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Investigating the effect of PH on the activity of the enzyme catalase.

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Investigating the effect of PH on the activity of the enzyme catalase.

Hydrogen Peroxide will be the source of substrate, and the catalase enzyme breaking down the hydrogen peroxide will be from potato’s. The products formed when H202   is broken down will be water and oxygen.
The word equation and balanced equation for the reaction would be:                                

                                (catalyse)
Hydrogen Peroxide  
   Water + Oxygen    

2H20 2  2H20 + 02image00.png

 Hydrogen Peroxide bond -

Catalase

Catalase is a common enzyme found in all living organisms. Its function is to catalyze the decomposition of hydrogen peroxide to water and oxygen. Catalase has one of the highest turnover rates of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. The optimum pH for catalase is around neutral (pH 7.0). It is a protein of four polypeptide chains, each over 500 amino acids long.
All
animals use catalase in every organ, its highest concentration is mainly in the liver. Catalase is also present among plants, but not so much fungi, however there are some species which have been found to produce the enzyme when growing in an environment with a low pH and warm temperatures.                                                               It can also be used in the food industry for removing hydrogen peroxide from milk prior to making cheese. Another use is in food wrappers, where it prevents food from oxidizing. Its also used in the textile industry, removing hydrogen peroxide from fabrics to make sure the material is peroxide-free. Recently, they have used catalase to be used in several mask treatments which combine the enzyme with hydrogen peroxide on the face with the intent of increasing cellular oxygenation in the upper layers of the epidermis.

Hydrogen Peroxide
H
2O2 is a powerful oxidizing agent and is potentially damaging to cells. By preventing excessive H2O2 to build up the enzyme catalase allows important cellular processes which produce H2O2 as a byproduct of water and oxygen to take place safely.
It is a very pale blue liquid which appears colorless in a dilute solution, slightly more
viscous than water as it is a weak acid. It has strong oxidizing properties and is therefore a powerful bleaching agent that is mostly used for bleaching paper, and can also be found in rocketry. The oxidizing capacity of hydrogen peroxide is so strong that the chemical is considered a highly reactive oxygen species.
Above roughly 70% concentrations, hydrogen peroxide can give off vapor that can detonate above 343 K (Kelvin) at normal atmospheric pressure. This can then cause a boiling liquid expanding vapor explosion of the remaining liquid.
Distillation of hydrogen peroxide at normal pressures is therefore highly dangerous.

Our aim in this investigation is to found out how different PH’s affect the catalase rate of activity. To find out how the different pHs work, we will be measuring how much oxygen has been released, this will give us an indicator as to which pH catalase works best with. Measuring the rate at which oxygen is evolved reflects the activity of enzyme catalase.

Factors affecting enzymes.

During our experiment we will be keeping one independent variable which will be pH. The rest of the factors will be controlled variables, these being temperature, concentration of substrate and concentration of enzyme. These 3 variables will be kept the same through out the whole experiment to ensure it becomes a fair test.

Temperature - The rate of reaction starts off slow due to insufficient kinetic energy from low temperature. Few collisions between enzyme and substrate. More enzyme + substrate complexes are being formed at around 30°C. The increase in temperature causes particles to collide more due to more kinetic energy.
The optimum temperature is at about 40°C this is due to many successful collisions therefore more products. After around 45°C the rate of reaction slowly starts to decrease again, the increase in kinetic energy causes molecules
within the enzyme to vibrate. This may result in the hydrogen bonds breaking. It can also cause the enzyme structure to change (not a permanent change), however not all enzyme are affected at this point as some products are still being made. When the temperature reaches 60°C more energy causes the molecules to vibrate more. Hydrogen bonds and (s-s ionic) begin to break. The 3° structure is permanently altered, this means that the active site and substrate are no longer complementary to each other. As a result no more product is being formed causing a decrease in rate.image01.png

image02.png

PH - Enzymes are affected by changes in pH. The most favourable pH value is where the enzyme is most active. Extremely high or low pH values generally result in complete loss of activity for most enzymes. Most human enzymes work at an optimum temperature of around 7.3/7.4. The majority of these are intracellular enzymes (An enzyme that remains active only within the cell in which it is formed). Extracellular e.g. Digestive enzymes work at more extreme pH’s e.g. Protease pH2 and Arginase pH11.
Rate of reaction is affected by changes in pH, the charges on the R groups maintaining the structure of the active site are essential for the formation of an enzyme substrate complex. If the properties change, the substrate can no longer bind (increase in protons will repel the substrate). PH can also affect individual amino acids as formations of zwitterions are common. Looking at the graph you can see as the PH increase the rate of reaction starts to increase with it. At around a pH of 7 the enzyme is working at its optimum pH resulting in more products being formed. After a pH of 7 the rate of reaction starts to decrease, base conditions begin to disrupt some of the hydrogen bonds between loops of the protein chains. If the disruption occurs at or near the active site, the active site becomes altered and the substrate can not fit perfectly. Therefore not all enzymes in the solution will be able to catalyze their reaction. With increasing pH, more enzymes become denatured, and fewer enzymes are able to form that enzyme-substrate complex. The reaction rate continues to decrease. At some point, all the enzymes are denatured, and the reaction rate falls to zero.

Enzyme concentration - The substrate binds to the active site of the enzyme to form an enzyme substrate complex. So as you increase the concentration of enzymes it increases the number of successful collisions and so to a point the rate of reaction is directly proportional to enzyme concentration and the higher the concentration of the enzyme the faster the reaction.
However, at some point this will plateau and reaches equilibrium where the reaction reaches its maximum rate regardless of the enzyme concentration because the substrates have run out and so the excess enzyme has nothing to react with.

Substrate concentration - when the concentration of enzyme is maintained constant, the reaction rate will increase as the amount of substrate is increased. However, at some point, the graph shows that increasing the amount of substrate does not increase the reaction rate. The line begins to level off and stay level at B on the graph. We call this Vmax or the maximum velocity of reaction. At first there is very little substrate and a lot of enzyme. An increase in the concentration of substrate means that more of the enzymes can be used to form a reaction. As more enzymes become involved in reactions, the rate of reaction increases. At B, all the enzymes are being involved in reactions. When this happens, some of the substrates have to wait to for a complex with the enzyme, as there are no enzymes free to work with. After point B, the reaction rate remains flat because the increase amounts of substrate must wait before they can fit with their enzyme.image03.png

There are two theories on enzyme function -

  • Lock and key – the shape, structure and charge of the active site in the enzyme is complementary to the shape, structure and charge of the substrate
  • Induced fit – The substrate and enzyme are not complementary. During collision the substrate induces a change in the active site’s shape so it becomes complementary.

Prediction – I predict that as we increase the value of pH, the rate of reaction will increase with it, however as the pH value reaches around 7 or 8 the rate will start to level off and then gradually decrease and the enzymes become denatured. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot go through catalysis.

Validity and reliability

To make the investigation valid and reliable we need to make sure we only use one variable as if we add more variables in to the test we would not know what had increased the rate of reaction. So in our experiment we will only be changing the values of pH which will be our input variable. The rest of the limiting factors will be kept the same, so the temperature and the substrate/enzyme concentration.
The investigation will be done using pH’s from 3 to 9. This gives us a good rang from a strong acid to neutral to a weak alkali hopefully giving us some successful results.
We will accurately measure and set the same volume and concentration of substrate for each test i.e. 10cm3 hydrogen peroxide. The enzyme will react with the same molarity of substrate each time so this will make it a fair test.
However keeping the temperature the same throughout each test will be difficult to control, we need to record the temperature to minimise fluctuations. To keep it the same, we could do the experiment in a water bath or do the experiment all in the same day. However this would be difficult, so we will just try and keep the test done at room temperature and become aware of this affect.
We also need to keep the same mass of enzyme (Potato), keeping the concentration the same for this controlled variable will be too difficult as every potato will be different. Although we could use the same potato for each test, to maximise validity. To collect the potato we will use a cork borer, this puts the potato into small cylinders, and these will be then cut around 1mm for each disc. It is important we try and keep each disc the same for each test otherwise the surface areas will differ and become unreliable.

We need to repeat the experiment more than once to get reliable results, so we shall do it three times.  This will allow us to eliminate anomalous results which could have been caused from simple mistakes. Also by doing more repeats we can create a mean set of results which are more accurate and valid.
Also as we are using pH’s 3-9 we get a wide range of acid- neutral – slightly alkali, this allows us to see what pH enzyme catalase works more efficiently at. However this is a limitation as we can’t use pH values of a strong acid and strong alkali, so we wouldn’t fully be able to work out what pH would be better.

Keeping the apparatus the same is highly important; we need to use the same equipment for each experiment to ensure validity and reliability. Also they need to be washed to make sure no excess of substrate will be added from previous tests.
 

Safety

Hydrogen peroxide is highly corrosive and irritant therefore precautions need to take place, safety goggles should be worn all the time throughout the whole experiment to prevent any reaching your eyes.  It also has to be stored in a brown bottle as it decomposes in the light.
It should also be handle with care, if spilled on clothing (or other flammable materials), will preferentially evaporate water until the concentration reaches sufficient strength, and then clothing will spontaneously ignite. Leather generally contains metal ions from the tanning process and will often catch fire almost immediately
.
Scalpels also need to be handled with care, if misused they can seriously damage people and furniture. If you cut yourself any infection could occur as the scalpel may not have been cleaned properly from previous experiments.
Also glass beakers need to be dealt with carefully so they wont smash or crack.
Any liquid or other solid’s such as potato or glass need to be cleaned up if dropped on the floor as they can cause serious accidents from slipping.






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