Biology AP – LAB #2 – ENZYME CATALYSIS
I. Introduction
In this lab exercise you will use catalase, an NZ found in peroxisomes of most cells and most eukaryotic organisms. You
will test for conditions that optimize the NZ’s activity. You will also test for conditions that hinder its activity.
II. Your mission
Work in groups and develop an assay for NZ activity. An assay is a recipe for an experiment that gives the same results
every time, if nothing changes. Changing something gives a new result. If your assay measures rate, then you should be
able to assess (detect) changes in rate from your assay.
The volume measurement apparatuses I showed you last week will come in handy. You will consider questions such as,
“what is the best concentration of H2O2?” and “what is the best amount of liver extract?”
Once you have developed your assay, choose two or more treatments to test for their effects on NZ activity.
III. Plan and Execute
Take notes in your lab journal. Write down all thoughts, initial tests (decisions made in developing the assay), and
results. Decide on a table format to record your results for the experiments. Once your assay is fine-tuned, pick two or
more treatments to apply to your NZ or reaction mixture (NZ + H2O2). Write down a hypothesis for each treatment and
do your experiment to see if your hypothesis is substantiated.
IV. WRITEUP
You will compile all results onto a single sheet of graph paper. Assign each experiment a color. On separate paper, for
each experiment, write the hypothesis, discuss the data, and justify your conclusion.
GRADING
This lab writeup will be completed at home and handed in tomorrow. It will count as ½ of a quizzam/test.
Example lab write up:
Enzymes catalyze reactions. In this lab we used _______ from liver extract. The reaction it catalyzes is____________.
The purpose of this lab exercise was to develop an assay for measuring the rate of the reaction, and then test various
treatments for their effects on the reaction rate.
I.
Development of the assay
We tested a number of H2O2 volumes and enzyme amounts to find a reaction rate that was reliable and very
similar between trials.
Here is a summary of our trials in table form
Time (s)
5
10
15
20
25
30
35
40
45
50
Volume of O2 produced with H202 (ml) + liver (drops)
10ml + 1 drop
20 ml + 1 drop
10 ml + 2 drops
20 ml + 2 drops
5
20
10
50
15
50
30 popped apart
25
80
50
35
70
45
90
55
65
75
85
95
And graph form:
100
90
80
70
60
10ml + 1 drop
50
20 ml + 1 drop
10 ml + 2 drops
40
20 ml + 2 drops
30
20
10
0
5
II.
10
15
20
25
30
35
40
45
50
Based on the results above, we chose 10 ml H2O2 and 1 drop liver extract for our assay.
Testing various treatments
a. Ammonia – raises pH (makes alkaline)
i. Hypothesis: Since enzymes have an optimum pH that is close to physiological pH of 7.2-7.4, we
believe that making the reaction mix alkaline by adding NH4 will slow down the reaction.
ii. Experimental results in graph form
100
90
80
70
60
10ml + 1 drop (control)
50
With 1 ml Ammonia (pH 8.5)
40
With 2 ml Ammonia (pH 10)
30
20
10
0
5
10
15
20
25
30
35
40
45
50
iii. Discussion
1. Addition of ammonia lowered the rate of the reaction, as is evidenced by the decreased
slope of the red and green plots. Presumably, adding more ammonia raised the pH above
the optimum pH for catalase, leading to reduced activity, and hence, reduced O2 production
over the course of the reaction.
b. Vinegar – lowers pH
i. Hypothesis
ii. Results
iii. Discussion
…………………………………….. fill in more treatments
III.
Conclusions
a. Development of the assay could be improved by altering the volume measuring contraption that Dr. Trexler
built. His poor design led to inaccuracies in our measurements.
b. Catalase has an optimum pH of 8 …. Yada yada because the rate of the reaction was highest for this pH
c. Etc………