen peroxide to react with, therefore speeding up the reaction due to the increased successful collisions. a smaller surface area would reduce the rate of reaction, and therefore change the amount of gas liberated.
Fair Test:
In order to make my results as precise as possible, I will use the stop clock to help get the exact time and readings and I will use a measuring cylinder that has accurate markings. I will measure all my readings on the same scale - ml. and I will make sure that I only start the stop clock once the bung has sealed off the boiling tube. I must also make sure that I have diluted the substrate precisely using a measuring cylinder and not a beaker in order to get accurate and reliable results. When measuring the volumes of Hydrogen Peroxide, the measurement should be the taken from a 90-degree angle to avoid parallax error. When measuring the volume of O2 take the reading from the bottom of the meniscus, and reading it at eye level.
In order for my investigation to be a worthwhile and conclusive experiment, I have to make sure that it is a fair test. Certain factors that are able to affect the rate at which catalase speeds up the reaction of hydrogen peroxide must remain the same throughout the entire experiment.
The surface area of the source must also be kept the same throughout so that there are the same amount of active sites available to the substrate. If there is a larger surface area and more active sites, the reaction will be speeded up and the experiment will not be a fair test in relation to the other times that I will be performing the experiment.
Washing of equipment: When washing for example test tubes it is essential that they are dried thoroughly because any water left in them would dilute the solutions.
Syringes: we must make sure that when syringes are used, they are used for the same substance each time because if we mixed them up, it would contaminate either the source of enzyme or the hydrogen peroxide. Also when measuring the solutions, always use syringes and where possible to use 5cm3 syringes instead of 10cm3 syringes.
When using measuring cylinder use the same measuring cylinder to measure the oxygen collected each time. I will try to use the most accurate measuring cylinder with measurements to every 0.1 cubic cm.
Use electronic scales when measuring the liver and peas as they are more accurate. Also use them to accuracy to the nearest 0.01g where possible.
The experiment must be carried out twice in order to get an average set of results and rid of any anomalous results I may have got.
