Investigating how the Concentration of a Substrate affects the Rate of Activity of immobilised Catalase

GCSE Biology Coursework:

Investigating how the Concentration of a Substrate affects the Rate of Activity of

immobilised Catalase

Planning:

“Catalase”

Catalase is an enzyme; enzymes are protein based biological catalysts (substances which increase the rate of reaction but don’t take part in it themselves). Catalase is found within many living cells, and is used to break down hydrogen peroxide (highly toxic) into water and oxygen (more useful substances). One molecule of it can deal with six million molecules of hydrogen peroxide in 1 minute.

Formula: Hydrogen Peroxide Water + Oxygen

2H2O2 2H2O + O2

Enzymes are also found speeding up reactions in our bodies as well as in industrial processes.

There are 2 primary types of enzyme:

Intracellular enzymes, which control reactions that occur within cells.
Extra-cellular enzymes, which control reactions that occur outside cells (for example, digestive enzymes work outside cells in the gut).

Such examples are:

Amylase which turns starch into maltose.
Lipase, turn fats into fatty acids and glycerol.
Proteases, such as pepsin, turn proteins into amino acids.

To do this, Enzymes help to break down the substrate, the substrate being a molecule of the substance that the enzyme is designed to break down, and turn it into the product.

To achieve this, each enzyme has an active site, which has a unique shape into thus only a substrate of the exact same unique shape can fit.

When this substrate fits into the active site it forms an enzyme-substrate complex. The substrate binds into this site, is held by a particular charge pattern, reacts and then leaves as the product. This process is called the “lock and key theory”

Diagram:

Factors to consider when using enzymes:

pH level: Each enzyme has an optimum pH level; this level can vary between the enzymes however. Some enzymes prefer a more acidic ph environment such as within the stomach as it has hydrochloric acid residing within it. Therefore the optimum pH level for these enzymes is 2. Others prefer a whole range from neutral at 7 to a more alkali Ph at around 9.

Graph:

Concentration: If you have more enzymes, more active sites are made available to react with the substrate. So the rate of reaction increases and therefore it is directly proportional to the enzyme concentration when the substrate is in excess.

Temperature: This is a factor of collision theory; as you increase the temperature, the rate of reaction increases also. This is because heat energy is given to the enzymes and the substrate, which causes more collisions between them. The optimum temperature for an average enzyme is 37oC. Enzymes stop working if the temperature rises above 40ºC; this is due to the enzyme’s active site changing shape. And therefore it is unable to work properly, as the substrate can no longer fit the active site and therefore the enzyme becomes “de-natured”

Graph:

“Denatured”

“Denatured” is where the bonds that hold enzymes together are broken due to them being quite weak, this causes the enzyme and therefore the active site , to deform. Certain environmental conditions can procure this such as the pH level and the temperature as described above.

“Immobilisation”

This occurs when enzymes are attached to a suitable support.

e.g.

Immobilised glucose (isomerase)

used in the making of high fructose syrups, this enzyme can be used continuously for over 1000 hours at temperatures between 60oC and 65oC

This obviously has many advantages;

They have increased stability – many enzymes become less sensitive when immobilised.

They are easily recovered and reused – a feature of great economic importance.
They can be readily collected from the process.
They have more favourable catalytic properties – in other words, they are able to operate efficiently in an extended range of reaction conditions.

Preliminary Work:

The effect of temperature on enzymes:

We placed 4 test tubes in differing temperatures and every 2 minutes we put a glass rod into the tubes and then placed this in iodine to test for starch.

We found that the test tube at 35 degrees worked best and did not denature as some did.

The effect of Ph on pepsin:

We put 2cm of egg-white into each test tube then;

1cm of pepsin into the first test tube (A)

3 drops of HCL into the second (B)

1cm of pepsin and 3 drops of ...

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The effect of temperature on enzymes:

We placed 4 test tubes in differing temperatures and every 2 minutes we put a glass rod into the tubes and then placed this in iodine to test for starch.

We found that the test tube at 35 degrees worked best and did not denature as some did.

The effect of Ph on pepsin:

We put 2cm of egg-white into each test tube then;

1cm of pepsin into the first test tube (A)

3 drops of HCL into the second (B)

1cm of pepsin and 3 drops of HCL in (C)

1cm of boiled Pepsin and 3 drops of HCL in (D)

We found that only test tube (C) worked – it changed from a cloudy solution to a clear solution proving that the conditions were correct for the egg-white to be broken down.

The effect of Catalase on Hydrogen Peroxide:

We placed 10ml of H2O2 into 5 labelled test tubes;

Tube A had boiled potato

Tube B had raw potato

Tube C had orange juice

Tube D had Yeast

Tube E had liver.

Then we immediately lit a spill then blew it out and placed it in the 5 tubes to see if the flame would relight. If it did, oxygen would be present – proving that the Catalase was working as it breaks down the hydrogen peroxide into hydrogen and oxygen.

Test tubes B, D and E worked:

Tube B worked due to the potato being uncooked and the fact that it has enzymes residing within it and tube D worked because Yeast contains catalase. Tube E worked because liver is found in the stomach where catalase is present.

Prediction

I predict that as you increase the concentration of Hydrogen Peroxide, the rate of reaction will increase; this is due to more H2O2 being able to react with the catalase and the substrate and enzyme will be able to combine quicker. Therefore more O2 will be produced so the bead will rise more quickly.

I predict that the bead does this due to H2O2 diffusing into it; it goes into the active sites of the catalase. A reaction occurs, producing O2 and H2O. The H2O dissipates into the surrounding solution and the O2 sticks onto the surface of the bead, making the bead lighter so the bead rises.

Prediction Graph:

Variables:

Independent (the variable that will be changed throughout the experiment:)

I will change the concentration of substrate (VOL).

Dependant (the variable that will be measured:)

We will be measuring the time taken for the bead to reach the surface (seconds)

Controlled (the variables that will remain the same. I will keep the following the same:)

Temperature

As temperature increases, molecules move faster (kinetic theory). The H2O2 will react with the catalase and they will form into the lock and key position quicker. Therefore, more O2 will be produced; resulting in a greater rate of diffusion so the beads will reach the surface quicker. It is the opposite if the temperature decreases. This would mean the test is not fair.

Action: I will carry out the investigation at room temperature, which is the optimum condition for catalase.

Volume of H2O2 column (10ml)

If the columns have different heights and widths, the beads will either have longer or shorter to travel to the surface therefore this will not be a fair test.

Action: Using the same 10ml measuring cylinders will allow this variable to remain constant.

Concentration of Catalase and Alginate in the Bead

When there is more catalase than there should be there will also be more active sites. More H2O2 will go into the bead, and more oxygen bubbles will be released. This results in the bubbles reaching the surface quicker therefore and unfair test. And the opposite would happen with less catalase (the bubbles would rise slowly), and therefore again an unfair test.

Action: I must ensure alginate and catalase are combined accurately.

Size of Bead

A large bead would allow more H2O2 to enter so that the reaction will be fast and the bead will rise quickly. A smaller bead would not allow much H2O2 to enter so the reaction will be slow and the bead will rise slowly.

Action: when creating the beads with the syringe and when releasing beads via the forceps into the H2O2 column I must use the same amount of pressure; to ensure that the beads will come out the same size. If there are abnormal beads created, I will choose roughly the same size beads from the batch.

pH Level

Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters its shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in the enzyme denaturing and a slower rate of reaction.

Action: I will use the litmus paper to indicate the pH is the same every so often.

Preliminary Experiment

Safety:

Hydrogen Peroxide is an irritant and could blister or redden the skin.

Therefore safety goggles must be worn during the investigation to prevent chemicals from damaging eyes.
Care should also be taken whilst handling the chemicals.
Wash area that has been in contact with any chemicals after the experiment.

Method:

I sucked up-using a syringe, 1ml of catalase and 9ml of sodium alginate and shook it to mix the two. I then made up a beaker of 200ml of CaCl2 and then I gently released the contents of the syringe drop by drop into the beaker; making sure that the drops were of even consistency and size.

I then filtered out the contents of the beaker using a sieve, making sure no beads escaped. After washing the beads with distilled water, I took 3 measuring cylinders and measured 10ml of each H2O2 concentration (1, 5, and 10 VOL) into each cylinder. To actually create this concentration, we had to pour the correct ratio of the hydrogen peroxide to distilled water for example, 1ml Hydrogen Peroxide, 9ml distilled water, for the 1 VOL reading.

I then took a single bead using the forceps and dropped it into the measuring cylinder with the concentration I was testing. When the bead hit the H2O2 I had to start the stopwatch, and then stop it when the bead re-surfaced.

Results:

Time taken for bead to reach Concentration of H2O2 (VOL)

surface of H2O2 (sec)

18.3 1

11.5 5

06.2 10

Conclusion:

As I predicted, the concentration of hydrogen peroxide increases the time taken for the bead to rise decreases.

Modifications:

I will test a larger number of concentrations as to ascertain more accurate results and I will also test each concentration 5 times so I can be more accurate again and to get the average rate-this will help to eliminate any anomalous results. I will use concentrations at intervals of 2 but will start at 2 rather than at 0, as such a concentration will get me no results.

e.g. 2, 4, 6, 8 and 10 VOL

Actual Experiment

Apparatus List:

1 x Sieve

1 x Stopwatch

1 x 10ml Syringe

5 x 10ml measuring cylinders

1 x 200ml Beaker

1 x Forceps

1 x 200ml Calcium Chloride solution

1 x 1ml Catalase

1 x 9ml Sodium Alginate

5 x Hydrogen Peroxide (2, 4, 6, 8, 10 VOL)

1 x Distilled water dispenser

Diagram:

Method:

Suck up-using a syringe, 1ml of catalase and 9ml of sodium alginate and shake it to mix the contents. Then get a 200ml beaker and add to it 200ml of CaCl2. Then gently release the contents of the syringe drop by drop into the beaker; Making sure that the drops are of even consistency and size.

Then filter out the contents of the beaker using a sieve, making sure no beads escape. After washing the beads with distilled water, take 5, 10ml measuring cylinders, in each put the following solution combinations;

Concentration H2O2 _ Distilled Water

2VOL 2ml 8ml

4VOL 4ml 6ml

6VOL 6ml 4ml

8VOL 8ml 2ml

10VOL 10ml 0ml

Then take a bead using the forceps and drop it into the column with the concentration that you desire. When the bead hits the surface of the H2O2, start the stopwatch, when the bead rises to the surface stop it. Repeat the results 5 times for each concentration and at the end work out the average time in seconds. Repeat this for the next 4 concentrations and compare the results.

Results:

The anomalous results are generally only in the first concentration (attempts 1 and 4) – I determined these by saying that any result with a more than 0.5 second margin, is anomalous.

Another table to show the rate of reaction, which is what, is actually being measured.

Analysis:

From the results, I can say that my prediction was correct; I predicted that as the bead is sinking, H2O2 diffuses into the bead, and goes into the active sites of the catalase. A reaction occurs, producing O2 and H2O. The H2O disperses into the H2O2 surrounding it and the O2 sticks onto the surface of the bead, making the bead lighter therefore the bead rises.

I also stated that as you increase the concentration of Hydrogen Peroxide, the rate of reaction will increase; this is due to more H2O2 being able to react with the catalase and the substrate and enzyme will be able to combine quicker. Therefore more O2 will be produced so the bead will rise more quickly and vice-versa.

These predictions have been clearly proven; because the averages increase from the lower concentrations to the higher concentrations within the results tables and can be seen easier in the graphs. For example, the average of the 2VOL concentration is 18.6, and the average for the 10VOL one is 3.7 – a difference of 14.9 seconds.

When looking for trends in the results, I can see that as the concentrations get higher, the time taken for the beads to fall and rise decreases: from 2VOL – 6VOL the difference of time is around 5 seconds where as from 6VOL – 10VOL, this difference goes down to around 2 seconds.

When studying the graphs, the best-fit lines are completely different. The graph comparing the “concentration of H2O2 and the average time taken…” has a negative correlation gradient and decreases as the concentration increases; proving that that the reaction will be faster in a high concentration of H2O2: Due to more H2O2 being able to react with the catalase and form into the lock and key position faster: therefore more O2 will be produced so the bead will rise quicker.

And the graph comparing the “Rate of reaction against the concentration of H2O2” has a positive correlation gradient and increases as the concentration increases. This proves again how the reaction will be quicker in a high concentration of H2O2.

To conclude: what has happened in the experiment was that the bead entered the H2O2 and sank, As the bead sank H2O2 diffused into the bead, and went into the active sites of the catalase. A reaction occurred, producing O2 and H2O. The H2O dispersed out of the bead and the O2 stuck onto the surface of the bead in the form of bubbles, making the bead lighter so the bead rose.

Evaluation:

I regard this experiment to have run efficiently giving reliable results. This was because we repeated each concentration 5 times, and we recorded each of the results to 1 decimal point. The beads were also made carefully as to not contaminate them so that they would work properly.

I came up with 2 anomalies in my final results but previously in my preliminary trials, I came up with many more. They varied to about 10 seconds at first, but then I discovered that the beads had been contaminated with oxygen before their testing so we fixed that. Another problem was that sometimes the beads did not rise at all and others stayed at the bottom for a long period of time. Some of this was also due to contamination but I deduced that another reason was that the final sinking point was lower than the base of the measuring tube so that they would stay on the bottom: Where as if we had used a taller measuring tube then this would not have occurred.

The results themselves are obviously not entirely accurate as they differ each time for one concentration but to try and eliminate outliers, I did 5 readings and worked out the averages. These final results I think are much more accurate and my graphs seem to prove so, but there is a discrepancy at the 6VOl reading, this causes a “bump” in my curve unfortunately.

The reasons for such anomalies are the following;

Human reaction time for the stopwatch

Improvement: Use some sort of sensor equipment that can detect when the bead hits the surface of the H2O2 it will start a timer, and when it re-surfaces, it stops the timer.

The syringe contents may not be mixed very well

Improvement: Use some form of mechanical mixer; that will mix the solutions far more effectively.

The beads may have varied in mass, size and shape

Improvement: Use a mechanical regulator that ascertains whether the bead is of the right mass via weighing it and uses a form of sensor to detect the size and shape of the bead.

The H2O2 becomes less and less concentrated as you test each bead so the beads may rise and fall at different rates.

Improvement: Re-fill the measuring column with the H2O2 after each bead is dropped into it.

The beads may be released at differing heights from the surface of the H2O2 solution. (so some beads may have a higher/lower speed when hitting the surface, meaning that they may be faster/slower).

Improvement: Use a form of delicate clamp that is set at a certain height as to keep the height constant.

Whilst these improvements would cause the experiment to be rather more accurate, the majority would also be expensive and in effect a waste of money. You need to look at the experiment in context of what you are trying to achieve and it what environment: in this case to prove a point not a specific result and in a school rather than a research lab, so they do not really need to be made.

I could increase the number of concentrations I do, so that I do every 1VOL rather than 2. But I would probably not go past 12 VOL as I can see that the best fit line will eventually turn into a horizontal straight line. This is because, even though there are still plenty of particles left, all of the active sites have been filled so there are no free ones left. You could also increase the number of readings you take for each concentration, but to make it even better, you could try out how different enzymes respond to this experiment (with their respective substrates). And find out whether the results to this experiment are shared throughout, plus you could add other factors to find the rate of reaction with differing concentrations and temperature and pH levels.