Enzyme Activity

To begin, we will determine the baseline for the peroxidase enzyme reaction. First, make the substrate by adding seven milliliters of distilled water to a clean test tube and then add 0.2 milliliters of guaiacol, a color-changing indicator which becomes more yellow-orange as the enzyme reaction progresses. Finally, add 0.3 milliliters of 0.1%hydrogen peroxide.

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 milliliters of distilled water and 1.5 milliliters 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 just shown. 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 zero, one, two, three, four and five 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 figure.

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. Now that you have your baseline data, you are ready to begin the experiments.

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. To investigate the effect of pH on peroxidase activity, you should first label six clean test tubes for each of the six pH buffer levels three through eight and the word enzyme.

Next, add six milliliters of the corresponding pH buffer solutions provided by your instructor to each of the test tubes. Then add 1.5 milliliters of the turnip peroxidase to each tube, being careful not to contaminate the dropper with the buffer solution. Then cover the enzyme tubes with sealing film and gently invert each tube to mix the tube contents.

As demonstrated in the baseline reaction experiment, make the substrates for these reactions by adding seven milliliters of distilled water, 0.2 milliliters of guaiacol and 0.3 milliliters of 0.1%hydrogen peroxide to six new clean test tubes. Next, label six additional clean test tubes with the pH levels three through eight and the word solution and get a timer ready. Working quickly, pour a substrate tube and a pH tube into the corresponding solution tube.

For example, you'll add one substrate tube and the pH six tube into the test tube labeled solution six. Repeat this step for each of the different pH levels. Then cover the tubes with sealing film for mixing by four inversions.

Place each tube in a tube rack, as they are mixed. Observe the color in each tube at time zero and immediately start the timer. Continue to observe the color in each tube at every minute interval, up to five minutes.

At each interval, use the color code in figure one to record the observed colors in your table. In this exercise, the experimental hypothesis is that the reaction rate will reach an optimum above room temperature, or 23-degrees Celsius, 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.

To investigate the effect of temperature on peroxidase activity, fill an 800-milliliter 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-milliliter beakers with the same temperatures as your substrate tubes and add 150 milliliters of tap water to each.

Using a hot plate and a thermometer, bring the beakers to 25, 35, 45 and 60 degrees Celsius as per their labels. Prepare five more pairs of substrate and enzyme tubes, as demonstrated for the baseline reaction. using distilled water in the enzyme tube, instead of a pH buffer.

Label the substrate tube as ice, 25 degrees Celsius, 35 degrees Celsius, 45 degrees Celsius and 60 degrees Celsius. When all of the water baths have reached their appropriate temperatures, mix the substrate ane 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 tubes. Cover the substrate tubes with sealing film and observe the colors at time zero.

Then stand the tubes in their corresponding beaker baths, based on the temperature label each tube was given and start a timer. For each one-minute interval, up to five minutes, remove the tubes from the water and record their color, using the chart as a reference. Then replace them into their baths until each time point has been recorded.

To determine the baseline for the reaction, graph the change in color intensity against time. The number of unites that the color increased by over the five-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. What was the optimum pH for the reaction? Did the reaction occur at each pH?

The optimum pH for the peroxidase enzyme is typically in the range of six to seven. By exposing the enzyme to a substrate with different levels of pH, you will have noticed 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.

What was the optimum temperature for the reaction and how did it compare to the baseline? Did the reaction occur at all of the temperatures? In this temperature experiment, the reaction appears most efficient around 45 degrees, and above this, the peroxidase enzyme begins to denature.

For this reason, it should not produce a color change in the 60-degree-Celsius beaker. And 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.