Inhibition of catechol oxidase enzyme in bananas by heavy metals

Does varying concentrations of the heavy metal solution lead ethanoate (0% , 1% , 2% , 3% , 4% , 5%) effect the rate of enzyme activity of catechol oxidase in Musa acuminata Cavendish(bananas), measured using optical density ?

Growing up chopped apples were a regular in my snack box and at the end of the day it would return looking exactly like how it looked when it was packed in the morning , as I found the brown apples to be unappetizing and so I would ask her to pack the fruit whole so I could enjoy it . One day I was eating some chopped apples and they tasted sour but they looked perfectly alright and I found out that if you squeezed some lime on them it would stop them from browning. This intrigued me. I wondered what else would help stop this browning process and what exactly caused the browning .

Growing up in Kerala , bananas were a native fruit and I noticed a similar browning in them too except it took much longer . During middle school I came to learn that this process was due to an enzyme-substrate reaction that took place in the presence of oxygen and it helped protect the fruit from getting spoiled. I wondered what else would slow down this enzyme activity apart from enzyme concentrations , substrate concentrations and extreme temperatures and pH. This is when I stumbled across the idea of inhibitors and the different types of inhibitors ranging from competitive , non-competitive , reversible and irreversible. I found this extremely fascinating.

Catechol oxidase is an enzyme found in plant tissues . It oxidizes (process of removal of hydrogen) its substrate catechol which is also found within the plant . Under normal conditions both catechol and catechol oxidase do not come together. However , when a plant cell is damaged catechol oxidase is released and comes in contact with its substrate catechol (colorless compound) in the presence of oxygen to form benzoquinone (orange compound) which is the compound that causes the brown color.

This benzoquinone is toxic to bacteria and helps prevent and slow down the decay within the damaged plant tissue .Heavy metals act as competitive inhibitors and are reversible inhibitors . The heavy metal competes with the substrate catechol to bind to the active site of the enzyme catechol oxidase and can even bind in between an enzyme-substrate complex. This binding is however reversible sosome substrate molecules will eventually bind to the active site of the enzyme and be converted to its product. In case of a reversible inhibitor once removed it will allow the enzyme’s active site to bind with the substrate ,catechol, as the conformation of the active site remains the same unlike irreversible competitive inhibitors. Heavy metals inhibit enzymatic activity by reacting with the S-H group of cysteine bonds,forming a covalent bond with sulfur atom and displacing the hydrogen ion.This causes the enzyme to loses it ability to catalyze reactions. I chose to use lead as my source of heavy metal as lead compounds are much less toxic in comparison to other heavy metal compounds and are easier to dispose safely.

Catechol oxidase being an enzyme its activity is inhibited by heavy metals (lead ethanoate) which are toxic in nature . The heavy metal binds to the active site resulting in the formation of fewer enzyme-substrate complexes , hence reducing rate of enzyme activity. As a result the browning effect reduces.

Catechol oxidase is a photochemical belonging to the class of polyphenol oxidase. It behaves as an enzyme in the process of oxidizing aromatic compounds especially bi phenolic rings to quinines which finally polymerises to form melanin pigments in cut fruits and plants. In plants they are mainly found within the chloroplasts and are often released during ripening stage. Catechol oxidase is mainly responsible for the dark brown colouration of fruits and vegetables which often leads to loss of nutrients.

The term heavy metals used here refers to certain specific metals in particular like Lead, silver, Copper and Arsenic. These metals are poisonous and are found to behave as non competitive inhibitors for certain enzymes. They bind to the enzyme in a site other than the active site. As a result, the shape of the active site of the enzyme changes and it is no longer capable to bind the substrate.

The basic aim of the investigation is to measure the effect of concentration of lead ( as heavy metal) on the catalytic activity of catechol oxidase. Catechol oxidase oxidizes catechol, a colourless product into a brown coloured compound called benzoquinone. This brown coloured compound polymerizes to form a brown pigment known as melanin.

The development of orange colour is the main tool to monitor the rate or effectiveness of this reaction. As the enzyme becomes more effective, more substrates (catechol) oxidizes to benzoquinone and thus more melanin is formed. Hence the orange colour formed gets deeper. The intensity of orange colouration can easily be measured using the optical density. Higher the optical density, more the amount of benzoquinone formed and thus more the effectiveness of the enzyme catechol oxidase.

Null hypothesis:

The activity of catechol oxidase has no correlation with the concentration of heavy metal solution (Lead ethanoate) used.

Alternate hypothesis:

The activity of catechol oxidase bears a correlation with the concentration of heavy metal solution (Lead ethanoate). The correlation is not an outcome of a random error.

Buffer solution (pH 7) preparation

• 4.20 g of powdered citric acid was weighed and placed into a beaker.
• 220 cm³ of distilled water was measured using a measuring cylinder and transferred into the beaker and stirred with a glass rod to form 0.1 M citric acid.
• 19.31 g of powdered disodium hydrogen phosphate was weighed and placed into a beaker.
• 680 cm³ of distilled water was measured using a measuring cylinder and transferred into the beaker and stirred with a glass rod to form 0.2 M disodium hydrogen phosphate.
• 200 cm³ of 0.1 M citric acid, 680 cm³ of 0.2 M disodium hydrogen phosphate and 220 cm³ of distilled water was mixed to form pH 7 buffer.
• Buffer was stored in reagent bottles.

Catechol solution preparation

• 1g of Catechol flakes was weighed and placed into a beaker.
• 100 ml of distilled water was poured into a beaker and stirred to form 1% catechol solution.
• Solution is sensitive to light hence it was stored in a reagent bottle in a dark cupboard.

Enzyme extraction

• 8g of banana was weighed.
• Banana was grinded using a blender to form a paste.
• Mixture was strained through a muslin clothes to help remove seeds and clumps in the paste.
• This is the source of the enzyme catechol oxidase.

Experimental procedure

• 10 test tube was placed in test tube rack in a water bath at 30.0 °C.
• 1 cm³ catechol solution was measured using a pipette and poured into the test tube.
• 1 cm³ of the catechol oxidase solution was measured using a pipette and poured into the test tube.
• 5 cm³ of the pH 7 buffer solution was measured in a 10 cm³ measuring cylinder and poured into the test tube.
• 1 cm³ of the lead ethanoate solution was measured using a pipette and poured into the test tube.
• Stopwatch was started and the test tube was allowed to rest for 10 minutes
• Spectrometer was set to 450 nm wavelength (Orange).
• Distilled water was poured into the cuvette and was used as a blank and was poured out.
• After 10 minutes, the contents of the test tube was poured into the cuvette with the help of a funnel lined with filter paper.
• Cuvette was placed into the spectrometer and the optical density value was recorded.
• The process was repeated for 9 more trials for each concentration.
• A control was done by following the steps above and omitting the 1 cm³ of Lead ethanoate solution.

There are no ethical concerns in the following experiment .

The average values are calculated for each concentration by using the formula:

Average =  All trial values for a particular concentration÷ Number of trials(10)

The graph shows a clear downward sloping trend. As concentration of lead ethanoate increases the optical density decreases. This can be explained as by increasing the concentration of the inhibitor, fewer enzyme substrate complexes form hence enzyme activity reduces as seen by the lower optical density due to a lesser browning effect which is originally caused due to the formation of enzyme-substrate complexes.No anomalous data is to be seen.

The R² value (0.998) is very close to 1 denoting a strong negative correlation between the concentration of lead ethanoate and the average optical density. The error bars are plotted to show to standard deviation on each data point. Its small size denotes there is a low chance of error. Therefore, indicating a relatively high sense of accuracy.

Sample calculation:

For 1.00 % Lead ethanoate solution:

Percentage inhibition = \(\frac{(0.72-0.63)}{0.72}\) X 100 = 12.50

The scattered plot above depicts the variation of percentage inhibition against the concentration of Lead ethanoate used. The concentration of Lead ethanoate being the independent variable is plotted along the x axes while the percentage inhibition being the dependent variable is plotted along the y axes. As clearly indicated in the graph, there is a positive correlation observed between the percentage inhibition and concentration of Lead ethanoate used. As the concentration of Lead ethanoate used increases from 1.00 % to 5.00 %, the percentage inhibition increases from 12.50% to 54.17%. The magnitude of R² (0.999) indicates a strong positive correlation between the percentage inhibition and the concentration of Lead ethanoate used.

A t test helps determine if there is any significant change between the means of 2 groups of data. It is used to test the hypothesis.

At significance level 0.05 and a degree of freedom of 18 the t critical value is 2.101 .

The calculated t value is larger than the critical table t value at 0.05 significance level with an 18 degree of freedom. Therefore the null hypothesis can be rejected. The alternate hypothesis is accepted : As concentration of lead ethanoate increases , the rate of enzyme activity of catechol oxidase decreases , optical density decreases.

The table below shows a summary of the results in our experiment as well as an ANOVA test.

This test summary shows us the change in optical density with change in concentration of lead ethanoate. The value of P in the test is 3.83249E-46 which is lesser than 5 which shows that the data we have taken are significantly different. From the above table, it can be seen that the F value (607.4136461) is greater than the F critical value (2.386069862); this means that the null hypothesis (As concentration of lead ethanoate increases , there is no effect on the enzyme activity of catechol oxidase , optical density remains the same) . The alternate hypothesis is accepted : As concentration of lead ethanoate increases , the rate of enzyme activity of catechol oxidase decreases , optical density decreases.

From the results of the experiment, I can come to a conclusion that as the concentration of lead ethanoate increases, the average optical density decreases as there is a decrease in the browning effect. The browning effect is a protective mechanism resulting due to the formation of enzyme-substrate (catechol oxidase- catechol) complexes in the presence of oxygen. With an increase in concentration of lead ethanoate, there is a greater number of lead ethanoate molecules in a given volume. Therefore as there are greater number of lead ethanoate molecules there is a higher probability of these competitive inhibitors colliding with the active site of the enzyme with the right orientation. They then occupy the active site of the enzyme and inhibits the typical enzyme-substrate complexes from forming. These successful collisions lead to the formation of enzyme- inhibitor complexes. This leads to fewer active sites of the catechol oxidase enzyme to bind with the substrate - catechol-and give rise to the browning effect. With fewer active sites left for the substrate, as the inhibitor concentration increases the browning effect reduces. When the inhibitor is not present (control) all of the enzyme’s active sites are free to be occupied by its substrate giving rise to a significant browning effect as seen by the high optical density. As these are reversible inhibitors, with an increase in concentration of the substrate there would be more substrate molecules in a given volume resulting in a higher probability of the substrate colliding with the active site of the enzyme in the proper orientation to form a greater number of enzyme-substrate complexes as a result of these successful collisions. This will reduce the effect of the inhibitor on the particular reaction.

Systematic errors are those caused by incorrectly calibrated or tared instruments. This experiment has a relatively low level of systematic error as the uncertainty of the measuring scale used is ±0.01. The error bars in the graph are small, indicating a low chance of error. This makes the data collected quite accurate. A sufficient number of trials (10) were taken in order to ensure the results are consistent and are not greatly varied by random errors. This helps us get a more accurate average value.

A possible extension to this experiment would be to conduct a further study on the effect of browning when natural inhibitors such as : lime juice, lemon juice, pineapple juice and salt solution are used in varying concentrations over an extensive period of time (24 hours) by recording the optical density in hourly intervals. This experiment can draw a conclusion as to which natural juice would be most effective in reducing the browning effect and as to how long it remains to be effective.