BIOS 100 Laboratory:  Determination of the Properties of the Enzyme Turnip Peroxidase

The key to completing this enzyme analysis lies in utilizing proper graphic techniques to determine enzyme activity. This web page will provide a step-by-step procedure to assist you in completing this task.

Exercise 1 - Standardization of Enzyme Concentration

The purpose of this exercise was to determine which concentration of enzyme is most suitable for the analysis. The concentration of enzyme within the turnips varies due to turnip size, age, and season of harvest. Therefore, we must perform a standardization to determine which concentration on enzyme extract will yield enough enzyme to make the analysis possible but not so much as to exhaust all of the peroxide in the solution.

To determine the most appropriate concentration, you measured the absorbance of three amounts of enzyme extract (0.5 ml, 1.0 ml, and 2.0 ml). When these are plotted versus time, the graph may look something like this:

You can see that all three enzyme aliquots produced a visible reaction. The 0.5 ml was linear, but the slope is very shallow. The 1.0 ml was also linear, but with a steeper slope. The 2.0 ml was linear for the first portion, but it leveled off at the end. This leveling off is due to the enzyme exhausting all of the peroxide in the solution. In this situation, the most appropriate amount to use is the 1.0 ml as it had the greatest reaction rate without running the reaction to completion in the allotted time.

In the example below, you can see that the most appropriate amount of enzyme to use would be 2.0 ml as this concentration did not exhaust the peroxide.

Determining the Rate of the Enzyme-catalyzed Reaction (the Enzyme Activity)

In order to complete the analysis, you will be required to determine the activity of turnip peroxidase under various environmental conditions. Enzyme activity can be easily determined by graphical means. First, you must prepare a graph of absorbance vs. time. Next, you must determine the best straight line that can be drawn for your data. You may do this by using a linear regression function spreadsheet. After you have drawn the best-fit straight line, enzyme activity can be determined from the slope of the line . 

Exercise 2 - Determining the Optimal Temperature for Turnip Peroxidase

Once you have established the most appropriate amount of enzyme extract to use, it is time to get down to business and determine some of the properties of turnip peroxidase. The first analysis we will perform will be to determine the optimal temperature. From the class data, you should have obtained five sets of "absorbance vs. time" data from samples at 4oC, 23oC, 32oC, 48oC, and 72oC. These data should then be graphed, which should look like this:

Next, you need to calculate the slopes for each of these lines to determine the enzyme activity of turnip peroxidase at the five temperatures. Finally, you will construct another graph, this one plotting enzyme activity vs. temperature. You should have five points for this graph. The x-coordinate will be the temperature, the y-coordinate will be the enzyme activity at that temperature. Connect the points with a smooth flowing curve. When you are done, the graph should look like this:

Note that the Topt is about 45C, NOT 48C. Even though the temperature with the greatest enzyme activity measured was 48C, the graph indicates that it is actually about 45C.

Exercise 3 - Determining the Optimal pH for Turnip Peroxidase

This analysis is performed in a similar manner to the determination of the temperature optimum. You should have four sets of "absorbance vs. time" data from the class, one from each pH. When you plot these data, the graph should look like this:

Next, you need to calculate the slopes to determine the enzyme activity. You should then prepare another graph relating enzyme activity to pH. You will only have four points for this graph, but do your best to prepare a smooth flowing curve for these data. The finished graph should look like this:

Determining the Effects of Hydroxylamine on Turnip Peroxidase Activity

For this analysis, you will only have to prepare one graph (whoopie!!!). First, you must prepare a graph of the "absorbance vs. time" data. It should look like this:

Next, you need to calculate the enzyme activity from the slope. Finally, you need to calculate the percent inhibition by the hydroxylamine by using the following formula:

% inhibition = [(normal activity - inhibited activity) / (normal activity)] * 100%

Hopefully, this step-by-step guide will help you prepare this lab. If you have any further questions, fell free to contact your TA or the lab coordinator.

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