To investigate the effect of substrate concentration on the activity of an enzyme.

Biology Investigation: Enzymology- Catalase

Aim:

To investigate the effect of substrate concentration on the activity of an enzyme.

Background Information:

 The image above shows a 3D structure of catalase from E.coli. The structure was solved using Xray Crystallography.

Enzymes are biological catalysts which speed up biochemical reactions, they lower the activation energy. Enzymes are globular proteins which catalyse metabolic reactions.

In an enzyme- catalysed reactions, the substrate binds to the active site of the enzyme. The enzyme and the substrate are held together in an enzyme-substrate complex by hydrophobic bonds, hydrogen bonds and ionic bonds. The enzyme then converts the substrate to the reaction products which are released leaving the enzyme to form another enzyme-substrate complex. The enzyme is recycled again and again. One enzyme molecule can carry out 1000 of reaction cycles every minute. Eventually the enzyme will breakdown.

Each enzyme is unique for a certain reaction because its amino acid sequence is arranged in a certain way. The active site has a specific shape so that only one or a few of the thousands of compounds present in the cell can interact with it. Any substance that blocks or changes the shape of the active site will interfere with the activity and efficiency of the

The diagram below shows the breakdown and the building up of a reaction:

There are several factors that will effect the enzyme’s activity:

Salt Concentration: If the salt concentration is very low, the charged amino acid side chains of the enzyme will stick together. The enzyme will denature and form an inactive precipitate. If the salt concentration is very high, normal interaction of charged groups will be blocked and the enzyme will precipitate. An intermediate salt concentration such as blood (0.9%) or cytoplasm are optimum for most enzymes.

pH: Enzymes amino acids contains group such as –COOH and NH2 that gain or lose H+ ions. When pH lowers, enzymes tend to gain H+ ion and eventually the side chains will be denatured. Many enzymes have an optimum in the neutral pH range, some enzymes (which act in the human stomach) requires a low pH has a optimum at low pH.

Temperature: As temperature is increased, so does rate of reactions. More molecules have enough kinetic energy to undergo the reaction. Enzymes are catalysts for chemical reactions therefore will proceed faster when temperature is increased. But when temperature is above the optimum point, the kinetic energy of the enzyme and the reactants are so great that the enzyme molecule is disrupted.

Inhibitors: Many molecules other then the substrate may interact with an enzyme. If a molecule increases the rate of reaction it is an activator, and if decreases the rate it is an inhibitor. Many inhibitors act by reacting with the side chains in or near the active site to change or block it. Others may damage or remove the prosthetic group.

For a better understanding of this topic, I discovered that for a reaction to occur, particles of the reactants involved must collide with enough energy- this is called the ‘Activation Energy’ or Ea.

For this investigation, I will be focusing my topic on ‘how the varying of the substrate concentration effects the activity of an enzyme’. I will be conducting a simple experiment, using hydrogen peroxide solutions to see how it effects the enzyme’s activity as concentration varies. Here is an example of an experiment that I have conducted through previous lessons, it is also related to the topic ‘rates of reaction’. This experiment will help me to understand the bases of the rate of reaction theory and how it is effected:

The Different Sized Marble Chip Experiment:

In the experiment of seeing how rate of reaction differs as surface areas varies, I conducted the ‘Marble Chip’ experiment, by using different sized marble chips and observing how long it takes for the reaction to end. The different sized marble chips were placed into hydrochloric acid and timed, the times were noted down and compared. From this experiment I learnt that powered marble chips reacted faster than the larger marble chips. Therefore I can justify my results and provide evidence that as you increase the surface area of a reactant you are also increasing rate of reaction.

From previous experiments, I learnt that rate of reaction can be affected with the presence of a catalyst, the varying of temperature, the surface area of the reactant and the concentration.

In this project I am going to investigate the enzyme Catalase, which accelerates the breakdown of hydrogen peroxide (a common end product of oxidative metabolism) into water and oxygen:

Hydrogen Peroxide + Catalase → Water + Oxygen + Catalase

2H2O2 (aq) + Catalase → 2H2O (l) + O2 (g) +Catalase

Catalase is extremely important in the cell because it prevents the accumulation of hydrogen peroxide. Catalase is found in animal and plant tissues and is especially abundant in plants storage organs such as potato tubers, corns and fruits.

Hydrogen peroxide is a topical antiseptic. When you get a cut, all the bacteria that live on your skin see a new source of nutrients. If bacteria invades before a scab forms, your immune system may not be able to kill them all. Pouring hydrogen peroxide on a cut, then, is an attempt to ill a bunch of bacteria. It will also kill some of your cells. This is why Hydrogen peroxide is sometime used as an antiseptic, but as diseases and infection become better understood, cytotoxic methods are used less frequently. This is because of antibiotics and therapeutics.

Hypothesis:

I predict that as the concentration of hydrogen peroxide increases so does the rate of enzyme-substrate complex being formed, therefore there will be more Oxygen (gas) produced and the also the rate it is produced. I predict that if the concentration of the hydrogen peroxide solution is increased the time taken for 10 cm3 of oxygen to be collected will decrease. The rate of reaction in other words will increase. I predict that as the concentration of hydrogen peroxide doubles, the amount of gas produced also doubles and the rate of reaction also doubles.

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Hypothesis:

Linking Hypothesis to Theory:

The collision theory states that as concentration in a solution increases, so would rate of reaction. This is because as you increase the concentration, you are increasing the number of molecules and particles therefore there will be more particles and successful collisions. So by increasing the concentration you are increasing the number of collisions per unit of time.

Therefore this explains why an increase in concentration leads to an increase in rate of reaction. If a substrate solution has a high concentration there will be a large number of the substrate particles in a certain volume then there is more chance of the Catalase enzyme colliding with a H2O2 molecule. A higher concentration of hydrogen peroxide will mean more of its particles being broken down by the rate of reaction occurs and oxygen collects more quickly in the gas syringe.

Apparatus:

Hydrogen peroxide 100cm3

Liver

Gas Syringe

Beakers

Stop clock

100cm3 water (distilled)

Bung and Delivery tube

White tile

Knife

Weight

Tweezers

Disposable plastic gloves

Measuring Cylinder

Test tube holder

Goggles

Labels

Thermometer

2 Stands

Diagram of apparatus:

Concentration Range:

Method: Safety regulations are checked before experiment starts. The apparatus and the reactant and Catalase are collected and set up. The different concentrated solutions of hydrogen peroxide are measured out by using a measuring cylinder and they are diluted with water depending on the decided concentration that will be investigated. I will check again to see that all the solutions are the same in volume. The Liver (containing the enzyme Catalase) is cut into small identical sized pieces with a knife (using a white tile to cut on) and weigh to make sure the same quantity of liver is being use (0.2g). When all the liver and solution has been cut and measured out accordingly, I will pour the solution into a beaker and use a thermometer to measure its temperature, I will make a note of it. I will then set out the apparatus as shown in the diagram. The liver will be put into a test tube and held by a stand, the stop watch is then zeroed and started once the measured hydrogen peroxide is poured in. The bung is placed immediately on the mouth of the test tube. I will observe the reaction until 10 cm3 of oxygen gas has been collected in the gas syringe, then I will take note of the time for it to be produced. I will check the temperature in the room constantly as well as checking the temperature of the reaction, if there is a change I will explain it in scientific terms (e.g due to the exothermic reaction, therefore thermal energy was given out, therefore it increased rate of reaction) and explain how it altered my results.

I will repeat this for the other different concentrated solutions, making sure it is as fair as possible. After completing the experiment, I will repeat it for a second time, to make sure the second set of results agree with my first, if it does not, I will repeat the experiment for a third time.

I must bear in mind to keep the factors that will effect rates of reaction constant. Therefore I must be aware to keep the temperature constant and the surface area of the liver constant.

Regulations to keep it as a Fair Test:

I will keep the volume of the concentration of hydrogen peroxide the same. The only variable will be the concentration.
I will keep the bits of liver the same.
I will check the temperature of the hydrogen peroxide constantly, to ensure that it is the same throughout the experiment.
I will use distilled water to ensure that other soluble minerals/ substances do not affect the results of the experiment.
I will start the stop watch once I put the liver in, to keep it fair.
I will wash the conical flask after every change of concentration, to get accurate result.
I will repeat the experiment at least two times to ensure my results are as expected, if the first two sets of result differs dramatically, I will repeat the experiment for the third time and work out an average from my results. This ensures consistency and it allows me to spot anomalous results.
When measuring out quantities of liquids, the reading must be read off at the base of the meniscus of the liquid.

Accuracy:

I will be repeating the experiment a second time to justify and back up my first results. If the first two set of results differs dramatically, I will be repeating my experiment for a third time. And depending on my results, I will be either working out an average from all my obtained results or simply select the two that are the most similar and work an average from them. The readings I will be taking from the stop watch will be to two decimal points to keep the results as accurate as possible.

Safety Precautions:

Goggles will be worn during the experiment- protecting the eyes from any splashes of chemicals.
Hair will be tied back to prevent contact with chemicals.
Chemicals will be handled appropriately.
Chemicals will be disposed off carefully.
If for whatever reasons, the chemicals come in contact with skin, then wash and rinse hand thoroughly with cool tap water.
After an experiment, wash and cleanse hand thoroughly.
Listen to teacher instructions. If advised not to do a certain thing, then follow the given advice.
Always behave accordingly in a lab, do not run during an experiment or with equipment.
Concentration is acquired to the maximum during an experiment. Always be alert for any unexpected accidents.
Hydrogen Peroxide must be handle with care, as it is a bleaching agent.

Table of result:

This will be the table where I will store my results in. I have displayed the range of concentration I will be experimenting with in the table below.

Obtaining:

I carried out my experiment accordingly to my plan and check that I am following my safety and fair test regulations. I discovered that there were some things that I will have to change to ensure that my experiment was as fair as possible.

I used a variety of differently sized measuring cylinders rather than having one big cylinder (which was very difficult to measure out small concentration), a small was later required to measure out the small concentrations.
I also used different cylinders for the different reactants, I used one cylinder to measure out the volume of distilled water and another cylinder for measuring out the hydrogen peroxide.

Observations:

I am measuring the time in which 10cm3 of oxygen is produced when a piece of liver is placed in differently concentrated hydrogen peroxide and water solution. The time in which 10cm3 of oxygen is produced will be timed by a stop watch, the oxygen will be measured by using a gas syringe.

Results:

I followed my method and carried out the experiment three times to justify my first two sets. After obtaining the results, I displayed my results into tables. Below is the table of results, which displays the time taken for 10 cm3 of oxygen to be produced.

From looking at my results, I need to display it onto graph, but to be as accurate as possible I will need to find an average from the times. I selected two times that were closest to each other and worked an average from them. (The times typed in italic bold were not selected.)

I measured the results to the nearest two decimal points to keep the results as accurate as possible. After working the average I will know need to plot my results onto graph.

Analysis of Results:

The graphs shows that as the concentration of Hydrogen Peroxide decreases the rate of reaction decreases and the time taken for the reaction to finish increases. The faster a reaction take place the smaller the time taken for the reaction to finish. My graph reflects this ‘concentration theory’, as the solution becomes diluter, there are less reactants for the Catalase to form enzyme substrate complex with, therefore rates of reaction decreases.

This causes the difference in rates. Also the reaction is exothermic therefore heat was produced during the reaction, therefore rates of reaction is increased. As temperature is one of the factors that affect rates of reaction, this alters the results by varying the rate. Therefore makes the results inaccurate in terms of this.

I have controlled the pattern and trend by using graphs, as you can see from my graph that as the concentration is diluted, the slope becomes steeper, indicating a slower reaction and a lengthy time for the reaction to complete. But if the solution becomes more concentrated then the slope will become more shallower, indicating a smaller time for the reaction to complete.

I found out that as I double the amount of concentration, the time taken for 10cm3 of oxygen to be produced is halved. E.g. 196.82s was required to produce 10cm3 of oxygen with 1 cm3 of Hydrogen Peroxide. And 329.34s was required to produce 10cm3 of oxygen with 2 cm3 of Hydrogen Peroxide. But because rate of reaction is not doubling at a constant rate, the curve displayed on the graph will be steep towards the concentrated end and shallower towards the dilute end.

In dilute solutions, there are less particles therefore this means less successful collisions. This is because there is a large space between the particles, therefore collisions are not frequent. I noticed that the difference in speed becomes smaller as concentration is increased. This is because there are a lot of particles colliding to have a major difference.

Also I noticed that as concentration is increased, less time was required for 10 cm3 of oxygen to be produced. This is due to the fact that there are less reactants to react with therefore less products are produced per second. My results agrees with my prediction ‘as the concentration of hydrogen peroxide increases so does the rate of enzyme-substrate complex being form…’. Therefore I can conclude that increasing the concentration also increases the rate of enzyme-substrate complex produced per second.

Conclusion:

The reaction was exothermic therefore heat was produced in the reaction, hence temperature was changed during the experiment. Therefore it altered my results, as temperature is a factor that effects rates of reaction. Increasing the temperature means providing the reactants particles with energy to move faster, therefore successful collisions is more likely. Therefore more reactants are colliding with the enzymes therefore more oxygen was produced. This was unfair on the diluter solution as there was little heat produced, therefore it was a slower reaction.

There was one obvious fact…that concentration also effect the activity of an enzyme, the more reactants there are, the faster is produces and form enzyme-substrate complex.

As shown below, particles are colliding more often as there are little space between them. Therefore there are more successful collisions.

From my evidence, I have proven that rates of reaction increases as concentration is increased. Therefore I have obtained results that shows me that reactions with a higher concentration finishes earlier than the reactions with a lower concentration. Therefore I come to conclusion that my prediction was correct and that as you increase the concentration you are increasing the rate of reaction. I also conclude that if you double the concentration of a solution in an experiment, you are also doubling rate of reaction. The graph shows that if you double the concentration the rate would also double. If you increase the number of particles in the solution it is more likely that they will collide more often.

Also, the surface area of the liver was not reliable and accurate enough, it was difficult to keep the surface area of the bits of liver equally the same. It was impossible to manually cut them exactly the same, so this will explain the anomalous results.

Evaluation:

The procedure used was appropriate to a certain extend. It tested my hypothesis and prediction. The evidence obtained was reliable, therefore it backs up my hypothesis that, ‘as the concentration of hydrogen peroxide increases so does the rate of enzyme-substrate complex being formed, therefore there will be more Oxygen (gas) produced and the also the rate it is produced’. My results shows this, so therefore it must be valid. The aim of this investigation was to investigate the effect of different concentration on the enzyme Catalase, graphs was drawn to show how rate of reaction varied as concentration varied.

The accuracy of the observations and measurements were valid and backed up my prediction. It reflects the concentration theory. There was some anomalous results, as shown n my graph. The anomalous results could be explained by:

There was a small change in temperature within a small period of time that I didn’t observe it.
There was a human error in the beginning and at the end of the experiment.
The measurements were not accurate.
There might be some solution still left in the cylinders and were not entirely emptied out.
The surface area of the liver was different.
I noticed that as the test tube bung was pushed in, it causes a slight pushing of air inside the gas syringe. Causing the reading to be wrong.
Also as it was an exothermic reaction, the temperature of the solution changed, altering the results.
Sometimes during the experiment the solution will froth into the gas syringe, causing a mess and an inaccurate reading.

A fair test was carried out as shown in the planning. The ‘fair test’ regulation was taken at the beginning, during and at the end of the experiment to ensure the results were fair. The same volume of hydrogen peroxide solution was used for each experiment and the same amount of liver was used. The only thing I changed was the concentration of the Hydrogen peroxide. I also checked the temperature of the solution at the beginning of the experiment and ensured that it remained the same throughout the experiment. The stop watch was started when the solution was poured into the test tube with the enzyme in it; and it was stopped once 10cm3 of oxygen gas was produced. Therefore a fair test was carried out and hence my results were reliable.

To improve and check the results, I had repeated the experiment several times to ensure consistency and to ensure that my results are similar and valid and that there were no major differences. To improve my results further, I could repeat the experiment for a fourth time and work out an overall average for all my results- this will make my results more accurate.

There are some things that I had found hard during the experiment and it may be because of this that there are differences between my obtained results. I found it was difficult:

I can’t really see through the goggles as they were scratched. Also I discovered that my vision was reduced by the goggles, I could see better without the goggles, but I kept them on because of the hazards.
It was hard to pour out all of the solution, some small amount of the solution remained inside the cylinders and I couldn’t empty it all out.
It was difficult to cleanse the inside of the test tubes.
It was difficult to get the inside of the test tubes to dry thoroughly, some water droplets remained within. This may have diluted my solutions.
It was hard to time exactly when the Hydrogen peroxide comes is contact with the enzyme and it was hard to stop the clock at the exact point of when 10cm3 of oxygen gas was produced. The speed of each individual experiment varied, therefore it was harder to time the faster reactions.

Although these problems I have encountered exists, they are minors and does not effect my conclusion in anyway, therefore I am confident that my results are sufficient enough to be reliable. But to ensure a better obtaining of results next time, I will need to figure a way to minimise these problems.

To improve the experiment further, I could monitor the temperature throughout the experiment and repeat any experiment that results do not fit in with my rest. I could:

Use a conical flask with a divider rather than a test tube, this method will allow me to start the experiment quickly and accurately.
Use a burette to measure small volumes accurately.
Because there is a human error due to the small delay of starting and stopping the stop watch, as I had to pour the solution and start the clock at the same time. This could be minimised by asking a someone to start the clock for me. There will be less human error and more reliable results.
I could use a computer to plot out my graphs for me- therefore I can analyse it more accurately.

If I had more time, I would have researched further into the issue by using the Internet and other sources of information (Library). I will use my improvements to make the experiment better and repeated it to get more reliable results. I would have done some independent research and also plot my graphs out on the computer to display my results clearly.

I will make addition work to research further about rate of reaction and enzymes. This will provide me with additional evidence to back up my conclusion. Also I will do dome more experiment to extend my enquiry about rates of reaction. I will research and perform experiments around these areas:

How the activity of the enzymes and rates of reaction are affected by:

Temperature
Surface area
Physical state of the reactants
Applying pressure

To back up my prediction, conclusion and obtained results, I will conduct other experiments that are about the activity of an enzymes. I will do an experiment on how temperature affects the activity of an enzyme. The theory is that as you increase the temperature, rates of reaction will increase. If I increased the temperature by 10°C then rates of reaction doubles. The reason why is because the activation energy Ea is increased as particles moves faster. To see if this theory is true I will do the same experiment. But this time I will keep:

The volume and the concentration of hydrogen peroxide constant.
The surface area, mass of the liver the same (using the same quantity of Catalase)

This is because I will not be experimenting the affects of rated of reaction as concentration varies; I will be looking at temperature. These are the factors I will change:

I will change the temperature- varying it depending the selected range.
I will be using a water bath to help me vary the temperature.
I will be using ice, warm and hot water.

Although this experiment seems safe, there are risks if the experiment is not carried out probably. For example I don’t want to boil the acid as it could be perilous. So my safety precautions will still apply. I also have to consider the fair test regulations and so forth.

Here is a diagram of my apparatus for this experiment:

Here are the range of temperature I will be using:

0°C
10°C
20°C
30°C
40°C

Readings will be taken every three minutes into the experiment. The results will be analysed by using graphs to compare and justified whether it backs up my prediction. To back up my results I will repeat the experiment again.