HOW DOES TEMPERATURE AFFECT CATALASE ENZYMES?

Aysha Sattar, 10b, 10/12/03

Aim.

To investigate the affect of temperature on catalase enzyme activity, using a potato as a catalyst. The source of catalase is in the potato cells.

Theory.

I have done some research to find out about enzymes in general and about catalase.

Enzyme is a protein molecule that speeds up chemical reactions in all living things. Without enzymes, these reactions would occur too slowly or not at all, and no life would be possible.

Many enzymes break down complex substances into simpler ones. Others build complex compounds from simple ones. Most enzymes remain in the cells where they were formed, but some enzymes work elsewhere. For example, the pancreas secretes the enzyme lipase, which travels to the small intestine, where it breaks down fats.

An enzyme's structure can easily be destroyed by heat, or PH. For example, scientists believe that a high body temperature, such as 42 °C, may cause death because the heat makes vital enzymes inactive.

Enzymes have many uses in addition to their natural functions in the body. Manufacturers use enzymes in making a wide variety of products. For example, some detergents contain enzymes that break down proteins or fats that cause stains. Enzymes are also used in the manufacture of antibiotics, beer, bread, cheese, coffee, sugars, vinegar, vitamins, and many other products. Doctors use medicines containing enzymes to help clean wounds, dissolve blood clots, relieve certain forms of leukaemia, and check allergic reactions to penicillin. Doctors also diagnose some diseases by measuring the amount of various enzymes in blood and other body fluids. Such diseases include anaemia, cancer, leukaemia, and heart and liver ailments.

All living cells make enzymes, but enzymes are not alive. Enzyme molecules function by altering other molecules. Enzymes combine with the altered molecules to form a complex molecular structure in which chemical reactions take place. Enzymes do not undergo any permanent chemical change of their own. The enzyme, which remains unchanged, then separates from the product of the reaction. Enzymes therefore serve as catalysts. A single enzyme molecule can perform its entire function a million times a minute. The chemical reactions occur thousands or even millions of times faster with enzymes than without them.

The basic enzymatic reaction can be represented as follows:

S+E→P+E

E represents the enzymes catalyzing the reaction.

S is the substrate, the substance being changed.

P is the product of the reaction.

Enzymes owe their activity to the precise three-dimensional shape of their molecules. According to the 'lock-and-key' mechanism, the substances upon which an enzyme acts (which are known as substrates) fit into a special slot (space) in the enzyme molecule: the active site. A chemical reaction takes place at this site and the products are released, leaving the enzyme unchanged and ready for re-use. Below is a more detailed explanation.

Enzymes are globular proteins; their molecules are round in shape. They have an area - usually thought of as a pocket-shaped gap in the molecule, which is called the active site.
Some enzymes are found inside cells (intracellular enzymes), and some - especially digestive enzymes - are released so they have their effects outside the cell (extra cellular enzymes).

The accompanying diagrams are intended to illustrate a generalised account of the action of digestive enzymes.

2 (Only) the substrate (or substrates) fit into the active site.

The enzyme speeds up the process of conversion of substrates (reactants) into products - usually so much that the reaction does not take place in the absence of enzyme.
Although the enzyme obviously joins with the substrate for a short while, the enzyme and substrate split apart afterwards, releasing the enzyme. Thus the enzyme is not used up in the process (unlike the substrate(s)), so it can continue to react if more substrate is provided.

3 Within the normal range, changes in temperature, pH, and concentrations of substrate and enzyme affect the rate of reaction in accordance with predictable interactions between enzyme and substrate molecules.

- The effects of temperature may be explained on the basis of collision theory - increased temperature increases the speed of molecular movement and therefore the chances of molecular collisions, so within a narrow range (often 0-45 °C), the rate of reaction is proportional to the temperature. It is often said that an enzyme's rate of reaction doubles for every 10° C rise in temperature.

- Changes in the pH probably affect the attraction between the substrate and enzyme, so the efficiency of the adaptation process. Often, there is an optimum pH - near to pH 7 (neutral) in intracellular enzymes, and either in the acidic range (perhaps pH 1- 6) or in the alkaline range (pH 8-14) for different digestive enzymes.

- Some enzymes work better if other substances are also present. Some enzymes (pepsin - from the stomach) works better if acid is present, and some, e.g. lipases are more effective if emulsifying agents are present because they break up the substrate into smaller ...

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4 Above normal temperatures (say 60 °C); heat alters irreversibly the enzyme molecule. This denaturation is due to molecular vibrations (caused by heat) which change the shape of the protein, altering the folding and internal cross-linkages in its polypeptide chains. These changes - especially in the region of the active site - mean that the enzyme is inactivated, even when returned to normal temperature.
It would be wrong to say that an enzyme is KILLED by heat, since it is only a molecule, not an organism.
However, misconceptions of this sort are easily spread.

The higher the temperature to which the enzyme is subjected, the longer the heating is continued, the greater the proportion of damaged enzyme molecules and the result is that the conversion process becomes less and less efficient.

Below normal temperatures, enzymes become less and less active, due to reductions in speed of molecular movement, but this is reversible, so enzymes work effectively when returned to normal temperature.

Below is an optimum temperature graph it shows how the enzyme avtivity would change according to the temperature.

Enzymes are sometimes adversely affected by other chemical substances which combine with them, either at their active site or by altering the overall shape of their molecule. Many drugs and poisons have their effect in this way.

A catalase is defined as an enzyme found in blood and tissues that decomposes hydrogen peroxide in to water and free oxygen.

Hydrogen peroxide hydrogen + oxygen

A catalyst is a substance serving as the agent in catalase. A catalyst is able to increase the rate of a chemical reaction without itself being consumed or changed by the reacting chemicals. The action of a catalyst is called catalysis. Catalysts are used by chemists to speed up many chemical reactions that otherwise would take place too slowly to be practical. The exact mechanisms of catalytic actions are unknown, but some types of catalysis are thought to occur when reacting chemicals are adsorbed onto the surface of the catalyst. Good catalysts are usually highly porous solids with surface areas of several hundred square meters per gram.

Catalysts may be solids, liquids, or gases, and their catalytic activity is classified as either homogeneous or heterogeneous.

So to summarize a catalyst is a substance which changes the rate of a chemical reaction without undergoing chemical change itself. Positive catalysts increase the rate of reaction; negative catalysts (inhibitors) decrease the rate of reaction. Catalysts make it possible for the reaction to proceed by an alternative pathway with different activation energy. Activation energy is the minimum amount of energy which must be supplied to the particles before they react. Enzymes speed up many biological reactions because they lower the activation energy of the reaction.

Catalysts play an important part in many biochemical and industrial reactions. Enzymes are biochemical catalysts that enable specific reactions to take place.

Preliminary work

I decided to do some tests before the actual investigation to ensure accuracy of the results.

Firstly I decided to grate the potato and then measure out 1g of potato instead of using discs because I thought that if I used discs it would be harder to use the same mass of potato. I also decided to grate it in the small sized grater because then the surface area would increase, therefore resulting on more frequent collisions. And so there would be a greater rate of reaction.

I also decided to test out the catalase in the following temperatures; 10ºC, 30ºC, 40ºC, 50ºC and 60ºC. This would make sure there is a good range of temperature and it would be easier to conclude which is the best and worst temperature for catalysts to be working at.

I also tested to see how much of each substance to put into the flask. I put a specific volume of hydrogen peroxide and mass of potato into the same flask to see if there was enough potato to make a reaction take place. I realized that 1g of potato was not a good amount because it was a very little bit of potato, and with 10ml of hydrogen peroxide very little gas was released. I kept increasing the volume of hydrogen peroxide and potato, until I thought that 30ml of hydrogen peroxide and 5g of potato was a good amount of each because gas was released and it was easy to measure this volume of hydrogen peroxide and potato.

I also tested to see which type of equipments to use, a conical flask was necessary to allow the bung of the gas syringe to fit.

I think that the preliminary work allowed me to get more accurate results. The usefulness of the preliminary work was that I got the idea of using instruments like a measuring cylinder for example, with more accuracy by measuring below the meniscus.

Apparatus/equipments.

Conical flask
Safety goggles
Gas syringe
Measuring cylinder
Clamps stand
Stop watch
Grater
Water Bath
Hydrogen peroxide
Electrical weighing scale
Potato
Spatula
Pipette
Thermometer
Beaker
Ice bath

METHOD:

1. First collect all the necessary equipment needed to carry out the experiment.

2. Put your safety glasses on.

3. Set up the water bath / ice bath

3b. fill the water bath up with water and have a thermometer on the side of it. The thermometer should be touching the water.

3c. the water bath should be on the lowest temperature for the first experiment and should gradually be increased.

3d. wait for the water in the water bath to be on the selected temperature.

4. Set up 2 clamp stands. One should have a gas syringe tightly secured on it. It is important that the gas syringe is pushed in all the way, and is on the marking 0.

5. The second clamp stand should have a conical flask attached to it. The conical flask should be in the water in the water bath.

6. Position the conical flask directly next to the gas syringe. IMPORTANT NOTE: check that the bottle top of gas syringe fits selected conical flask.

7. Grate a potato using a grater on a tile. The potato should be grated on the medium sized grater. The potato should not be in contact with the table in case it is mixed with any other substances already on the table.

8. Leave this grated potato in a large beaker.

9. Measure out 5g of grated potato and put it in the conical flask using a spatula.

10. Using the measuring cylinder, measure 30ml of hydrogen peroxide.

11. When pouring hydrogen peroxide into the conical flask, make sure you don’t spill any of it on your hand. If you happen to accidentally spill some, wash it off quickly under cold, running water.

12. Get the stop watch ready.

13. Reset the timer.

14. Pour in the 20ml of hydrogen peroxide into the conical flask.

15. Push the gas syringe into the top of the conical flask instantly and start the stopwatch immediately.

16. Repeat this with six other temperatures for the hydrogen peroxide to get more accurate results and also to make it a fair test.

The different temperatures should be 0ºC, 10ºC, 20ºC, 30ºC, 40ºC and 50ºC. So we can see which temperature the enzymes work best at.

17. For each experiment, take down the results after every 30 seconds.

Below is a diagram show how the experiment should be presented.

Safety

It is vital that while I am doing this experiment I am safe, the environment around is safe as well as people around me so I have made a list of safety precautions that I will take.

Wear safety goggles at all times to protect my eyes.
Keep my fingers away when grating the potato to avoid harming my fingers.
Not run with equipment especially equipments made of glass e.g. beakers.
Be careful when using the hydrogen peroxide as it is a corrosive chemical, so overcome this problem by wearing goggles and gloves.
Not spill hydrogen peroxide on your hands if you do wash immediately under cold running water.
Put bags and stools under the table do prevent tripping over them.
Place equipment in the centre of the table and not on the edge. This will decrease the likeness of the equipments falling on the floor.
Not touch the water in the water bath as it will be hot.

FAIR TESTING

It is important that when I do the experiment, I make it a fair test in order for it to produce accurate and reliable results. So, in order for it to be a fair test I will make sure that I will do the following. I will;

Use the same mass of potato (5g)
Use the same sized potato to ensure the surface area is always the same.
use the same volume of hydrogen peroxide(30ml)
use the same conical flask(this will mean that I am certain that the bottle top of the gas syringe will fit)
use the same thermometer
Make sure that the weighing machine is on zero-error before I put the potato on it.
Make sure I read the bottom of the meniscus when reading the measuring cylinder.
I will test out 5 different temperatures; they are 10ºC, 30ºC, 40ºC, 50ºC and 60ºC. And does each test twice so I will end up doing the experiment 10 times.

VARIABLE:

Key Variables:
Temperature
pH
Concentration of enzyme of substrate
Volume of hydrogen peroxide
Mass of Potato
the surface area of the potato

I am going to only vary the temperature from above, and control all of the other variables.
I shall only be altering the temperature, as that is my main variable, and so I will therefore keep the others the same in order to make it a fair test, and a comprehensive study without many variables. I will keep my variables the same by making sure that I have implied the same strategy for each test taken. The volume of Hydrogen Peroxide is carefully measured out using the measuring cylinder; enabling me to make sure it is kept constant throughout the experiment. The mass of potato will stay constant too.

PREDICTION:

I predict that the enzyme will work at a slower rate after 40-45ºC because the enzymes would have denatured by then. My reason for this prediction is because every enzyme has a temperature range of optimum activity. Outside that temperature range the enzyme is caused to become inactive. This occurs because as the temperature changes, enough energy is supplied to break some of the molecular bonds. When these forces are disturbed and changed, the active site becomes altered in its ability to hold the substrate molecules as it was intended to catalase. Most enzymes in a human body shut down beyond certain temperatures. This happens if the body temperature gets too low (hypothermia) or too high (hyperthermia).

I have predicted this because from my background research I have done, it is evident that since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with increasing temperature. However, if the temperature of an enzyme catalysed reaction is raised still further, an optimum is reached: above this point the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme molecules starts to be disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of denaturing more and more enzyme molecules. Also, the stability of the protein also decreases due to the thermal degradation. Holding the enzyme at a high temperature for a long period of time may cook the enzyme and therefore denature it.

Hypothesis:

My prediction is supported by the kinetic theory in that if I apply twice as much heat, there will be twice as much particle vibration, therefore the reaction will take place twice as quickly. It is also backed by collision theory in that if I apply twice as much heat there will be twice as much collisions taking place, therefore the reaction will double.

Results

Graphs

Analysis of results

From the graph, I am able to back up my theory. The enzymes work at a slower rate after 40ºC. From the graph, I have found out that, enzymes work at the fastest rate at 10ºC, even though I my theory says that the optimum temp should be 40 because, this is where the greatest number of collisions takes place between the enzyme and the substrate and therefore the highest rate of reaction is. My results show that the optimum temperature is 10C, but is has to be said that this is incorrect and this is probably because I made errors while the experiment was taking place.

The enzyme denatured at about 60°C because the weak bonds, which hold the molecule into the specific shape for one substrate, are broken. The increase in molecular collisions and vibrations at higher temperatures is great enough to permanently change the shape of the active site. The enzyme is said to be denatured because it can no longer form an enzyme-substrate complex as its active site has been unalterably changed.

This was a very big problem in the experiment and is all explained in my evaluation.

Evaluation

I think that I worked well to achieve my aim. I worked safely and achieved accurate results. I did have anomalous results but when I did I repeated the experiment. For e.g. when I did 30 C for the first time I had very strange results. All 3 of the experiments gave different results. After I had finished testing out all the other temperatures, I did the 30C experiment again. I then had accurate results and all the experiments gave similar results.

I did have some minor problems, for most of these I had found a solution too. Firstly, when I did the experiment for the first time I started the experiment but realised that the hydrogen peroxide and potato did not release any gas. And when it did it was so little it was unnoticeable. I then realised that the gas syringe we used was not reliable as it was very tight I decided to change the gas syringe to an ordinary one which I did. So I had overcome this problem.

The second problem I faced was that I had to carry out my experiment over 4 lessons which meant I had to use different potato every lesson. This meant that each potato might have had different concentration of catalyse, therefore it would give different results. I think that I could have overcome this problem by carrying out all the experiment on the same day.

Another problem I faced was that I had wasted a lot of time when repeating the 30ºC experiment again. The problem was that the water in the water bath was not increasing temperature. It took 20 minutes for the water to be 10ºC and I needed it to be 30ºC so I used another water bath which was working properly.

Other than these problems I worked well and achieved accurate results. I worked efficiently too, as I was able to carry out all the experiment intended to do in time.

I also think it would have been better if I had used the same potato from the whole experiment but was unable to due to the time restrictions. I had to conduct the experiment over a number of days and could not therefore use the same potato. This is a source of error because the concentration of catalase in the potatoes may have been different which may have produced an inconsistent rate of reaction. This might be one of the reasons in which my results were inaccurate. To remove this problem, I could repeat the experiment not only with three readings at each temperature, but also with three different potatoes, which would provide an even more accurate reading, as I could calculate an average.

I would also carry out more experiments with a range of 5ºC. I would do 0ºC-70ºC. By 70ºC the enzyme would have denature. I would have carried out more experiments because this would give me more results and allow me to plot a more accurate graph. Also, it would be easier to tell when the enzyme got to the optimum temperature and denaturing point.

So next time I would do all the things below to ensure even more accurate results;

Tighten the bung on the flask.
Use a good gas syringe (not too tight or loose)
Use the same equipments apart from the flask.
Do all the experiments on the same day using the same potato.
Use three different potatoes for each of the experiments so; I could calculate an even better average.
Make sure the water bath is working.
Carry out more experiment, with a temperature range of 5ºC.

Conclusion

To summarize I found out that enzymes work fastest at 10ºC even though according to my theory it should be 40ºC. I also found out that it is correct about how the enzyme denatures after 40ºC.

So enzyme activity is affected by temperature. The optimum temperature for enzymes should be 40ºC, and after this they should not work well as they are denatured.

HOW DOES TEMPERATURE AFFECT CATALASE ENZYMES?

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