EXPERIMENT 3 Potato Polyphenol Oxidase (I) 1 BACKGROUND Polyphenol oxidase, also known as tyrosinase (EC 1.14.18.1) is an enzyme responsible for one of the steps in the conversion of tyrosine into melanine, a pigment molecule. The first step in the conversion is oxidation of tyrosine to 3,4 dihydroxy phenylanine. Humans lacking polyphenol oxidase/tyrosinase exhibit albinism. In plants the same enzyme is responsible for browning. The potato form of the polyphenol oxidase can react with a number of mono or di-hydroxy substituted benzenes. The enzyme contains a copper ion in its active site. Phenylthiourea can bind to copper ion, effectively removing this metal from the enzyme. If oxygen is removed from the immediate environment of the enzyme catalysis will be inhibited. Ascorbic acid (vitamin C) can nonenzymatically scavenge oxygen, thereby lowering the rate of catalysis. Of course polyphenol oxidase is one of thousands of enzymes in the potato. To inhibit a class of enzymes known as phosphatases, fluoride, in the form of sodium fluoride, is added to the potato preparation. PROCEDURE Part A: Preparation of polyphenol oxidase extract 1 This experimental procedure was modified from Horowitz, S. and Manatt,, M.R., Chemistry 353 Lab Manual pp 18-24. 1
1. Set up a funnel lined with six layers of cheese cloth in a funnel rack placed over a 100 ml beaker immersed in an ice bath. 2. Peel a small, or one-half of a large potato. 3. Dice potato and add to homogenizer. Add 50 ml of NaF solution. Note that NaF is a poison. 4. Blend in homogenizer for 1-2 min. 5. Pour homogenate through cheesecloth-lined funnel and collect soluble lysate in beaker. You will need 20 ml of lysate for your experiment. 6. Wrap waste in paper towels and throw away. 7. Keep lysate on ice to minimize proteolysis of enzyme, and the production of colored products within the lysate. Some particulate matter will settle to the bottom of the beaker. For your enzyme studies only use the supernatant. Part B: Time course study of enzymatic activity 1. Set up the following test tubes: a. Enzyme (1 ml of lysate) b. Substrate (1 ml of water) c. Enzyme + substrate (1 ml of lysate) 2. Prepare 37ºC water bath. 3. Prepare timer 4. While the test tubes are still on ice, add 1 ml water to test tube a, add 1 ml 0.01 M catechol 2 to test tube b, add 1 ml 0.01 M catechol to test tube c. 5. Quickly vortex and record initial color of test tube solutions. Then place the tubes in the water bath and record color changes every 5 min up to 15 min total. Shake tubes every 5 min to replenish dissolved oxygen. Use the following key as a guide: Key: 0 = no color change, 1 = slight color change, 2 = strong color change, 3 = intense color change. You may use video clips to record the changes. 6. Fill in Table 1 below (this is to be included in your lab report). The first line of the table has been filled in for you. Time (min) Enzyme alone Substrate alone Enzyme + substrate 0 0 (no color change) 0 (no color change) 0 (no color change) 2 Keep catechol solution shielded from light. 2
5 10 15 Part C: Properties of the enzyme catalyst 1. Label six test tubes A, B, C, D, E, F and keep on ice. 2. Add 1 ml NaF solution to A (this is the negative control). 3. Add 1 ml lysate to B through F. 4. Add 1 ml water to A (this is the negative control) and 1 ml water to B (this is the positive control) 5. Add 1 ml 5% (w/v) trypsin solution to C. 6. Add 1 ml trichloroacetic acid (TCA) solution to D. (Do not get TCA on your skin). 7. Add a few crystals of phenylthiourea to E. 8. Vortex all tubes. Incubate the tubes at the following temperatures for 15 min (shake every 5 min): Tube Temperature (ºC) A 37 B 37 C 37 D Room temperature E 37 F 0 9. Record any changes to the solutions. 10. Add 1 ml 1% (w/v) ascorbic acid to F. Gently mix. 11. Record initial color of solutions. 12. Add 1 ml catechol solution to all tubes, vortex, and place at 37ºC. 13. Record color changes after 10 min of incubation at 37ºC (see Table 2 below) Tube Color change Other observations A (neg. control) B (pos. control) 3
C (trypsin treatment) D (TCA treatment) E (phenylthiourea treatment) F (ascorbic acid treatment) What is the effect of the treatments on the enzyme activity? At the molecular level, what is happening? Part D: Effect of temperature on the enzyme-catalyzed reaction. 1. Label four test tubes 0, 37, 100, control. 2. Add 1 ml lysate to each 0, 37, 100. 3. Add 1 ml NaF solution to control. 4. Keep 1.2 ml of catechol in a shielded test tube on ice and keep 3.4 ml of catechol in a shielded test tube at room temperature. 5. Incubate the tubes at the following temperatures for 10 min. Tube Temp (ºC) 0 0 37 37 100 100 control 37 6. Add the following 1 ml of catechol solutions to the tubes. Tube 0, add ice-cold catechol. Tubes 37, 100, and control, add room temperature catechol. 7. Shake tubes, record color, and return tubes to their respective temperatures for 10 min. 8. Record colors and color changes. 9. Incubate tube 0, tube 100 and tube control at 37ºC for 10 min. Record colors and color changes. 10. Record your observations in Table 3 below. Tube Initial Temp (ºC) Color change 2 nd Incub Temp (ºC) Color change 0 37 ----- ----- 100 control 4
Plot a column graph with enzyme activity (0,1,2,3) on the y axis and temperature on the x axis using results in columns 2 and 3 of Table 3. Questions: 1. What is the optimum temperature for enzyme activity. 2. Explain the observed changes in enzyme activity at 0ºC and 100C. 3. What conclusions do you draw from the results in columns 4 and 5 with regard to the reversibility of enzyme activity? 5