Enzyme Function Worksheet Name

The enzyme peroxidase was isolated from fresh turnips. This enzyme is a slight variation on the catalase you

have used in your protein study. In both cases each enzyme breaks down hydrogen peroxide to oxygen gas and

water. In this case, however, the oxygen produced reacts with guaiacol, bringing about a color change.

( ) ( )colorless brownGuaiacol + Oxygen Tetraguaiacol By using a spectrophotometer (or colorimeter), the amount of oxygen produced can be quantified by measuring

the increasing amount of brown tetraguaiacol produced. Thus the amount of oxygen produced can be recorded

by measuring the absorbance of the brown pigment. Using a standard curve, this can be converted to moles of

oxygen. You have done similar measurements using colorimeters in chemistry.

Experiment A

In this initial experiment, 1 ml of a fresh peroxidase-containing solution was mixed with 0.1 % guaiacol and 0.5

% H2O2 in a 50 ml beaker containing. Every minute a sample was taken and its absorbance measured. The

experiment was stopped after 10 minutes.

Time 0 1 2 3 4 5 6 7 8 9 10

O2 (moles) 0 0.16 0.39 0.48 0.53 0.6 0.6 0.615 0.62 0.612 0.614

Graph these data.

1. Describe the changing shape of the curve.

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0 20 40 60 80 100 120

Title

Title

2. Calculate the initial rate of reaction as well as the rate at 7.5 minutes. Explain why these rates are different.

Experiment B

This study was repeated, but this time the amount of substrate was altered. 20 ml of peroxidase solution were

mixed with different concentrations of substrate. The oxygen produced was recorded for 6 minutes. The data

from these trials are graphed below. Complete the graph by putting in best-fit curves.

Rate of reaction, product/ time, is typically a more meaningful parameter when discussing and describing

enzyme action. Using your best-fit curves, determine the initial rate of reaction for each substrate concentration

and record them in an appropriate table on the next page. Using these results, construct a graph showing the

relationship between substrate concentration (% H2O2) and peroxidase rate of reaction.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 1 2 3 4 5 6 7

Ox

yg

en

Pro

du

cti

on

(m

ole

s)

Time (minutes)

6.25%

12.50%

25.00%

50.00%

100.00%

[H2O2]

Explain, in terms of the number available of active sites and number of substrate molecules present, why the

slope of the curve is different at low, medium, and high substrate concentrations.

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0 20 40 60 80 100 120

Title

Title

Experiment C

This study was repeated, but this time the amount of enzyme was varied by the same dilution process.

The oxygen produced was recorded for 6 minutes. The data from these trials are graphed below. Complete the

graph by putting in best-fit curves.

Using your best-fit curves, determine the initial rate of reaction for each substrate concentration and record

them in an appropriate table on the next page. Using these results, construct a graph showing the relationship

between enzyme concentration and peroxidase rate of reaction.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 1 2 3 4 5 6 7

Ox

yg

en

Pro

du

ced

(m

ole

s)

Time (minutes)

6.3%

12.5%

25.0%

50.0%

100.0%

[Enzyme]

Explain, in terms of the number available of active sites and number of substrate molecules present, why the

slope of the curve is different at low, medium, and high enzyme concentrations.

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

0 20 40 60 80 100 120

Title

Title