Lab Report: Effect of pH on Enzyme Activity
Introduction
Enzymes are important in human life because they control almost every biochemical reaction in the body. The enzyme activity is influenced by several factors such as temperature. Another important factor is the environmental condition determined by the concentration of hydrogen in solution. The concentration of hydrogen is a measure of its pH. In a similar way to temperature, pH also interferes with the three dimensional geometry of the enzymes. This geometrical disorientation regulates the function of the enzymes. Individual enzymes operate best at optimum pH (Brooker et al., 2011). Different enzymes have their operating pH in the basic or acidic range. However, most enzymes actively function at near neutral pH. At conditions above or below the optimum pH, the enzyme activity decreases. (Audesirk, Audesirk & Byers, 2008) The optimum pH of an individual enzyme corresponds to its natural environment’s pH.
This experiment aims at investigating the range of pH over which catechol oxidase enzyme catalyses its substrate. The experiment also determines the optimum Ph at which the catechol oxidase functions best. This experiment is based on the hypothesis that extreme pH values denature enzymes, therefore, making them less active.
Materials and Methods
The materials used in this experiment include seven clean dry test tubes, a metric ruler, and china marker, wash bottle containing 1% catechol and ice bath with wash bottle of potato extract containing catechol oxidase. In addition, a water bath at 40 degrees Celsius and phosphate buffer series with pH range between 2 to 12. In this case the phosphate buffer has pH values of 2, 4, 6, 7, 10, and 12.
Seven test tubes are labelled from 3A to 3G with china marker. The test tubes are then laid against the metric ruler and lines indicating 4cm, 5cm, and 6cm marked from the bottom of each tube. Each of the test tubes is filled with the phosphate buffer series to the four centimetre mark. Each of them has a different pH value. One centimetre of potato extract containing catechol oxidase is added to each of the seven test tubes to bring the total volume to five centimetres. The tubes are agitated with the hand to ensure regular mixing. Another one centimetre of 1% catechol is further added to bring the total volume of the test tubes’ content to six centimetres. At time zero, immediately after adding the 1% catechol, the colour intensity of each test tube is noted, and the corresponding pH value recorded in table 1. The tubes are placed in the water bath at 40 degrees Celsius. For the next ten minutes, the test tubes are agitated periodically to ensure uniform distribution of heat. The hypothesis addressed by this experiment at this point is that the pH of a substrate and an enzyme determine the amount of product formed (Pascale et al., 2005). After ten minutes, the test tubes are removed from the water bath.
Results
Relative colour intensity
Time
(minutes) Tube 3A
(pH 2) Tube 3B
(pH 4) Tube 3C
(pH 6) Tube 3D
(pH 7) Tube 3E
(pH 8) Tube 3F
(pH 10) Tube 3G
(pH 12)
0 2 3 4 5 3 3 1
10 2 3 5 4 4 2 1
Discussion
From the results, it is observed that the rate changes in pH levels affect the speed of reaction. At time zero, the rate of reaction as observed fro the colour intensity of the product shows a gradual increase in the activity of the enzyme as the ph level increases from pH 2 to pH 7. However, as the ph increases further, the colour intensity decreases showing a decrease in the enzyme activity because less products are formed. The lowest rate of reaction is at a ph level of 12. This shows that the enzyme activity is very low at extreme basic conditions.
The results obtained after raising the temperature of the solution to 40 degrees Celsius shows only a slight difference with highest rate of reaction at pH 6. The optimum pH at which the catechol oxidase enzyme can operate is nearly neutral. This further supports the hypothesis that enzymes operate best at neutral pH levels, but are denatured at very low and very high pH levels.
In this experiment, there are variations in the rate of reactions that may have been as a result of human error. Slight delay in transfer of the test tubes from the hot water bath, or non uniform agitation to ensure equal distribution of heat is some sources of errors in this experiment.
Conclusion
From the experiment, it can be concluded that a change in pH alters the rate of reaction. The optimum pH of catechol oxidase appears to be between Ph 6 and pH 7. The results obtained shows that the enzyme catalase present in potatoes is affected by extreme pH. However, at temperatures higher than the normal body temperature, the activity decreases. At forty degrees Celsius, the enzyme is denatured and the rate at which it catalyses the reaction decreases.
References
Brooker, R. J. (2011). Eric, P., Graham, L. E. & Stilling P. D. (2011). Biology, 2nd Ed. New York: McGraw Hill.
Pascale, T., Noirot, M. & Kochiko, A. (2005). The Mechanism of Action of Alpha-amylase from Lactobacillus Fermentum on Maltooligosaccharides. Journal of Chromatography 42-47.
Audesirk, T., Audesirk, G., Byers, B.E. (2008). Life on Earth, 5th Ed. San Francisco: Pearson Benjamin Cummings.
Suffolk County Community College. (2005). Principles of Biology Laboratory Manual. Boston: McGraw Hill Publishing.
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