The study of the rate at which an enzyme works is called enzyme kinetics. Let us examine enzyme kinetics as a function of the concentration of substrate available to the enzyme. This theory came from Michaelis-Menten. This applies to the experiment I am doing by varying the substrate concentration. At time zero, a fixed amount of the enzyme preparation was added. Over the next few minutes, the concentration of product formed was measured . Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observe is the initial velocity of Vi.
Plotting Vi as a function of [S], we find that at low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S]. But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola). The asymptote represents the maximum velocity of the reaction, designated Vmax. The substrate concentration that produces a Vi that is one-half of Vmax is designated the Michaelis-Menten constant, Km(named after the scientists who developed the study of enzyme kinetics).
Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the similarity (so the lower the concentration of substrate needed to achieve a given rate). This theory can be used to predict the initial rate or reaction.
Based on this scientific knowledge I would make a hypothesis of the experiment, which should follow these theories of induced fit, collision and lock and key. I will do this in my prediction.
As the concentration of the substrate decreases, I think that the rate of the breakdown of hydrogen peroxide will become slower. I think that when the concentration of the hydrogen peroxide halves, I think the rate of reaction will half as well. This is because when the concentration of H2O2 halves, the number of molecules of H2O2 will halve as well, which in turn will halve the rate of reaction. This prediction is based on the results of the preliminary experiment, where the concentration of the substrate, along with other variables, was tested.
In theory this also makes sense scientifically because if there are a lot of substrate molecules for the active site of the enzyme to latch onto and digest to produce two products in this case water and oxygen. One idea about enzyme activity is that enzymes are configured such that only one type of substrate molecule can be broken down in its active site. The shape of its active site is complimentary to the shape of a substrate; therefore it can break it down. The chances of this occurring will be affected by the proportion of enzyme to substrate to water- both the amount of H2O2 and catalase, the catalase concentration remains constant, so it is logical to suppose that the higher the concentration of Hydrogen Peroxide, the greater the likelihood that enzyme and substrate will come into contact with each other, so the initial rate of reaction will be faster. However, it must take an amount of time for a Catalase molecule to breakdown a Hydrogen Peroxide molecule, so their must be a maximum amount of Hydrogen Peroxide molecules which a Catalase molecule can handle per second, i.e. a maximum rate of activity. This maximum rate is termed Vmax, this is where the graph levels off as the rate of reaction is constant because every enzyme is working with a substrate molecule and cannot break it down any quicker so the rest of the substrate molecules have to wait in a queue to be broken down, therefore there assuming that the concentration of catalase is constant, no matter if you keep adding substrate molecules the enzyme will only be able to breakdown a certain amount per second once it has reached Vmax.
The explanation for this is quite straightforward. When the enzyme and substrate are first mixed, there are a large number of substrate molecules. At any moment, virtually every enzyme molecule has a substrate molecule, which it attaches to through substrate via the collision theory, the substrate molecule will attach itself to the enzyme’s active sites which contain the R-groups wit these groups the active site and substrate bond together by ionic interactions, hydrophobic interactions, hydrogen, and in some reactions, covalent bonds. The rate of reaction at this point will depend on how catalase molecules there are and the speed of which catalase can breakdown hydrogen peroxide into water and oxygen, release it and start again by successfully colliding into another substrate molecule via the collision theory and so on.
However as more hydrogen peroxide is converted into water and oxygen, there is less substrate molecules to bind with the catalase. Enzymes may be waiting for a substrate coming to the end of the reaction.
This can be illustrated by this graph, which is what I expect my graph to look like. At the beginning of the reaction the graph is at its steepest as the reaction is happening quickly because there are many hydrogen peroxide molecule for the catalase to break down and a lot of oxygen is collected, but the number of substrate molecules get fewer and fewer and so the reaction get slower as the enzyme has to wait for the substrate molecule, this is why the graph gets less steep after some time. At the end of the graph the curve almost levels of because at this point there are no more substrate molecules to be broken down so there is no more gas being collected so it is constant.
In this experiment there are certain variables that should be kept constant and some that should be changed. Below I have shown in the table the variables that should be changed and the reason for the change. Cm3
To make sure that the final experiment is well thought of and planned I decided to do a preliminary experiment which will enable me to complete the final experiment to the best accuracy and precision, which I may not have thought of beforehand, which could lead to the experiment not being a fair test, leading to imprecise results. My preliminary experiment is very similar to the actual experiment apart from the things that I learn that I need to improve on.
Safety is a key issue that must be considered prior of proceeding with the experiment. If these certain safety rules are not considered they could consequence in inaccurate results due to being unprepared, you have a good chance of wasting time and making a mistake. As well as this you are being hazardous to the rest of the class as well yourself while performing the experiment. Below are the rules that you should comprehend:
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Before proceeding with the experiment
Keep your lab bench organized and free of books and other clutter
Know how to use the eyewash, fire blanket and first aid kit
Pay close attention to any information give at this time
Ask questions if you are not sure of anything
- Long hair and bulky clothing are dangerous in the lab
There is a chance of catching fire, as well as being drawn through chemicals
Wear appropriate clothing
Tie long hair back
- Rings, watches, and jewellery are dangerous in the lab
Corrosive or irritating liquids may get underneath a ring or watch and produce irritation
Dangling jewellery may catch on a piece of labware and cause an accident
Should be worn to avoid any chemicals getting in the eyes, inform teacher if this does occur IMMEDIATELY!!
Glassware should be handled with care avoid leaving it near the edge of the table.
Notify the teacher if any glass is broken, it should be disposed of in and appropriate container
Sharp instrument such as the scalpel should not be used in any other activity apart from when needed
Often labs involve the use of chemicals. Please be careful to keep chemicals off your skin and out of your mouth and eyes. If you get any chemical on your skin or in your eyes, INFORM your teacher immediately!
Hydrogen Peroxide is an oxidizing agent and extremely hazardous substance because it is corrosive. Not flammable but may start fire in contact with organic materials. Avoid skin contact, serious burns may result.
Below is a table of the equipment that I have chosen to use for the experiment, I have commented why I have chosen that particular piece:
I will use a simple technique to find out the rate of reaction using varying concentrations of hydrogen peroxide.
- Make sure the work surface is clear from books and clutter
- Set up the equipment like shown in the diagram below
- Make all the concentrations, which are shown in the dilution table, using one 5ml measuring cylinder for the distilled water and the other 5ml measuring cylinder for the 2 mols hydrogen peroxide and pour into each boiling tube and make sure to label them
- Collect one of the boiling tubes with some hydrogen peroxide in it and place it under the bung firmly
- With the 5ml syringe collect 5ml of catalase making sure there are no air bubbles in the syringe
- Insert the syringe in the hole of the bung and release it in to the boiling tube
- Be prepared with the stopwatch to start it as soon as the first bubble reaches the pipette
- Every 30 seconds read the pipette and record it into the table on the next page, continue this for the next minute
- Repeat the steps 4-8 for all the different concentrations
Also the whole experiment has to be done twice to ensure a fairer set of results, which can be found by finding the average of the two results and then plotted into a graph.
Here is a diagram of how I will set up my equipment:
Once I have completed the experiment I will record the data in a table like below:
Below are the results of my experiment, which I have organized into this table and worked out the average of both experiments:
From this table it is not possible to see how the result look visually and whether they are reliable or not and if they are close to the trend lines or not so I have decided to plot my results in a graph like below: I have plotted concentration of hydrogen peroxide (H202) solution against the time (seconds)
My experiment was very successful well considering there were only a few anomalous results when I constructed my graph. However I still feel that my results could have been closer to the line in my graph. This means that I will now need to assess the maximum possibility of the error of the apparatus I used as well as other errors which would affect the whole set of results, this is called a continuous error, an example of this is change in temperature. Where as on the contrary you could also get errors, which affect one result causing an anomalous result, this is called a single error, an example of this is an incorrect mixing of concentrations due to error reading the values. It is these sort of errors that I could improve on to ensure the accuracy of the experiment and hence the results.
My method was very accurate, as I followed the procedure very easily; and it was very easy to understand. The method was a step-by-step process, which made it difficult for any human errors to occur. The main errors occurring in my experiment were due to inbuilt errors, which I have shown in the tables on the next pages, and parallax errors. Parallax errors occur due to the equipment being slanted and therefore measurements were read wrong. Also there were major errors, which are errors that occurred in more than once and minor errors, which only occurred once. These are also shown in the table on the next pages. Those highlighted in blue are single errors, which affect single results causing anomalous results and those highlighted in red are continuous error which affect the whole set of results.
I have put the errors of the experiment in a rank according to which would affect the results more that the others to the errors that would affect the results by a small amount.
- Enzymes: http://www.ultranet.com/~jkimball/BiologyPages/E/Enzymes.html
- Cambridge Advanced Sciences Biology 1
