How does catalase work?

Introduction/How does catalase work?

Our brief for this experiment was;

“To see how concentration of a solution affects the enzyme activity.”

To perform this practical experiment, we are going to use Hydrogen peroxide, and alginate (which is basically a yeast ball containing catalase, an enzyme). Enzymes exist in all living things. They are composed of polymers of amino acids and are produced in living cells. Each cell contains several hundred enzymes, which Catalase a vast number of chemical reactions. Enzymes are known as Biological Catalysts as they dramatically increase the rate at which reactions occur within living organisms, without being ‘used up’ or effecting the reaction in any other way. Enzymes catalysis saves the need for an increase in temperature in order to speed up reactions within living things. Such an increase in temperature would be lethal to the organism. Enzymes such as Catalase are protein molecules, which are found in living cells. They are used to speed up specific reactions in the cells. They are all very specific as each enzyme just performs one particular reaction.

Catalase is an enzyme found in food such as potato and liver. It is used for removing Hydrogen Peroxide from the cells. Catalase speeds up the decomposition of Hydrogen Peroxide into water and oxygen. It is able to speed up the decomposition of Hydrogen Peroxide because of the shape of the Hydrogen Peroxide molecule. This type of reaction where a molecule is broken down into smaller pieces is called an anabolic reaction.

The image above is the 3D structure of catalase from E. coli. The structure was solved using xray crystallography.

Catalase behaves as a catalyst for the conversion of hydrogen peroxide into water and oxygen. Catalase is an example of a particularly efficient enzyme. Catalase has one of the highest turnover numbers for all known enzymes (40,000,000 molecules/second). This high rate shows an importance for the enzymes capability for detoxifying hydrogen peroxide and preventing the formation of carbon dioxide bubbles in the blood.

The reaction

Hydrogen peroxide (catalase) Oxygen + Water

Prediction

As the concentration increases, why will the yeast ball rise quicker?

To start off with, I predict that the general flow of events will look something like this;

Yeast ball enters hydrogen peroxide
When it’s sinking though the solution, it then starts to do it’s work and it holds the oxygen on the surface, [after it’s created from the collision between the yeast ball and the hydrogen particle(s)] leaving just the water, therefore creating the divide of hydrogen peroxide into water and oxygen.
The additional oxygen gained, is held on the outer skin of the yeast ball, and as there are more and more oxygen particles joining onto the ball, it begins to defy gravity. Slowly at first, but then it gets faster as it travels up the test tube, because it’s gaining more oxygen as it goes. Finally it stops floating up any further, when it reaches the top of the liquid.
This time should become quicker and quicker, the more concentrated the solution, because if the solution is more concentrated, then it has more oxygen particles made quicker so the yeast ball can pick up more particles easier and quicker, therefore resulting in a faster time for it to get to the top.

The yeast ball will therefore rise quicker, as the concentration increases because there are more hydrogen particles to collide with the yeast balls in order to produce oxygen, for it to gather and then rise to the top with in a richer concentrated solution. This is also because, as there are more particles, the yeast ball, has more of a chance of colliding with one, so it significantly lowers the margin of error involved when you up the concentration. And with a greater chance of hitting one, it will get the same amount quicker, and rise to the top ...

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½ the concentration = 2x the amount of time taken to rise to the top,

Or,

2x the concentration = ½ the amount of time taken to rise to the top.

This sort of equation works out to mean it’s inversely proportional. In other words, the concentration is inversely proportional to the amount of time taken to rise to the top (the yeast ball), and it works the other way around too. Simply then, if you half one, you double the other, and if you double one, you half the other.

As the concentration is decreased, why will the yeast ball rise slower?

The yeast ball will rise slower, if the concentration is decreased because there is less hydrogen in the solution for the alginate beads to collide with to make oxygen, and even though it will rise eventually, there will be a much longer time to wait, as it struggles to gather the oxygen from the collisions.

Prediction graph

Apparatus

For this experiment you will need;

1x test tube

1x measuring cylinder

1x stopwatch

1x piece of filter paper

1x tweezers

1x syringe (if necessary)

15x alginate beads

A small amount of;

1%, 0.8%, 0.6%, 0.4% and 0.2% of hydrogen peroxide solution.

It should be set up as shown below;

Method

Initially, make sure all the equipment is clean and dry, then set it up, as shown above.
Measure out the first 8cm3 of hydrogen peroxide, with the syringe or the measuring flask, into the test tube.
With tweezers, pick up a few yeast balls, and place them onto a piece of filter paper to dry out, so that they do not stick to the sides of the test tube on their fall into the solution
Prepare the stopwatch (i.e. reset it to read zero seconds), and pick up the yeast ball with the tongs
Drop the yeast ball into the solution
As soon as it hits the bottom of the tube start the timer
Stop the timer, as soon the ball reaches the top of the solution
Empty the contents of the test tube into a sink, or a suitable nearby depositing area
Clean all the equipment and dry it
Repeat twice more with the same concentration to get an average
Finally, re-measure a different concentration of hydrogen peroxide into the test tube, and start the experiment all over again.

Fair test

For the fair test, I will have many different and differing factors to think about.

For example, my hand eye coordination might have varied during the practical, which may have resulted in some inaccurate timing. The volume of the alginate bead is not an issue, as it’s always around 17.18mm cubed. The question is not the volume of it, but how much oxygen it will gather and hold on its sides. This can be influenced by concentration, temperature and many other factors, but the only variable we can control in this experiment is concentration, therefore the chance of all the other factors, is going to have to be left down to science, (except for temperature, which will be kept at room temperature).

Safety

As always, goggles should be worn, when working with science equipment and solutions.

Results

Here are the results of the experiment when I performed it between the times of Wednesday the 14th to Friday the 23rd of May 2003.

Analysis

From the results table and graph, it’s quite clear to see that as you increase the concentration, you increase the rate of reaction, which ties in rather nicely with my prediction, and proves that I was correct with my presumption, that making the concentration greater, you make the time taken for the alginate ball to reach the top smaller, therefore creating an inversely proportional situation, with the two values measured. It gets quicker because, as you increase the concentration of hydrogen peroxide, the amount of hydrogen particles within the solution increase, which means there are more particles to collide with the active sector of the Catalase, and produce oxygen. This oxygen then collects on the outer skin of the alginate bead and helps is rise up through the solution. With all the oxygen going straight the alginate bead, that means it’s extracting the oxygen from the hydrogen peroxide, and leaving the water which is left after the reaction in the test tube, thus creating the divide between oxygen and water. Catalase within this experiment is displaying its true traits as a catalyst, by speeding up this reaction amazingly well.

If we look at the graph, you can obviously see, that during phase 1, the rate of reaction is increasing rapidly because more and more hydrogen particles are present; therefore increasing amounts of oxygen are being produced.

In phase 2, the rate of reaction is still increasing, but not as rapidly. This is because at 0.4, 0.6, and 0.8% concentration, there are always plenty of hydrogen particles within the solution to keep producing oxygen at a steady speed. This means that the turning point is between 0.3, and 0.4%, as to at which point the alginate beads are no longer struggling for oxygen, and therefore hydrogen particles.

Finally, at phase 3, by this time the reaction is so quick it won’t get much quicker, but it certainly isn’t going to decrease in speed, therefore a more stable, level line is formed.

Basically it’s just one continuous cycle;

Increase concentration Increase H202 particles Collision more likely

Ball rises quicker Oxygen is made quicker

Evaluation

I felt the investigation went well, and my partner and I used our time wisely. I didn’t find great parts of the experiment really testing, and I especially found the stop clock operation was very easy, however, to time the exact moment the ball hits the top or the bottom of the solution, I found increasingly difficult as the times became shorter and shorter, and the timing became more crucial. This could’ve quite possibly hampered some of my final results, and therefore might’ve produced a few oddities within the graph. Otherwise, however, my results are seemingly relatively reliable, when I compare them to those of my peers.

Three results that I am not so sure about are test 2, for 0.3, and 0.8% concentrations, and also test 2 for 0.4% concentration. To improve these possibly inaccurate results I think I should’ve used plastic tweezers, as I believe that the metal ones were too harsh on the poor innocent little yeast balls. They could have been squashed by the hard metal tweezers, altering the active sector of them, in turn making them collect less oxygen, so taking longer to get to the top, or shorter if one was particularly well handled. Another problem might have been that H202 breaks down in daylight, and it might have been left in daylight for a bit, which would, consequently make it less efficient in its tasks to collect oxygen, i.e. destroy its active sector.

Finally, the yeast balls may have been of different sizes, which would produce un even results, however, we didn’t have enough time to measure each and every yeast ball before it went in, as that would take ages, so we automatically assumed that the average size of each yeast ball was around 17.16mm3. Hand eye coordination, is obviously another factor, which would need further investigation if we had the time, or the equipment, which we didn’t.

Seeing as my predictions are correct and tie in with my results, and the results of fellow students I have spoken to, I would be confident in saying, that, yes, my results are sufficient to support my conclusions.

To provide myself with additional evidence to support my conclusions, I could have experimented with different volumes, with which to compare my concentration results with. Or, I could have introduced 2 yeast balls, and experimented with how long, that took to rise, as the 2-alginate beads have to compete for the hydrogen particles.

If I did the experiment again I would prefer better equipment, e.g. an oxygen probe with a computer to measure accurately the concentration of o2 left in the solution after the yeast ball had risen to the top, and a device able to measure accurately the volume of the yeast ball before it enters the solution. This would be speaking hypothetically, however, as it would take weeks to do all that and we have just a few 40 minute slots in which to complete the entire experiment within, so to look at it realistically, using exactly the same equipment with an extra few minutes, the first thing I would’ve done, before setting up the equipment would have been to set out a the amount of yeast balls I was going to need onto a piece of filter paper and let them dry out well and truly before dropping them into the solution, as the main problem we had was the alginate beads sticking to the sides of the test tube and getting stuck before actually entering the solution. Also, doing this would prevent any dilution of the solution by addition of extra-unknown liquid amounts from the wet alginate beads.

If I did the experiment again, I could choose a different variable/factor to change, for example;

I could change the temperature. To achieve this I would have to use a water bath, and with this method I could measure the temperature, in a much more accurate way, than simply by using a flask and a Bunsen burner. Using this method, I could also investigate at what temperature Catalase denatures, because after a certain temperature the enzyme denatures and from then on cannot function properly.
Or I could change the ph level. I could investigate the result I get from change the ph from an acid, to a neutral, to an alkali. Obviously, to keep the experiment as a fair test, I would have to make sure that the ph of solution I use was at equal ends of the ph scale, e.g.

Ph 3 (medium strength acid), then ph 7 (neutral), Then finally ph 11 (medium strength alkali) because then both the acid and the alkali would both be 4 ph from neutral, in opposite directions, as apposed using a very strong acid, but a very weak alkali, or visa versa, because that would not a fair test on one of the solutions.

Another factor I could change would be the surface area of the yeast ball. It would require some very accurate, and probably quite expensive measuring equipment, but it would be quite an interesting factor to investigate because you could involve a quantitative prediction, and then try and prove your prediction to be correct. You could try predicting, for example, that if you double the surface area of the yeast ball, then you half the rate/speed of reaction, or if you half the surface area of the yeast ball, then you end up doubling the rate/speed of the reaction.

By Ben Hibberd