Abstract
Enzymes are biological catalysts and work by speeding up the rate of chemical reactions. The factors that affect enzyme activity include enzyme concentration, temperature, pH, and the substrate concentration. This experiment aimed to determine the effect of lemon juice, which is acidic on the activity of catechol oxidase enzyme. According to the results, high acidity inactivates the catechol oxidase enzyme. The optimum pH level for catechol oxidase enzyme can, therefore, be said to be pH 6.
Introduction
Enzymes are biological catalysts and work by speeding up the rate of chemical reactions. The enzymes achieve this by reducing the amount of activation energy that is needed for a given reaction to proceed (Ophardt, 2003). Enzymes are very specific as they react with one or several similar substrates to produce one or more products. The rate of an enzymatic reaction is usually affected by several factors. These factors include enzyme concentration, temperature, pH, and the substrate concentration (Worthington Biochemical Corporation, 2010). Increasing or decreasing these factors in a reaction may cause a change in the rate at which an enzymatic reaction occurs.
Catechol refers to an organic compound that has a molecular formula C6H4(OH)2. The compound is also called 1,2-dihydroxybenzene or pyrocatechol. There are small quantities of catechol that are found naturally in vegetables and fruits together with an enzyme known as polyphenol oxidase, catechol oxidase or catecholase (Sallee & Clapper, 2007). The enzyme catalyzes the oxidation of the colorless catechol to form a reddish-brown orthoquinone and water. Catechol oxidase enzyme is usually inactivated by acidic conditions such as lemon juice, and the activity reduced by low temperatures. The orthoquinone is a derivative of benzoquinone, which is found in many plants and is one of the major phenols found in argan oil (Charrouf & Guillaume, 2007).
The catechol oxidase reaction is responsible for the browning that occurs when an apple or a potato is cut due to the production of orthoquinone. The brown colored product from this reaction may be used to determine the rate of reaction through the use of a spectrophotometer (Schultz, 2002). The experiment aimed to determine the effect of lemon juice, which is acidic on the activity of catechol oxidase enzyme.
Methodology
The machine was given 15 minutes to warm up and using the % transmittance knob the transmittance was set to zero. The correct wavelength was set at 540nm, and the filter adjusted to make sure it was set to the correct wavelength. The lemon was cut, squeezed and filtered to obtain the lemon juice. Stock solution was made according to the stock solutions chart, and the solutions were allowed to settle for 5 minutes. The pH level of each stock solution was measured before beginning. For each solution, a blank was made by adding the amount of water, lemon and catechol as labeled on the chart. At least 15 small pieces of parafilm were obtained, and the blank was used to practice covering and mixing the solutions by inverting the cuvette once. Using a pipette 6mls of solution 1, 2, and 3 were added into cuvette 1, 2 and 3 respectively. The blank for cuvette 1 was used to zero the spectrophotometer and using a different pipette 2mls of catechol were added to Cuvette 1. The cuvette was covered quickly using parafilm and inverted once to mix. The absorbance of the content was measured and recorded at time 0 for tube 1. The blank for cuvette 2 was used to zero the spectrophotometer and 2mls of catechol added to cuvette 2. The cuvette was covered quickly using parafilm and inverted once to mix. The absorbance of the content was measured and recorded at time 0 for tube 2. The blank for cuvette 3 was used to zero the spectrophotometer and 2mls of catechol added to cuvette 3. The cuvette was covered quickly using parafilm and inverted once to mix. The absorbance of the content was measured and recorded at time 0 for tube 3. After every minute for four minutes, the machine was re-blanked and new absorbance measurements taken for all the three cuvettes. The cuvettes were covered using parafilm and inverted to mix the content. The absorbance readings obtained were recorded. After the four minutes, the pH level of each cuvette was tested and recorded.
Results
The standard solutions were made using a combination of water, potato extract, and lemon juice as shown in Table 1 below.
The absorbance readings at different time intervals were given in the Table 3 below. In the cuvette with low lemon, the absorbance reading increased at a very slow rate when compared to the cuvette that had no lemon juice. On the other hand, the cuvette that had a high amount of lemon showed no change in absorbance. After the four minutes, there was a slight change in color in the cuvette that had slight lemon while the one with no lemon had changed to a golden brown color. There was no color change in the cuvette with high lemon content. There was no pH change in the three cuvettes.
A graph of absorbance against time was drawn (Figure 1) to show the trend in absorbance.
Figure 1: Relationship of absorbance and time for the different lemon concentrations
Discussion
The experiment aimed to determine the effect of lemon juice, which is acidic on the activity of catechol oxidase enzyme. From the results, increase in the amount of lemon juice added reduced significantly the activity of catechol oxidase enzyme. The lemon juice has an acidic nature that affected the enzymatic activity of the enzyme. When the acidity is high there is an alteration of the three dimensional configuration of the enzyme affecting the active site which is involved in catalyzing a reaction (Worthington Biochemical Corporation, 2010). This causes the reaction catalyzed by the enzyme to fail as shown by failure to increase in absorbance reading in the reaction with high lemon. The cuvettes that had low lemon showed a slow increase in absorbance reading since the lemon presence did not affect the activity of the enzyme completely. The cuvette where no lemon was added showed a steady increase in absorbance reading since the pH level (pH 6) was optimum for the catechol oxidase. The rate at this pH was also not very high, and this may have been as a result of lack of optimal level in other factors such as temperature.
The experiment shows that pH has an effect on the enzymatic activity of catechol oxidase and pH 6 may be the optimum pH for the enzyme. Future experiments may test to determine how other factors affecting enzyme affect catechol oxidase and determine the optimum conditions for the enzyme.
Reference List
Charrouf, Z., & Guillaume, D. (2007). Phenols and polyphenols from Argania spinosa. Am J Food Technol, 2, 679-683.
Corporation, W. B. (2010). Introduction to Enzymes. Retrieved October 31, 2013, from http://www.worthington-biochem.com/introbiochem/effectspH.html
Ophardt, C. (2003). Role of Enzymes in Biochemical Reactions. Retrieved October 31, 2013, from http://www.elmhurst.edu/~chm/vchembook/570enzymes.html
Sallee, J., & Clapper, A. (2007). The Effects of Temperature and Chelating Agents on Catechol Oxidation. Retrieved October 31, 2013, from http://socrates8181nc.tripod.com/id9.html
Schultz, D. L. (2002). Biology 155 General Biology I Laboratory Supplement. Retrieved October 31, 2013, from http://www.nicholls.edu/biol-qcf/biol155lab/Labsuppleadobe.pdf
