Investigate how concentration of the enzyme catalase in celery tissue alters the rate of reaction with hydrogen peroxide.

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Problem: Investigate how concentration of the enzyme catalase in celery tissue alters the rate of reaction with hydrogen peroxide.

  An enzyme, a biological catalyst, accelerates a chemical reaction, without changing the reaction’s outcome and can be recovered from amongst the end products. For just about every reaction in a living organism there is an enzyme to catalyse it.

  For a chemical reaction to occur two conditions must be satisfied: the reacting molecules collide at the correct orientation to each other; and the second is that the reactants contain enough energy to bring about the breakage of appropriate chemical bonds. The energy can come from heat energy but the temperature needed is hard to obtain and maintain in an organism and is harmful to cells, amongst other things. In addition to raising the temperature, the rate of reaction can be altered by increasing the pressure, the presence of a catalyst, the concentration of reactants and the surface area across which the reaction occurs, although these factors may not apply to all reactions. This energy, required by the substrate to react and form an end-product, is called the activation energy. The diagram below shows how activation energy is changed by an enzyme-induced reaction.

Fig 1.1 Activation Energy and Enzyme-catalysis, influence of.

  An enzyme decreases the activation energy and therefore rate of reaction increases, because molecules now have energy greater than, or can easily acquire enough energy to reach, the new activation energy. An enzyme must combine with a substrate for it to catalyse the reaction. By certain analytical techniques, we can see that an enzyme is a globular protein molecule, which has a specific tertiary structure and shape. A protein molecule is made of polypeptides which is a series of amino acids linked together by peptide bonds. A protein molecule can consist of one polypeptide, linear chain, or several chains making a globular structure, maintained by Van der Waal’s forces (between all molecules) and by otherintermolecular forces (Nuffield Advanced Chemistry, p.209). Secondary, tertiary and quaternary-structured protein molecules (haemoglobin is an example) are in increasing order of complexity. A tertiary-structured protein molecule is recognised by the supercoiling of the α-helix (the secondary protein structure, an example is the DNA double helix). The three dimensional shape of the protein is stabilised by a “series of interactions between –R groups on the polypeptide chain”. The ‘–R groups’ are unspecified molecules which vary from protein to protein and are the ‘active site’ of an enzyme.

The active site of an enzyme is the only area where a substrate molecule can do this. It was originally thought that the substrate molecule must be an exact complementary shape to the active site, as shown by the diagram below, but this is not the case. The ‘Induced-Fit’ Theory, contrary to the lock and key hypothesis, illustrates that the substrate doesn’t have to be an exact match but can induce the enzyme to respond in such a way that it accepts the substrate molecule, as if the enzyme is ‘flexible’ only to the substrate. The outcome is the same either way; the enzyme binds to the substrate for a fraction of a second, before detaching and binding to another substrate.

                                                                        The products:

                                         Lipids                                         Fatty acids

                                  (substrate molecule)                                and

                                                                                             Glycerol

 Enzyme

  Lipase                                                                         

 (secreted by

Pancreas into

 small intestine)

                        Enzyme-Substrate Complex          

Fig. 1.2 LOCK AND KEY THEORY

  Catalase (alternately called Peroxidase) is one such enzyme and is very important to all living cells, and is found in all cells, in organelles called Peroxisomes. The name ‘Peroxisome’ name arose because this organelle was strongly involved in the formation and decomposition of Hydrogen Peroxide. It contains four enzymes, three of which produce H2O2 urate oxidase, D-amino oxidase, and alpha-hydroxylic acid oxidase whilst the fourth …. Catalase: causes the catalysis of the breakdown of Hydrogen Peroxide, an oxidising agent, which would otherwise cause irreparable damage to a cell. Hydrogen Peroxide can inactivate critical enzymes allowing toxic levels of substances to build up. It can also be converted to free radicals, which can damage cell components such as DNA, lipid and proteins, making Hydrogen Peroxide a potentially damaging, oxygen species. The decomposition of Hydrogen Peroxide produces water and oxygen, as shown by the equation below, but the vast amounts produced by those three enzymes in the peroxisome must be quickly decomposed, in order to eliminate the risk of damage to the cell.

2H2O2 → 2H2O + O2 

  Catalase has a turnover (T.O.N) rate of 50,000 molecules per second, which means that it catalyses the breakdown of 50,000 molecules of H2O2 to H2O + O2 in one second. It is said that Catalase has the highest turnover rate of all enzymes, so this enzyme has been specifically engineered to make sure that any Hydrogen Peroxide produced is purged from the cell as quickly and efficiently as possible. In this experiment we shall investigate how the concentration of catalase alters the rate of reaction with hydrogen peroxide.

  Through research and AS knowledge, I have found supplementary factors which affect the rate at which Catalase catalyses the decomposition of hydrogen peroxide into oxygen and water. Conversely, these factors can be associated with all enzyme-substrate ‘reactions’:

  • Substrate concentration: The rate of an enzyme-catalysed reaction increases in relation to the substrate concentration, until the velocity reaches a maximum level. At the maximum rate, at any given time, all the active sites of the enzymes are being implemented, so increasing the substrate concentration has no further effect on the rate of reaction. See below for a graph explaining this relationship.

Fig 1.3

  • Temperature: As temperature increases, more energy is being transferred to the molecules, increasing the amount of kinetic energy they have (velocity), which naturally leads to more successful energetic collisions, resulting in increased enzyme-substrate reactions. The substrate must be in close proximity to the active site, but the probability of this happening is increased as the molecules are moving more quickly. At higher temperatures, the enzymes’ tertiary structure is distorted, as bonds are broken, and denatured, so there is an optimum temperature at which reaction occurs. At lower temperatures there is less energy available to the substrate and enzyme molecules, so they collide more slowly with less energy. Enzymes optimal working temperature depends on the conditions in which they exist in. In relation to temperature, enzymes are classified into Thermophiles, those that prefer temperatures above 40oC, Psychrophiles, enzymes efficient below 20oC, and Mesophiles which are efficient between these temperatures. Catalase is mesophilic in nature; it operates in virtually all cells so

  • Incubation Time/Thermal denaturation: As time progresses, the rate of enzyme reaction decreases, even with an excess of substrate molecules, at its optimum temperature. This is due to the denaturing of the enzyme from the maintained heat, which means that the shape of the enzyme and therefore its active site is changed so that substrate molecules can no longer to bind to the enzyme. Denaturing is an irreversible process.

  • pH: If the pH is low, there are more Hydrogen ions, H+, which can “interact with the R groups of amino acids, affecting the way in which they bond with each other and therefore affect their 3D arrangement.” There is a structural change in the protein, due to the Hydrogen ions interacting with the active site, which means that the enzyme is denatured. Optimum pH value changes from one enzyme to the next, for example Catalase prefers pH 7 whilst stomach Lipase favours pH values from 4.0-5.0.

  • Enzyme inhibitors: Competitive inhibitors slow down the rate of reaction by competing with the substrate for the active site of the enzyme. Non-competitive inhibitors bind elsewhere on the enzyme, changing the shape of the active site, preventing the substrate from binding. They can be reversible or irreversible, depending on whether or not they bind briefly, or permanently, respectively.

Prediction

  I predict that at any given enzyme concentration, whilst the substrate amount and concentration is limited (not infinite), the initial rate of reaction will be relatively quick and eventually reach a maximum velocity, before which the rate of reaction appears to level off and decrease. The initial rate of reaction is quick because there is a large amount of substrate available for the Catalase enzyme to bind with; it acts in a similar way to the effect of Substrate concentration, as shown in the bullet points above. As the substrate is being converted to product, the amount of substrate is being reduced, so there is less and less for the enzyme to bind with. If there is less substrate for the enzyme to react with, as time progresses, then the rate of reaction will decrease. There is less chance of collisions, resulting in the binding of the enzyme with the substrate, because there is simply less substrate left for the enzyme to react with. Also, as time progresses (incubation time) the enzyme becomes denatured, even with excess substrate, so it is harder for the substrate to bind with the active site on the enzyme. The graph below displays this relationship.

Fig. 2.1

  At lower levels of enzyme concentration, the overall amount of oxygen gas produced (a measure of the rate of reaction) will decrease because at any given time there is less and less enzyme binding with the substrate molecule. As there is less enzyme molecules, the probability of substrate molecules coming into contact and binding successfully with the active site decreases. Increasing the enzyme concentration will increase the rate of reaction as this will increase the probability of successful collisions, but eventually it will level out because there is not an unlimited supply of substrate molecules. All my predictions are dependant that pH, pressure and temperature are constant and the amount/concentration of substrate molecules present remains unchanged. If any enzyme inhibitors are present then the overall rate of reaction would be much slower than a reaction without. The velocity-time graph would still show a similar correlation to the graph above, as the amount of enzyme inhibitors is finite and does not speed up with time.

Variables

Below are comments on the variables which must be controlled to ensure a fair experiment is being conducted.

Pressure: The pressure is effectively controlled in the classroom where we are doing the experiment in. If it was not, then we would do the experiment in a pressure-controlled chamber.

Temperature: It is possible that the temperature of the solutions may change throughout the experiment, depending on the heating of the room, weather conditions, wind from the windows etc. Constant checks, using a thermometer, are needed to make sure that the temperature of the reactants is constant right the way through the duration of the experiment. Any temperature fluctuations will be recorded and taken into account during the evaluation of the experiment. The pilot experiment showed to me that sometimes the temperature does change so I will have to observe and analyse the effects of temperature change. Ideally, I could use a thermostatically controlled water bath, but not everyone being supervised can have one. It is better to go without, as all the class results produced can be compared for accuracy, which could lead to better judgements on the extent of the effect of temperature.

pH: H2O2, Hydrogen Peroxide, could affect the pH level of the solution, but I have limited the consequences by using the same volume, concentration and source of H2O2.

The effect of Surface Area is controlled by the liquidising of the celery tissue. Bubbles can also change the surface area of reactants, so the pouring of solutions will be done in a subtle way to reduce the amount of bubbles and the tests will not be continued until all bubbles are popped inside the boiling tubes and syringes, the ‘Equipment’ passage explains how to do this.

Volume of Hydrogen Peroxide, and reactants, will not change. The total reactant solution will always be 10 cm3. The concentration of celery extract solution will be changed, by the increase in volume of water content and decrease of celery extract, but the total volume remains the same: 5 cm3 for the celery extract solution. So there will be equal volumes of substrate, hydrogen peroxide, and enzyme, found in celery tissue, but the concentration of the enzyme is the only independent variable. I will use this relatively large volume, because it makes it easier to observe when measuring out the volumes. Also with larger volumes, if there is a small mistake in measuring, the overall percentage error is smaller than the percentage error you could get using a smaller volume.

Three aspects constitute a fair test: reliability, accuracy and validity. Firstly, I must have confidence in the observations or measurements I record, and therefore have no doubts as to whether or not I judged something wrongly, or the equipment hindered from making a good judgement. Secondly, the degree to which the results are a reflection of the true outcome is critical; this can be the deciding factor in commenting on the experiment’s success. Finally, only one variable should be adjusted whilst others are constant, which will hopefully show that it is reasonable to have no doubt in the conclusion, as there are no alternative solutions. These two aspects can be promoted by the use of averages, obtaining the highest degree of accuracy possible with the apparatus available(e.g. rate of reaction is measured in seconds so the timing does not need to be accurate to within 1/10 sec), identifying anomalous results, the use of statistical techniques, appropriate equipment etc.

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Pilot Experiment: Before writing the complete method, I did a pilot experiment to discover what potential problems I may come across. These problems included lack of suitable equipment, the problem of how often to take recordings, timing, accuracy and reliability and the technicalities of the method. I shall write about how the outcomes of the pilot experiment affected my choices. I was interested in how much gas was produced over 5 minutes when the Celery Extract concentration was at its highest, so I recorded this value. I found that in one minute no more than 10 cm3 of oxygen was ...

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