Introduction
An enzyme is a protein with catalytic properties in biological systems. During an enzymatic reaction, a substance, called the substrate, binds to the active site of the enzyme and is then turned into one or more substance(s), called the product(s). The enzyme itself is not consumed or altered during the reaction. The enzyme activity depends on factors, including pH, temperature, buffer composition, enzyme concentration, substrate concentration, and the presence of inhibitors.
Enzymes have an optimal pH at which their activity is maximal. pH determines the ionization states of certain amino acids at the active site that are critical to enzyme activity (Nelson, 204). The changes in the ionization states may also affect the interactions that maintain protein structure (Nelson, 204). In addition, shift in pH may change the ionization state and structure of the substrate (Nelson, 204). The optimal pH varies from 1.5 to 10 for different enzymes. For example, pepsin, trypsin and arginase have the highest activity at pH 1.5, 7.7 and 9.7, respectively (Garrett, 397).
Tyrosinase is a copper monooxygenase that is widely distributed in living organisms (van Gelder, 1309). It has both cresolase and catecholase activity, turning the orthohydroxylation of monophenols and the oxidation of o-diphenols to o-quinones, respectively (Xuan, 6575). These tyrosinase-mediated reactions are responsible for the browning of potatoes and mushrooms (van Gelder, 1309). Here, I hypothesize that neutral pH is optimum for tyrosinase. In this laboratory, we focused on the catecholase activity of tyrosinase isolated from potato. We monitored spectroscopically the oxidation of catechol to benzoquinone by tyrosinase in the presence of dioxygen and studied the effect of pH on the tyrosinase activity.
Materials and Methods
2.25 g of potato (with skin) was weighed out, cut into small pieces, and ground to a fine paste with mortar and pestle. A little distilled water was added to help emulsify the potato. After mixing the potato paste with 30 mL distilled water, it was filter through cheesecloth in a funnel into an Erlenmeyer flask. The filter solution was stored on ice until use.
0.05, 0.10, and 0.20 mL tyrosinase crude solution were added to three cuvettes, respectively. Distilled water was added to each cuvette using the 5 mL serological pipette, so that the total volume of solution in each cuvette was 2.50 mL. Label each cuvette properly. The opening of the cuvette was covered with plastic wrap, and the solution was mixed by inverting the cuvette with finger.
The spectrophotometer was turned on and allowed to warm up for 10 minutes. It was set up to measure absorbance at 420 nm. The spectrophotometer was blanked at 420 nm using distilled water. 0.50 mL 0.75% pyrocatechol solution was then added to each cuvette. After 6 minutes, the cuvettes were inserted into the spectrophotometer, so the clear side was facing me. The lid of the spectrophotometer was closed, and the absorbance at 420 nm was measured and recorded.
When finished, the yellow enzymatic reaction solutions and any remaining catechol solution were disposed into the appropriate waste container located in the hood in the back of the lab. Cuvettes were rinsed well with distilled water and returned to their foam storage containers. The remaining tyrosinase solutions, paper towels, and plastic tips were thrown in the trash can.
Results
The absorbance at 420 nm after 6 minutes (Table 1) was plotted against the amount of the tyrosinase enzyme (Figure 1). Based on Figure 1, 0.05 mL of the tyrosinase enzyme solution was selected to determine the effect of pH on tyrosinase activity. The absorbance at 420 nm over time at pH 3, 7 and 11 was recorded in Table 2, and the kinetic curves were plotted and shown in Figure 2. At neutral pH (pH 7), the absorbance at 420 resulting from the generation of benzoquinone was much higher than that at pH 3 or 11 (Figure 2).
Discussion and Conclusion
Based on the fact that the absorbance at 420 resulting from the generation of benzoquinone was much higher at pH 7 than that at pH 3 or 11 (Figure 2), the tyrosinase is more active at pH 7 than at pH 3 or 11. Therefore, the results of the experiments have supported my hypothesis that the neutral pH is optimum for the activity of tyrosinase.
The kinetic curve at pH 7 in Figure 2 suggests that the enzymatic reaction is the fastest in the first three minutes, characterized by the rapid increase of the absorbance at 420 nm. The decreasing in the absorbance at 420 nm in the kinetic curves at pH 3 and 11 after 3 minutes could result from erroneous reading at time 0. Possible reasons for erroneous reading include air bubbles in cuvette and inadequate time between insertion of cuvette into the spectrophotometer and reading.
In sum, this experiment suggests that the neutral pH is optimum for the activity of tyrosinase.
Reference
Garrett, Reginald H., and Grisham, Charles M. Biochemistry. Boston: Mary Finch, 2010. Print.
Nelson, David L., Lehninger, Albert L., and Cox, Michael M. Lehninger Principles of Biochemistry. New York: W. H. Freeman, 2008. Print.
van Gelder, Celia W. G., FlurKey, William H., and Wichers, H. J. “Sequence and Structural Features of Plant and Fungal Tyrosinases.” Phytochemistry 45.7 (1997): 1309-1323. Web. 28 Apr. 2013.
Xuan, Ying Ji, Endo, Yasushi, and Fujimoto, Kenshiro. “Oxidative Degradation of Bisphenol A by Crude Enzyme Prepared from Potato.” Journal of Agricultural and Food Chemistry 50 (2002): 6575-6578. Web. 28 Apr. 2013.
