This lab was performed in order to discover the activity of the enzyme catecholase in different pH levels as well as its absorbance in differently concentrated solutions. A spetrophotometer was used to measure the absorbance of the enzyme catecholase in different pH solutions as well as to measure the absorbance of catecholase in solutions with different concentrations of potato juice and phosphate buffers. Absorbance of the enzyme catecholase was at an optimum level when pH was close to neutral. When pH was acidic or basic, the catecholase was less effective. Also, when there was a higher concentration of potato juice and a lower concentration of phosphate buffer, absorbance of the enzyme increased.
According to Edmund J. Stellwag, in his article "Enzyme" an enzyme is “a catalytic protein produced by living cells.” Enzymes function as catalysts by lowering the amount of energy required for a reaction to occur. “Enzymes already have a wide range of applications, including chemical synthesis, biodegradation of harmful chemicals, environmental and medical diagnostics, medical therapeutics, food manufacturing, detergents, and agriculture” (Kuchner). Because enzymes are biological molecules, changes in pH can greatly change their effectivity in chemical reactions. A change in the concentration of enzymes in a given area can also greatly affect the rate of reaction.
The purpose of this lab was to observe and to record the activity of the enzyme catecholase in different pH levels as well as its effectivity in different concentration levels.
Hypothesis A: If pH level increases (becomes more basic), then absorbance of the enzyme catecholase will increase.
Null Hypothesis A: If pH levels increase, then absorbance of the enzyme catecholase will decrease.
Hypothesis B: If the concentration of potato juice in an area increases and phosphate buffer decreases, then absorbance will increase.
Null Hypothesis B: If the concentration of phosphate buffer in an area increases and potato juice decreases, then absorbance will increase.
Materials and Methods
Laboratory number two was used as a reference for using the spectrophotometer. The wavelength of the spectrophotometer was set to 420 nm. Five test tube blanks were prepared, each containing 9mL of a different pH buffer, 1mL of potato juice, and 1mL of water. Each blank was labeled using the appropriate pH buffer followed by the letter ‘B’. Five more test tubes were prepared in the same manner except 1mL of catechol was added to each of these tubes. Parafilm was used as a cover and the tubes were inverted at one minute intervals for five minutes. After these five minutes, a cuvette was filled with the test tube pH 4B solution and was used to blank the spectrophotometer. Another cuvette was filled with the test tube pH 4 solution and was placed in the spectrophotometer to measure the absorbance of the solution. This procedure was done for all of the remaining test tubes. All results were recorded.
Four test tubes were labeled, and each was filled differently and mixed thoroughly using paraffin and introverting rapidly: one with 11mL of pH 7 phosphate buffer, one with 10.5mL pH 7 phosphate buffer and 0.5mL of potato juice, one with 10mL of pH 7 phosphate buffer and 1mL of potato juice, and one with 9mL of pH 7 phosphate buffer and 2mL of potato juice. All tubes contained 1mL of water (these tubes were the blanks). Four more test tubes were filled the same way except with 1mL catechol in place of the 1mL of water in each tube. The catechol was not added until absorbance was ready to be measured. All solutions were poured into separate cuvettes. Absorbance of the enzymes in the solutions was measured using the spectrophotometer in the same manner as in Exercise A (using blanks in between each solution measure). Each tube was measured seven times (once per minute) and absorbance was recorded for each...
Cited: Daniel Wellner, G. P. Royer, Edmund J. Stellwag, "Enzyme," in AccessScience, McGraw-Hill Companies, 2008, http://www.accessscience.com
Neil A. Campbell, Jane B. Reece, Lisa Andrea Urry, Michael L. Cain, Steven Alexander Wasserman, Peter V. Minorsky, Robert Bradley Jackson. Biology Ninth Edition. San Francisco: Pearson, Benjamin Cummings, 2011. Print.
Olga Kuchner, "Enzymes and directed molecular evolution," in AccessScience, McGraw-Hill Companies, 1998, http://www.accessscience.com
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