At the optimum pH, the reaction occurs very fast. The maximum catalytic rate for one catalase molecule is 6 million molecules of hydrogen peroxide converted to water and oxygen per minute. The reaction product is 6 million molecules of water and 3 million molecules of oxygen. (Because the oxygen molecule consists of two oxygen atoms, the number of oxygen molecules made in the reaction is half the number of water molecules.)
However as enzyme activity is affected by pH, I predict the optimum pH of catalase is approximately pH 7.0 (neutral) this is because if the buffer solution is too acidic (low pH value) or too basic (high pH value) the catalase will become denatured and consequently inactive – as it no longer functions as an enzyme; therefore will be unable to produce oxygen (the product used to measure the rate or reaction). Therefore if the enzyme is subjected to fluctuations in pH, the protein structure may lose its integrity (denature) and its enzymatic ability.
Apparatus:
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2 – 100 cm3 glass beakers
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2 – 10 cm3 syringes
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8 – 25 cm3 glass beakers
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Glass tube (approximate length ≈ 31 cm)
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Rubber connector tube (approximate length ≈ 2.3 cm)
- Stopwatch
Chemicals: Hydrogen peroxide (H2O2), potato extract (homogenate) solution containing the enzyme catalase, buffer solutions – pH 2, 4, 6, 7, 8, 9, 10
Procedure: A 100 cm3 glass beaker will be filled with 50 cm3 of potato extract solution (containing catalase enzymes) and labelled, while another 100 cm3 glass beaker will be filled with 50 cm3 of hydrogen peroxide and labelled H2O2 to make a clear distinction from the pH buffer solutions. Seven pH buffer solutions have been used to compare how very low pH concentrations (acidic); neutral (water) and extremely high pH concentrations (alkaline) affect the nature and rate of the enzyme-controlled reaction – oxygen production. Consequently through results an optimum pH value can be attained. However if a pH buffer solution provides anomalous value it may be isolated, so that the remaining results from the other six pH buffer solutions provide enough adequate data to form a sufficient analysis and conclusion.
When potato extract is placed into the beaker the enzymes may gradually settle at the bottom of the solution (base of the beaker), and due to this may affect results, as the solution will no longer be a homologous mixture – as the enzymes are concentrated in an isolated region, this means less enzymes will be available to produce oxygen; decreasing the rate of reaction.
Due to the many experiments that will be carried out to establish the enzyme’s optimum pH, a considerable quantity of each solution is required. Next, seven 25 cm3 glass beakers will be filled with different pH concentrations and labelled according to their pH value.
Subsequently 2.0 cm3 of a specific pH buffer concentration (acidic or alkaline) and 2.0 cm3 of hydrogen peroxide will be drawn up with a 10 cm3 syringe and deposited in a empty 25 cm3 glass beaker. The beaker will be gently oscillated to make certain the solution is a homogenous mixture, retaining the definite pH of the buffer solution. Moreover, 2.0 cm3 of each solution has been used, in order for clear measurements and observations to be constructed concerning the rate of reaction (quantity of the gas product; oxygen produced).
The syringe will then be rinsed off with tap water (H2O), so that any remaining traces of the buffer solution will not affect the pH of the potato extract solution causing possible future anomalies to arise when testing contrasting pH buffer solutions. Afterwards the syringe will draw up 2.0 cm3 of the potato extract solution and then draw up the separate mixture of hydrogen peroxide and pH buffer solution in the glass beaker. Then the syringe will be inverted several times to mix the contents; this enables the catalase enzymes to work efficiently in more regions (operate in a greater area) to break down the substrate (hydrogen peroxide) into the products of oxygen and water.
To achieve accurate results, the 25 cm3 glass beaker will be washed out with water and dried after each experiment to prevent any foreign ions, excess pH and/or water solution entering future pH buffer solutions, as it may have serious and damaging outcomes on future results.
An equal amount of each solution (2.0 cm3) has been used to gain an equilibrium or stability to which variables, such as enzyme concentration (potato extract) or substrate concentration (hydrogen peroxide) remain controlled, and therefore will not be able to cause serious problems when analyzing results and forming a final conclusion. As a result of this the only factor, which is altered while all others remain constant is the pH of the buffer solution to ensure reliability of results. Thus the variables of temperature, enzyme and substrate concentration (volume of solutions) must be kept at a constant while the experimental factor; the pH buffer solution is the only single factor that is changed. Another factor that must remain a constant is the temperature, which may be difficult as fluctuations occur due to excess heat produced by the body and by light, as well as the continual motion of air.
Furthermore the same potato extract solution must be used for the entire investigation, as another solution will inevitably contain different quantities of enzymes. Therefore due to this difference in catalase concentrations it will cause variations in results measuring the rate of reaction (product formation) when compared to the results from the initial potato extract solution.
To keep the temperature constant the beakers containing the solutions could be stored within a water bath. Therefore in order to minimize or restrict temperature change, each experiment must be performed in an isolated area where the factor remains a constant.
By carrying out these precautions a clear and precise conclusion can be drawn, so that an analysis of results and the essential problem – investigating the effect of “pH” do not contain limitations or errors.
When the extract has been inverted, a small rubber tube connected to a long glass tube will be directly fitted over the mouth of the syringe. The apparatus will then be positioned on a piece of paper, to which a mark will be sketched to represent the starting position of the liquid in the tube, when this has been achieved a stopwatch will immediately be started. After one minute a mark will be sketched on the paper to indicate where the liquid had reached in the allocated time. When the following process has been carried out the distance between the two marked points on the paper will be measured, to which the rate of reaction can be determined: X millimetres (mm) per minute (min–1).
The same glass tube must be used for the experiment as different tubes may vary in total cross sectional area. If the area of the tube were larger than that of the initial tube it would be more difficult to distinguish between the rate of reaction concerning the different pH concentrations; affecting the accuracy of observations and future conclusions. This is the reason why a tube that with a small total cross sectional area, will be used in the investigation as the rate of reaction if able to be observed and established more effectively, so results will be more apparent. Thus, if two different tubes are used within the investigation if would difficult to accurately determine the rate of enzyme-controlled reactions under specific pH concentrations.
To ensure the reliability of measurements, observations and results are the procedure must be repeated an addition two times, using the same pH buffer solution, in order for a mean to be calculated (a single number to summarize and represent the values of items in a set). Then the remaining six pH buffer solutions can be tested to gain measurements illustrating the rate of the enzyme-controlled reactions. Therefore if discrepancies are evident within the results, they may cause a false and inaccurate inference to be compiled, and consequently should be removed. If extreme results are recorded it is perhaps more sufficient to calculate the median of the values, as the median is less affected by extreme values and will be less than the arithmetic mean.
However as human error causes limitations to an experiment, it is difficult to mark the precise position as to where the liquid has arrived at after exactly one minute. Additionally the reliability of results may be affected due to excess water within the syringe, this could cause the pH buffer solution the become diluted resulting in an alteration in the rate of the enzyme-controlled reactions; providing erroneous conditions and situations to arise. Furthermore air bubbles within the glass tube cause problems and difficulties when measuring as well as observing the rate of reaction (product formation). In order to counteract these effects a different syringe will be used for each experiment and the glass tube may be warmed under a lamp to eliminate excess water or trapped air.
Safety: Before each experiment can be carried out a risk assessment must be completed.
Eye Protection: Concentrated vapours cause discomfort in the mucous membranes and the eyes. Contact of the eyes with hydrogen peroxide is particularly dangerous because corneal burns can occur very rapidly. Therefore, safety glasses or, preferably, goggles should always be worn when handling concentrated hydrogen peroxide. If, however, any hydrogen peroxide does get in the eyes, flush eyes thoroughly with water and consult a physician promptly.
Protective Clothing: Rubber gloves and suitable protective clothing such as aprons or coveralls made of polyester acrylic fibre, polyvinyl chloride, polyethylene, or neoprene should be worn when handling concentrated hydrogen peroxide.
Contact with moderate concentrations of hydrogen peroxide will cause whitening of the skin and stinging sensations. The whitening is due to the formation of gas bubbles in the epidermal layer of the skin. The stinging, in most cases, subsides quickly after thorough washing, and the skin gradually returns to normal without any damage. Highly concentrated hydrogen peroxide can cause blistering if left on skin surfaces for any length of time.
Inhalation of hydrogen peroxide vapours can cause irritation and inflammation of the respiratory tract. If inhaled, fresh air should be sought at once; if the inhalation has been prolonged, a physician should be consulted immediately.
Accidental Swallowing: Contact or concentrated solutions (over 3%) with the members of the mouth is to be avoided. Under no circumstances should hydrogen peroxide be taken internally. If hydrogen peroxide is swallowed, drink water immediately to dilute, and contact a physician but do not attempt to cause vomiting.
Maximum safety in handling hydrogen peroxide is assured through the use of proper materials of construction, recognition of the need for venting in storage, and overall avoidance of contamination. The oxygen and water by-products of decomposition are innocuous, but splashing, inhaling vapour, and ingesting hydrogen peroxide must be avoided. If by unusual circumstances an accident should take place, flushing with large quantities of plain water is the simple corrective action needed. By adhering to straight-forward common sense procedures, every aspect of your operation will be aimed toward safety and a clean environment.
Therefore to make the experiment safe, check the person you are working with knows the safety procedures. As there is the risk of skin cells or eye cells being damaged when working with acid or alkali buffer solutions, make certain eye protection and gloves are worn, also make sure a non-corrosive surface is used to do the experiment and that the investigation is not carried out near electrical equipment. Verify all the equipment is safe to use and efficient to carry out the experiment.
To reduce hazards occurring happening you must handle acid or alkali buffer solutions with as much caution and safety as possible. Make sure that when chemicals have been used they must be sealed to confine any toxic gases being released. Moreover suitable ventilation must be acquired to rid off any toxic fumes.
Results Table: Volumes will be measured to the nearest cm3, while the reaction of reaction will be measured to the nearest mm. min–1.
Analysis: A line graph will be drawn to highlight the rate of reaction – how far the liquid in the glass tube travels from its initial point to its new position after one minute (mm. min–1) when different pH concentrations are used. Therefore by producing a line graph results can be analysed appropriately and observed to determine any anomalous readings. Due to this the errors may be removed or isolated so they do not cause significant affect on the final conclusion. From the graph the optimum pH can be established (the peak of the curve) as well as the effects different pH’s have on the activity of catalase’s enzyme-controlled reactions. I believe the graph may bare similarity to the one show below:
A graph will be constructed according to the mean values (mm. min–1), which will provide theoretical values, whereas the results from the experiment provide the practical values. Thus as the rate of reaction values are mean values the curve will be a line of best fit. In addition it may be necessary to construct another line to portray the median values, if the medium values are too extreme or unreliable to form an adequate analysis.
A generally applicable schematic diagram of the variation in the mean (average value of three results) rate of an enzyme catalysed reaction (y-axis) with the pH of the solution (x-axis). The centre of this inverse parabola displays the optimum pH of the enzyme catalase; where a maximum amount of substrate molecules (H2O2) are broken down to yield the products water and oxygen. The oxygen is consequently measured to indicate the rate of enzyme-controlled reactions. Extremely high or low pH values generally result in complete loss of activity for a majority of enzymes, as it affects the stability of enzymes.