Enzyme Activity

To begin, the baseline for the peroxidase enzyme reaction must be determined. Make the substrate by adding 7 mL of distilled water to a clean test tube and then add 0.2 mL of guaiacol. Note: Guaiacol is a color-changing indicator that becomes more yellow-orange as the enzyme reaction progresses.

Next, add 0.3 mL of 0.1% H2O2.

Using a marker, label the test tube substrate and then cover the tube with a piece of sealing film.

Holding the cover in place, invert the tube four times to mix the tube contents.

In a second test tube, add 6.0 mL of distilled water and 1.5 mL of turnip peroxidase and label this tube “Enzyme”.

Cover this tube with a new piece of sealing film and mix these solutions by four inversions as previously described (step 4).

When both tubes have been mixed, place a timer on the bench, next to the test tube rack, then pour the contents from the substrate tube into the enzyme tube, being careful not to spill any liquid.

Next, pour the entire volume from the enzyme tube back into the substrate tube to thoroughly mix the solutions and cover the substrate tube with another piece of sealing film.

Put the test tube in the rack and start the timer.

Observe the color in the substrate tube at 0, 1, 2, 3, 4 and 5 minutes, post-mixing.

To quantify the relative change in color and the rate of the reaction, match the observed color to the closest color in the standard. NOTE: Taking a photo of the observed color intensity at each sample point may make it easier to record the results.

Record the colors you see in the table. Click Here to download Table 1

HYPOTHESES: In this exercise, the experimental hypothesis is that the reaction rate will reach a maximum or optimum at a mid-range neutral pH and that these rates will be higher than or roughly equal to the baseline rate. The null hypothesis is that there will be no differences in the reaction rates between the baseline and the pH treatments.

First, label six clean test tubes for each of the six pH buffer levels 3 – 8 and the word “Enzyme”.

Next, add 6 mL of the corresponding pH buffer solutions provided by your instructor to each of the test tubes.

Then, add 1.5 mL of the turnip peroxidase to each tube, being careful not to contaminate the dropper with the buffer solution.

Cover the enzyme tubes with sealing film and gently invert each tube to mix the tube contents.

As described in the baseline reaction experiment, make the substrates for these reactions by adding 7 mL of distilled water, 0.2 mL of guaiacol and 0.3 mL of 0.1% H₂O₂ to six new clean test tubes.

Next, label six additional clean test tubes with the pH levels 3 – 8 and the word “Solution” and get a timer ready.

Working quickly, pour a substrate tube and a pH tube into the corresponding solution tube. Repeat this step for each of the different pH levels.

Then, cover the tubes with sealing film and mix each tube with four inversions. Place each tube in a tube rack, as they are mixed.

Observe the color in each tube at time 0 and immediately start the timer. Continue to observe the color in each tube at every minute interval, up to 5 minutes.

At each interval, use the color code in the standard to record the observed colors in your table.

HYPOTHESES: In this exercise, the experimental hypothesis is that the reaction rate will reach an optimum above room temperature, or 23 °C, but below enzyme denaturation rate and that this maximum will be higher than the baseline rate. The null hypothesis is that there will be no differences in the reaction rates between the baseline and the temperature treatments.

Fill an 800 mL beaker about halfway to the top with ice and pour cold water into the beaker to create an ice bath. Label this beaker as “Ice”.

Then, label four new 600 mL beakers with the same temperatures as your substrate tubes and add 150 mL of tap water to each.

Using a hot plate and a thermometer, bring the beakers to 25, 35, 45 and 60 °C, as per their labels.

Prepare five more pairs of substrate and enzyme tubes, as described for the baseline reaction, using distilled water in the enzyme tube, instead of a pH buffer.

Label the substrate tubes as “Ice”, “25 °C”, “35 °C”, “45 °C”, and “60 °C”.

When all of the water baths have reached their appropriate temperatures, mix the substrate and enzyme solutions together by first pouring each substrate tube contents into an enzyme tube and then pouring the enzyme tube contents back into the substrate tube.

Cover the substrate tubes with sealing film and observe the colors at time 0.

Then, stand the tubes in their corresponding beaker baths, based on the temperature label each tube was given and start a timer.

For each 1-minute interval, remove the tubes from the water and record their color, using the stabdard as a reference.

Then, place them into their baths until each time point, up to 5 minutes, has been recorded.

To determine the baseline for the reaction, graph the change in color intensity against time. NOTE: The number of units that the color increased by over the 5-minute time period will serve as the baseline rate of reaction for relative comparison.

Now graph the difference in color intensity against the pH levels used in the first experiment.

Add in the baseline for comparison and then determine whether the optimal reaction rate was higher than, lower than or equal to the baseline.

Using this graph, determine the optimum pH for the reaction. Note: The reaction may not occur at every pH. The optimum pH for the peroxidase enzyme is typically in the range of 6 – 7. By exposing the enzyme to a substrate with different levels of pH, you will notice a drop-off in the reaction rate as the pH of the enzyme becomes more basic, making the enzyme more unstable.

Finally, graph the difference in color intensity over time for the different temperatures used in the second experiment, using the same baseline for comparison.

Using this graph, determine the optimum temperature for the reaction and compare it to the baseline. NOTE: The reaction may not occur at every temperature. In this experiment, the reaction appears most efficient around 45 °C, and above this, the peroxidase enzyme begins to denature. For this reason, it should not produce a color change in the 60 °C beaker. The reaction rate will also slow or stop in the ice bath, as there is not enough energy for the enzyme to catalyze the reaction.