Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799

SACE STAGE 2 BIOLOGY

THE EFFECTS OF SUBSTRATE CONCENTRATION ON THE ENZYME ACTIVITY OF CATALASE IN YEAST

Abstract:The purpose of this investigation was to explore the effects of varying substrate concentrations in hydrogen peroxide (2H2O2) on the reaction rate of catalase in yeast.Yeast was used as the source of catalase in this practical. The experiment consisted of small discs of filter paper being soaked in a yeast solution before being submerged in solutions containing varying concentrations of 2H2O2. The oxygen produced by the reaction between the substrates and enzyme will surround the disc allowing it to become buoyant and float to the top of the solution. This explores the effects of substrate concentration on reaction rate. The substrate concentration was manipulated by using varying concentrations of 2H2O2 solution, and the reaction rate was measured by measuring the time taken for enough oxygen to be produced to allow the discs to float. It was hypothesised that “If the concentration of 2H2O2 is increased, then the reaction rate will increase.” The hypothesis was supported as the trend seen in the results indicated that increased levels of 2H2O2 caused a faster reaction rate.

Introduction: Catalase is an enzyme found in yeast that acts as a catalyst to speed up the reaction that breaks down 2H2O2 into water and oxygen. Catalase, like every other enzyme is a protein molecule used to speed up a specific reaction. All enzymes are very specific as they perform only one reaction. This is due to the complementary substrate-enzyme binding process (Crierie & Greig, 2010.) Catalase is produced by the liver and is found in multiple food sources including potato and liver, but the specific source used for this experiment was yeast. Catalase is used to remove the toxic by product of metabolism, 2H2O2 (Reiner, 2010.) It speeds up the process of breaking down 2H2O2 into water and oxygen as shown in the equation below:

Catalase is able to asist in this specific reaction as its active site is complementary to the substrate (2H2O2). The reaction that takes place is known as an anabolic reaction (Crierie & Greig, 2010)

Because proteins are sensitive molecules, they can easily be affected by things such as pH, temperature, inhibitors and they operate at their specific optimum level of environmental variables. These variables either increasing or decreasing leads to the possible denaturing of the enzymes. This reults in destruction of the 3 dimensional shape and changes the shape of thie active site, making it impossible for that enzyme’s subrate to bind. There are 2 ways the enzyme binds to the subsrate: the induced fit model and the lock and key model. The induced fit model is when the enzyme’s active site changes shape to fit complementary to the substrate, whereas the lock and key model is

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799when the enzyme’s active site is already perfectly complementary to the substrate as shown in the diagrams below (Crierie & Greig, 2010.)

Fig. 1 The induced fit model (BiologyForkids, 2010)

Fig. 2 The lock and key model (BiologyForKids, 2010)

Hypothesis: As the concentration of 2H2O2 increases, the reaction rate will increase. Independent variable: the concentration of 2H2O2.Dependent variable: the reaction rate, measured by time taken for a paper disc to rise to the top of the solution. Constant variables:

thelvolumelofl2H2O2, thelsurfacelarealofl thelpaperldisc, timelbetweenlmixinglreactantslandlrecordinglobservations thelsunlightlexposurelandltheltemperatureloflthelroom Extentloflagitation:lkeptlconstantltolensurelthelsamelconcentrationloflcatalaselwas

administereditoieach trial.

The hypothesis will be supported if the time taken for the disc to rise decreases as the concentration of 2H2O2 increases.

Procedure: Materials--Paper filter discs (6.3mm width and 102 type paper from double rings)-Forceps-Yeast Sachet-Stop Watch-40mL of each 2H2O2 solution (0%, 0.1%, 0.2%, 1%, 3%, 5%)-Distilled water at 30 degree Celsius

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799-Stirring rod-1/2 teaspoon of sugar-50mL measuring cylinder-100mL measuring cylinder-3x50mL beakers-paper towel-Plastic spoon

Method-The yeast suspension was made by adding 1 sachet (7g) of yeast and ½ teaspoon of sugar to 100mL of distilled water warmed to 30 degrees Celsius. 40mL of each concentration of 2H2O2 solution was then measured into clean, separate 50mL beakers. The forceps were then used to dip 1 paper disc into the yeast solution, for 5 seconds. They were then wiped on paper towel and allowed to rest for 10 seconds to remove excess yeast solution. Using the forceps, the soaked disc was then placed at the bottom of the beaker containing 0% 2H2O2. The timer began once the disc was placed at the bottom of the beaker. The timer was then stopped once the disc reached the top of the solution. This time was recorded. If the amount of time exceeded 5 minutes without the disc rising, it was recorded as 300 minutes. This process was repeated to complete 3 trials of each concentration level (0%, 0.1%, 0.2%, 1%, 3%, and 5%)

Time taken for discs to rise (sec)

Concentration of H 2O 2

Trial 1 Trial 2 Trial 3 Average Time (sec)Taken for discs to rise

Reciprocal Average Reaction rate

(sec-1)0 300.0* 300.0* 300.0* 300.0 1/300.0 0.00330.1 161.0 268.0 110.0 179.7 1/179.7 0.00560.2 33.0 11.0 10.0 18.0 1/18.0 0.11111 6.0 7.0 5.0 6.0 1/6.0 0.16673 1.0 3.0 1.0 1.7 1/1.7 0.58825 1.0 1.0 1.0 1.0 1/1.0 1.0000

Results:

*Maximum Time (did not rise)

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799

Graph. 1:

RELATIONSHIP BETWEEN THE CONCENTRATION OF HYDROGEN PEROXIDE AND THE TIME IT TAKES FOR THE DISC TO RISE

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799Graph.2:

THE RELATIONSHIP BETWEEN HYDROGEN EROXIDE CONCENTRATION AND REACTION RATE OF CATALASE ENZYME

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799

Discussion: The investigation explored the effects of varying substrate concentrations in 2H2O2 on reaction rates of catalase in yeast. 2H2O2 is a common by-product of metabolism, which can become highly toxic if accumulated in the body. The enzyme catalase is produced by our liver and acts as a biological catalyst used to speed up the reaction that breaks down the 2H2O2 and releases the products as water and oxygen (Reiner, 2010.) (2H2O2 2H2O + O2).

It was predicted that the reaction of breaking down the 2H2O2 would increase as the substrate concentration increased because the higher concentration provides more freely available substrate molecules resulting in more collisions with the catalase enzyme and a faster reaction rate.

Graph 1 was indicative of a relationship between the concentration and average time for the disk to rise. As 2H2O2 concentration increases, time taken for the disc to float decreases. This is shown in the line of best fit on Graph 1. It can be seen through the range of 300 seconds between the lowest concentration (0%) and the highest concentration (5%) found in the data, as evidenced by the steep decline seen in Graph 1. This trend continues throughout the entire data set and can be shown by differences between the 0.1% concentration and the 5% concentration. The 0.1% concentration achieved results of 0.0056 sec-1, whereas the 5% concentration solution achieved results of 1.0 sec-1. This shows al197.77% difference. This is illustrated by the specified plot points on above graph 2.

An enzyme is a biological catalyst that speeds up a reaction by lowering activation energy.

Reactant Products A+B C+D AB A+B

In this case:

2H 2O2 -----> H 2O + O2

An enzyme-substrate reaction occurs when a specific substrate binds to the active sight of the enzyme. This reaction relies upon the active site being highly specific to allow complementary binding between enzyme and substrate, hence a successful reaction. (Crierie & Greig, 2010)

Fig 3: Process of catalysed reactions (ScientificAmerican, 2014.)

Enzyme

CATALASE

HYDROGEN PEROXIDE

WATER + OXYGEN

Enzyme + Substrate CATALSE + 2H2 O2

Enzyme Substrate Complex

Enzyme +ProductsCATALASE + WATER +OXYGEN

EnzymeEnzymeEnzymeEnzymeEnzyme

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799Ultimately the more particles of 2H2O2, found in the increased concentrations, led to more successful collisions per unit time therefore, the substrate enzyme binding occurred more frequently, allowing more oxygen to be produced, hence speeding up the time taken for the disc to reach the top of the solution, as demonstrated by the formation of bubbles.This decrease in the time taken for the discs to rise can be seen in graph 1. The vast decline represents the massive change in time, ranging between 1 and 300 seconds.

There are other factors that are known to affect the reaction rate of enzymes, including: Temperature and sunlight exposure. Enzymes have an optimum temperature for operation; these extraneous variables such as temperature have an influence on enzyme activity. Previous research papers have presented a theory of catalase saturation. This theory suggests that enzyme activity will get to a point and remain constant as each enzyme’s active sight will be full. (TermPaperWarehouse, 2013, ReviewMyLife, 2012.)This theory is supported when looking at the results found in the data table and graph 1. The results show a great decline in the time taken for the disc to rise but begin to level out slightly once it reached 1.0 seconds for the first time at 3% concentration. The results then begin to level out between 3% and 5% with little range of only 1-3 seconds compared to the overall range between 0% and 5% of 1-300 seconds. The easiest way to attempt to obtain the saturation point would be to increase the range of concentration, up to 10% with intervals of 0.2.

Random errors are statistical fluctuations in the measured data possibly due to the precision limitations of a measurement device, but mainly due to human error (Crierie & Greig, 2010.)These errors undermine the precision of an experiment and are not consistent or maintained throughout the entire practical.There are many factors that could have contributed to the effect of random error found in the results of this practical. A major error noticed whilst undertaking the practical was the misjudgement of the end point. With the necessity of 3 stop watches being in use to measure 3 separate discs being timed at the same time due to time limitations, proved to be extremely difficult and was most likely to be a major cause of imprecision seen in the results. This imprecision is evident when looking at trial 1 of the 0.2% concentration. There is a 23 second range within trials, thus causing significant scatter in the results. Due to the diversity found in teamwork, different perspectives and understanding of the instructions was a noticeable, probable error throughout the experiment. Slight misconceptions were frequently observed through the conflicting opinions of team members as to when the correct time to start and stop the timer was. More evidence of this imprecision and the effect of the aforementioned random errors can be seen in trial 2 of the 0.1% concentration. The results from the first and third trial are reasonably precise with a range of 51 seconds, whereas the range of the entire 0.2% test is 158 seconds. The massive difference in data evidences imprecision and suggest the effect of random error caused by the aforementioned human errors of measurement.

There are some slight fluctuations in data found in the 2nd trial of the 3% concentration test. The results for trial 1 and 3 for that concentration of 1 second not only correlate with each other but with the results found with the 5% concentration (this precision supports the theory of enzyme

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799saturation.) The similarity of results suggests random error most likely affected the data collected from trial 2. Possible and probable means of causation observed through the practical include damage to one of the paper discs through the use of forceps. This would have misshapen a paper disc, causing its buoyance to differ from other unaffected discs. Another observed error was the singular exposure of a disc to un-protected hands causing contamination and the possible presence of un-specified inhibitors. This causes the inability of some substrates and enzymes to undertake the process of complementary binding resulting in less enzyme- substrate reactions, slowing the production of oxygen and hence slowing the time taken for the disc to rise (Crierie & Greig, 2010.) The effect of random errors can be reduced by increasing the sample size by several trials. This experiment would achieve better results with reduced influence of errors by including at least 10 trials for each concentration. Averaging all 10 results from the collected data would help gain a more reliable dataset with a minimized effect of random error. This effect would continue to reduce as the sample size is increased.

Systematic errors are re-producible inaccuracies that are consistently in the same direction.Often equipment derived errors are carried throughout the entire practical, undermining the accuracy of the results (Crierie & Greig, 2010.) As this particular experiment did not involve the knowledge of a true value, accuracy cannot be analysed or discussed. This makes it hard to determine the effect of systematic error in the context of the results recorded for this experiment. Although the exact effect of systematic error and evidence of that effect cannot be discussed in relation to the results, experimental weakness experienced during the process of wiping the yeast soaked disc onto paper towel as an attempt to remove the excess yeast solution was a frequently observed and very probable cause of systematic error. Due to the fact that a measurement device was not used in the process of the removal of the excess yeast, the amount of solution on each disc was inconsistent. This inconsistency of yeast solution means that each trial for each concentration had varying concentration of the catalase enzyme. This affected the results as the higher concentration of catalase enzymes present means more reactions can take place resulting in faster oxygen production hence the disc rising faster. The chemical 2H2O2 is very sensitive and can become contaminated or unknowingly begin the breakdown reaction before the effects of catalase are in place. Each beaker was filled with the different concentrations of 2H2O2 at the same time and was left on the bench on standby until they were needed. Each was tested in ascending numerical order (0, 0.1, 0.2, 1, 3, and 5.) This method resulted in the 5% concentration having more sunlight exposure as it was left in the sunlight longest before use. Because of this, the reactions in the 5% concentration would have already begun, giving it a head start. This effect would have contributed to the results. Although it is a systematic error, as it affects all tests in the same way, the level of effect would have increased proportionately with each concentration. This pattern would have contributed to the trend seen in Graph 1 and Graph 2. There is also equipment or mechanical errors that were observed and would have influenced the results, such as the forceps being unclean or contaminated. Although it is impossible to minimize or reduce the effects of systematic error, it can be determined and considered when analysing results. The methods of error identification would be to repeat the same experiment with slight altercations in condition, this will highlight where the systematic error occurred which can then be considered and worked around.

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799

There are many different improvements that could be made to improve this experiment’s precision, reliability and validity. A great example of a good improvement is increasing the sample size. Increasing the amount of trials for each concentration and including more ranges of concentration will improve the data, mainly the analysis of results. Changing the different ranges in concentration to every 0.2% up to 10% will improve the results as the line of best fit will be more formed as there will be more data to input into a graph. This will therefore increase the evidence of a trend and help identify outliers. Increasing the amount of trials tested for each concentration to 10 or more and calculating the average of those results will begin to reduce the effect of random error. Another improvement could be changing the material used as the source of the catalase enzyme. There were significant problems in consistency when getting the enzyme yeast solution on the disc. It would be an improvement to use other sources of catalase that of which is found in liver or potato. These sources would be easier to maintain a precise enzyme concentration for each test. Other ways of improving this practical is by reducing the effect of random and systematic error. The effect of the random errors seen in this experiment would be reduced if the sample size was increased and averaged as previously mentioned. The effect of the systematic errors seen in this experiment would be identified and therefore considered by repeating the same experiment under the same conditions on a different day using different equipment. Another vast improvement would be to use a more sophisticated means of measurement. Although it is a valid method of measurement, timing how long it takes for a disc to rise is not the most resolute form a measurement. A much more sophisticated and valid form of measurement would be to use an oxygen probe attached to a sensor, this will improve the research method and reduce the effect of random error as it is deriving the actual concentration of oxygen rather than relying on human judgment.

Danni Schwarz, SACE# 928361xDate Conducted: 26/2/15Word Count: 2,799Bibliography:

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