In my Biology IB Class, I was introduced to enzymes and how they work in different conditions in different conditions, as well as, what inhibits them to function. Regularly, it interests me how what we consume daily impacts our health. But also, how changing our specific diets can improve or deteriorate different aspects of our health. Being an athlete, it's important to consistently maintain a balanced diet, not only to maximise performance levels, but also, promote muscle repair and prevent injuries from occurring. Consequently, I started inquiring about the possible health benefits if we decided to increase our intake of flavonoids and if they have a significant impact upon increasing energy generation and blood glucose concentrations to improve performance output. Hence, I chose to use different types of berries; to explore the effects it has on the breakdown of hydrogen peroxide. Even though there are many health benefits attributed to berries, I wanted to investigate if it has any specific drawbacks as being an inhibitor of the enzyme Catalase.
Enzymes are globular proteins with a specific function that acts as a catalyst in living organisms, thus, they speed up the chemical reactions inside and outside the cell without being altered by the process itself. On the surface level, an enzyme has a region known as the active site, which is where substrate molecules bind and a chemical reaction is catalysed. However, each active site is adapted to one specific substrate, meaning one reaction can occur, this is known as the Lock and key model. As seen in Figure 1, the substrate entering the active site of the enzyme must alter itself slightly as the substrate binds into perfect contact. As a result, an enzyme-substrate complex is formed, and the products are released.
Consequently, the presence of inhibitors can occur; a molecule that disrupts the normal pathway between an enzyme and a substrate (Brent Cornell, 2016). In this experiment, flavonoids are considered to be a competitive inhibitor; they bind to the same active site. As seen in Figure 2, though the shape of the substrates are structurally and chemically similar, the inhibitor blocks the active site and prevents the development of an enzyme substrate complex, thus, no reaction occurs. There are various different types of enzymes in living organisms, but his experiment specifically focuses on the enzyme catalase found in the Celery. The presence of catalase, an antioxidant enzyme found in all living organisms, is integral for the decomposition of 2 hydrogen peroxide to 2 water molecules (H2O) and 1 oxygen molecule (O2). Most importantly, it protects the cells from oxidative damage carried out by reactive oxygen species and maintains an optimum level of the molecule in the cell for cellular signal processes. Inevitably, if there is an exorbitant volume of Catalase, a disease called acatalasemia, a genetic disease formed when catalase level is too low, is activated and the catalase deficiency becomes a potential threat to the cell and the living organisms. Accordingly, the methodology developed attempts to quantify the different effects between fruit species that contain flavonoids, used by 5 different types of berries: Blueberry containing 1100mg/100g dry weight, Raspberries and strawberries containing 500 mg/100g dry weight (Jiyun Lun), Cranberries containing 7.9mg/100g dry weight and blackberries containing 38.8mg/100g dry weight (Croge et al.). They were extracted and converted to liquid form to investigate how each of these inhibitors will affect the volume of oxygen being released. Celery Juice was used to display the breakdown of hydrogen peroxide as it is rich in catalase to sustain health and inhibit the enzymes of transcription factors important in inflammation. This experiment seeks to reciprocate the natural procedure of catalase reactions in the liver through controlled means, creating the optimum medium in which it naturally occurs. As a result, the breakdown of hydrogen peroxide as displayed by the celery juice that is rich in catalase to sustain health, Each of the berry juice was mixed with the celery juice to see to what extent it has inhibited the function of Catalase. If a prominent volume of O2 is released, it indicates the berry used did not interfere significantly with the chemical action, conversely, if a minimal volume of O2 is released, it indicates the berry used worked effectively on slowing down the chemical reaction.
The Alternative Hypothesis (Ha) predicts that as the concentration of flavonoids increases in different species of berries, the inhibitor will become more prominent, decreasing the volume ((cm3) of oxygen (O2) being produced. The prediction is that blueberries will have the greatest effect on preventing the breakdown of hydrogen peroxide because they are anticipated to produce the lowest volume (cm3) of oxygen ( O2) as it has the highest concentration of flavonoids. As a result, they act similarly to mutagens, specifically these pre-oxidants act as inhibitors that prevent release of O2 molecules. of enzymes. Consequently, Blackberries, strawberries and raspberries should produce somewhat the same volume of oxygen as they contain similar concentrations of flavonoids. Whilst, cranberries will produce the highest volume of oxygen (cm3) as it has the lowest concentration of flavonoids. Nonetheless, a Null hypothesis (H0)can occur, whereas there is no difference in the rate of reaction when different concentrations of flavonoids are added. The topic of interest in this investigation is the impact on different species of flavonoids (Strawberry, Blueberry, Raspberry, Blackberry, Cranberry) have on the effect on the breakdown of hydrogen peroxide in Celery juice to supply the enzyme - Catalase. In numerous previous scientific studies (Skibola and Smith), flavonoids was seen as an competitive inhibitor to several enzymatic reactions, decreasing the breakdown of hydrogen peroxide.
Independent Variable -
The Different types of berries used ( Strawberries - Fragaria anassa, Blueberries - Vaccinium sect, Raspberry - Rubus Idaeus, Blackberries - Rubus, Cranberries - Vaccinium subg. Oxygccous). 5cc of each berry were added each trial throughout the experiment.
Dependent Variable -
The Volume of Oxygen (cm3) measured by the Gas Syringe (2 minutes)
A preliminary experiment was done to identify if there needs to be alterations to the method. It was important to determine whether tea or berries ( liquid form ) would work better as an inhibitor for the enzyme Catalase. A control variable of Celery juice ( Hydrogen peroxide ) was used to understand the effect the independent variable had on the dependent variable, as well as, to ensure the experiment results were unskewed.
The methodology developed attempts to quantify the effect of flavonoids on the breakdown of hydrogen peroxide using a Gas syringe to measure the volume of oxygen (O2) released. Hydrogen peroxide, consisting of water and extra oxygen atoms tacked on, is broken down by the enzyme catalase to water and oxygen. This test is composed of a mixture of flavonoids (berries juice), hydrogen peroxide and catalase (celery juice). As all components are mixed together, the hydrogen peroxide reacts with the catalase, but is inhibited with the mixture of flavonoids. Therefore, it's expected that with a larger concentration of flavonoids added in the 2 minute intervals, the lesser volume of oxygen (O2) wll be released.
Safety - Hydrogen peroxide can cause severe eye irritation, burns, and respiratory system when in contact with the skin, Therefore, goggles and lab coats must be worn when handling the acid to prevent any injuries. Additionally, the use of glass beakers and conical flasks must be handled with precaution to prevent any cuts to the conduct/peer. If an accident occurs, immediately rinse the contaminated skin and inform a teacher or supervisor.
Ethical - There were no ethical implications to be considered throughout the investigation as non living organisms were used in the procedure.
Environmental - The hydrogen peroxide used in this experiment was safely poured into waste bins to prevent pollution in the water supply. Furthermore, all berries used were disposed properly through composting, a conventional trash bin or rescuing the leftovers parts if possible. Only the minimum amount of each berries were used to avoid food wastage.
*All data are rounded to 2 significant figures
*cm3 = Volume of gas syringe
Qualitative Data - Throughout the experiment, there were many observations made on the amount of oxygen released by the reaction after being inhibited by berries (flavonoids).
In all trials, there was never any distinctive smell that was released from the solution besides the smell of the berries. The foam foamed in all trials was created because celery contains an enzyme called catalase, as it comes into contact with hydrogen peroxide (H202), this reaction happens so quickly, the bubbles formed are pure oxygen bubbles created by catalase. In general, there was an inverse relationship because as the concentration of flavonoids increased, the amount of foam decreased.
Decision for uncertainties - The uncertainty for the volume of oxygen released +/- 0.5cc, was used as the gas syringe used in the experiment changed by 1, not more or less, meaning, the volume oxygen released changed by 1, therefore it was unable to measure the volume of 17.6cc of 22.3 as an example. Therefore, this unit was kept constant throughout the mean and standard deviation,
*all data rounded to 0 decimal places
*The error bars represent the standard deviation for the different types of berries
T-Test - As the investigation's main objective is to determine if there’s a correlation between both variables, a t-test was used to determine the significance between two groups. In this experiment, the significance was determined on a 95% confidence interval.
Type of berries | T-Test Value | Significance difference/no significant difference |
---|---|---|
Raspberries and Strawberries | P = 0.027993 | Since 0.027993 > 0.05, there is insufficient evidence to reject the null hypothesis and thus, |
support the alternate hypothesis as there is no significant difference between both berries. | ||
Blueberries and Raspberries | P = 0.80274 | Since 0.80274 > 0.05, we reject the alternative hypothesis as there is sufficient evidence to support the null hypothesis as there are significant berries between both berries. |
Figure 11 - Table On Which Shows The T-Test With Error Bars That Overlap
*There were other overlapping errors but it was not calculated because it was prominent that the T-Test value was P< 0.05. *Rounded to 5 significant figures
Type of Berry | Anomalies Range (uncertainty +/-0.5cm3) | Anomalies present (uncertainty +/-0.5cm3) | Reason for anomaly present |
---|---|---|---|
Cranberry | Lower bound - 31 Upper bound - 35 | Trial 3 = 41 oxygen molecules (O2) released | Human error, accidentally forgot to immediately turn on the magnetic error after injecting the celery juice and cranberry juice. Thus, the rate of inhibition decreased because the solution was not mixed at the start. |
Figure 12 - Table On Which Shows Any Anomalies Present For Each Berry In The Experiment
he alternate hypothesis (H1) stated: as the concentration of flavonoids increases in different species of berries (strawberry, blueberry, raspberry, blackberry, cranberry), the inhibitor will become more prominent in restricting the breakdown of hydrogen peroxide by the enzyme catalase. As illustrated by the processed data and graph above, the results are mostly in accordance with the alternate hypothesis indicating a strong inverse relationship between the concentration of flavonoids and the volume of oxygen (O2) released by the enzyme reaction. As shown on the graph, all the data was according to the hypothesis with the controlled variable expected to release the greatest amount of oxygen at a mean volume of 45cm2 because there's no inhibition on the catalase enzyme, thus it proceeds to decompose the hydrogen peroxide (H202) into oxygen and water. Additionally, cranberries released a mean volume of 33cm3 oxygen molecules, this was anticipated because it states in the background hypothesis the concentration of flavonoids is 7.9mg/100g per dry weight. The qualitative data also indicates both these claims, with the fairly large amount of foam generated indicating pure oxygen bubbles being created by the catalase.
The hypothesis expected both Raspberries and Cranberries to have relatively similar results because they both have the same flavonoid concentration of 500 mg/100g per dry weight (Jiyun Lun, 2020). The results contradict this claim as illustrated in the graph, raspberries were proven to inhibit the enzymatic activity of the hydrogen peroxide by releasing a mean volume of 15cm3 of oxygen molecules. Whilst, strawberries worked sufficient enough as an inhibitor, releasing a mean volume of 21cm3 of oxygen molecules. But when taking into consideration that both fruits have the sameflavonoid concentration. it was more ineffective. On the other hand, blueberries and blackberries had somewhat unexpected results. Whilst having the lowest mean volume of oxygen molecules at 15cm3 for blueberries and 6cm3 for blackberries. These results differ from the prediction made in the Alternate hypothesis, suggesting that blueberries were supposed to inhibit the hydrogen peroxide greater because they had the highest concentration of flavonoids of 1100mg/100g in contrast to blackberries (Jiyun Lun) containing 38.8mg/100g per dry weight (Croge et al.). This may be explained through the biological variation of both fruits in terms of age as fruits undergo significant changes during ripening. Although they were kept in the same conditions, the raspberries had an earlier harvest date. Because of heat, oxygen and light, the nutrients (flavonoids) are lost because enzyme activity is slowed down and decreases the synthesis pathway of flavonoids. However, it's possible that the human errors made when starting the experiment, injecting the juice into the Hydrogen peroxide and turning on the magnetic stirrer contributed to these results. The reaction time and preciseness of measurement of both the berry juice and celery juice could also have been inaccurate. Likely, with a partner, these errors could be eliminated and blueberries will release a smaller volume of oxygen molecules (cm3)
The standard deviation identifies how to spread the recorded data from the average or expected value. It is represented in the error bars on the data point in the bar graph. The greatest standard deviation is at 6 for raspberries, exemplifying that majority of the data recorded as far away from the mean value. Strawberries also had a relatively large standard deviation of 4. This can potentially mean for both berries there were some consequential errors made in this independent variable. The standard deviation for Blackberries and cranberries had the smallest size error par and the same because they were the first two berries being tested, promoting there may have been fewer human errors made. Moreover, Blueberries and the control variable were also the same with a standard deviation of 2.
Outliers - Although no anomalies had been detected in the data besides the Raspberries due to human error as mentioned above because the variation is negligible to the average. There are some outliers in Trial 3 of the strawberries, all having unusually small volumes of oxygen released compared to the rest of the trials. As noted, this was due to a human error of mixing up the cranberry syringe with the blueberry syringe, mixing both juices. Therefore, because blueberries contain more flavonoids, they increased the flavonoids concentration and inhibited the catalase activity more.
It can be concluded that the results obtained support the alternative hypothesis and suggest that there is an inverse relationship between the concentration of flavonoids and the mean amount of oxygen released (cm3) for all berries. In comparison to the Null hypothesis which states there were no changes. There is conclusive evidence that blueberries had the greatest concentration of flavonoids and thus, are an inhibiting factor of the enzyme catalase by producing the least volume of oxygen molecules. Research has shown that computer inhibitors disrupt the normal pathway between an enzyme (catalase) and a substrate (hydrogen peroxide), limiting the decomposition of the substrate into water and oxygen molecules. Additionally, according to Pubmed (Skibola and Smith), the abstracted state aligned with the conclusion made in this experiment, that the inhibition is caused by the formation of hydrogen bonds between catalase and flavonoids. Hence, the greater the concentration of flavonoids, the less effect the inhibition has on the enzyme. However, the difference between the experiments was that Pudmed focused on the relationship between the molecule structure and the degree of inhibition.
Observing that higher concentrations of flavonoids may result in the lower inhibition of the enzyme catalase may institute that people should adjust their diet to consumer berries that have higher concentrations, such as blueberries and blackberries as investigated in this experiment. As flavonoids are an antioxidant, they may help your body remove hydrogen peroxide at a more efficient rate. Therefore they help protect cells, prevent damage related to ageing and chronic conditions, and most importantly it helps maintain an optimum level for the molecule in the cell for cellular significant processes to function.
Strength - Aside from finding all the berries in the local supermarket, all materials used are relatively easily obtained from any standard laboratory. The highly controlled lab environment helped minimise the risk of contamination and increased the accuracy of the data collected, forming a strong inverse relationship between both variables. The wide range of berries (blueberry, raspberry, blackberry, strawberry, cranberry) and multiple controls (ie. duration, correct volume/concentration, time, temperature) kept consistent, it contributed to more precise results further supporting the conclusion and making the data more reliable. Additionally, having 8 trials per berry helped reduce the standard deviation, whilst enabling more comparable results, increased precision and increased the confidence in the data.
Source of error | Improvements | Why it's significant |
---|---|---|
1) The time is taken between injecting the catalase (celery juice) or flavonoids (berry juice), turning on the magnetic stirrer and starting the stopwatch - random error | Ask a partner to help start the stopwatch whilst the conductor injects the catalase (celery juice)and flavonoids (berry juice). Start the magnetic stirrer prior to the trial. | Because the time between injecting both solutions and the stopwatch is different, this can increase/decrease the rate of reaction and thus, the amount of oxygen released. |
2) Slight difference in the berry to water ratio as it was difficult to measure the exactly 170g of berry to 10ml of water without a 10ml syringe as the juices were prepared at home, not a laboratory - Systematic error | Even though a 10ml syringe was not available at that time, using a measuring spoon can be a more accurate tool in measuring volume with less uncertainty attributed to it compared to a measuring cup. | Because if there's more water in one of the berry juice than in another, this can decrease the concentration of flavonoids. |
3) Limited time and number of trials - Systematic error | Plan more time and do as many repeats as possible. A 10 trials minimum would reduce the likelihood of methodological and measurement error impacting the final results. Any errors would have been indefinite and balanced out. | Due to time constraints, it was only possible to test 2 minutes per trial and only 8 trials were conducted per berry. The longer the time, the greater the amount of product will be formed. The more trials there are the more the standard deviation will be reduced allowing more precise results. |
4) The harvest date of each berry - Systematic error | Placing the berries in a cold environment to avoid them becoming soggy or mouldy. As well as placing it in a shallow container, spread over a single layer; loosely covered with a clean sheet of paper. However, this error cant be fully eliminated. | There was possible biological variation between the berries as all of them can't be harvested at the same time. The earlier the berry was harvested, the less amount of flavonoids it contained due to underlying factors before purchase (temp, light, air). |
5) Human error when returning the gas syringe to empty before the start of each trial - Random error | Setting a reminder after each trial to push the plunger back to the bottom of the barrel will limit this error | Because if the plunger was not returned to the tip of the syringe before each trial, this could mix up the volume of oxygen released between 2 trials and interfere with the accuracy of the data. |
Figure 13 - Table On Several Sources Of Error, Improvements And Why Its Significant
As flavonoids are a common antioxidant found in various types of berries, it would be useful to extend the research into a broader range of berries instead of simply the most common ones: blueberry, raspberry, strawberry, blackberry, cranberries. Moreover, there have been limited attempts made at researching the relationship between flavonoids as an inhibitor for the enzyme catalase, this extended research could enable a better understanding of the benefits of flavonoids on our body, thus by increasing the range it helps determine which berries are likely better/worse impactful for our health.
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