- The Effect of pH and Enzyme Concentration on Enzyme Activity
- Abstract
Enzymes help many chemical reactions to occur in living organisms. Enzymes are very specific molecules, each enzyme has a certain type of active site necessary for the reaction to happen. The reactions are very sensitive to changes in pH and enzyme concentration. Changes in the pH can drastically affect the solubility, structure and stability of the enzyme while the enzyme concentration correlates to the reaction rate. The objective of this experiment was to locate the pH where the absorption for the enzyme was the highest and how they can affect the reaction of a solution. After making solutions and adding catechol the reaction rate and the absorbance rate could be measured by using a spectrophotometer set at a wavelength of 420nm. The enzyme is catecholase and in exercise A it was discovered that a pH of 8 has the most efficient enzyme absorbance. In exercise B it was found out that over time the absorbance would increase in relation to the amount of potato juice in the solution. In conclusion the catecholase inside the potato juice was the cause of the increase in absorbance because the enzyme concentration was increasing.
- Introduction
Enzymes are characterized by two fundamental properties. First they increase the rate that chemical reactions happen and second they lower the activation energy necessary for the reactions to happen (Cooper 2000). However, enzymes are sensitive to changes in the environment around it, like changes to the temperature and pH. The goal of the exercise A was to observe the different absorbance rates at different pH levels. The hypothesis is, if the pH of the enzymes environment changes then there will be a direct result on the enzyme activity. The null hypothesis is, if there is a change in an enzymes environment then there wont be an effect on the activity of the enzyme. Exercise B wanted to support the hypothesis, the concentration of the enzyme has a direct affect on the rate on the reaction in the solution. Therefore the null hypothesis is that enzyme concentration in a solution does not have a relationship with the reaction rate.
- Materials and methods
In Exercise A 5 blanks were made by filling cuvettes with 1mL of Di H2O, 1mL of potato juice and 9mL of the correct pH buffer (4,6,7,8,10). Label the cuvettes according to Table 6.1 then calibrate the spectrophotometer.
Table 6.1 Enzyme pH Blank Setup
Test Tube | pH Buffer | Potato Juice | Water | Total Volume |
pH 4B | 9 mL of pH 4 | 1 mL | 1 mL | =11 mL |
pH 6B | 9 mL of pH 6 | 1 mL | 1 mL | =11 mL |
pH 7B | 9 mL of pH 7 | 1 mL | 1 mL | =11 mL |
pH 8B | 9 mL of pH 8 | 1 mL | 1 mL | =11 mL |
pH 10B | 9 mL of pH 10 | 1 mL | 1 mL | =11 mL |
After preparing the 5 blanks the 5 experimental test tubes must be setup. Make 5 more blanks following Table 6.2 by filling the cuvettes with 1 mL Di H2O, 1mL of potato juice and 9mL of the correct pH buffer. After all the solutions are prepared add the 1 mL of catechol to the test tubes and cover with parafilm.
Table 6.2 Enzyme pH Experimental Setup
Test Tube | pH Buffer | Potato Juice | Water | Catechol | Total Volume |
pH 4 | 9 mL of pH 4 | 1 mL | 1 mL | 1 mL | =12mL |
pH 6 | 9 mL of pH 6 | 1 mL | 1 mL | 1 mL | =12mL |
pH 7 | 9 mL of pH 7 | 1 mL | 1 mL | 1 mL | =12mL |
pH 8 | 9 mL of pH 8 | 1 mL | 1 mL | 1 mL | =12mL |
pH 10 | 9 mL of pH 10 | 1 mL | 1 mL | 1 mL | =12mL |
Give 5 minutes for reaction time, inverting the test tubes in one minute intervals. After the 5 minutes of reaction time fill a cuvette with solution pH 4B and blank the spectrophotometer and then fill another cuvette with pH 4 solution and measure the absorbance of the solution. Note the results and repeat the process for the remaining test tubes.