Lab Practical: Factors Affecting Enzyme Activity

Extracted Content from Question Files:

FACTORS AFFECTING ENZYME ACTIVITY.

Determining the effects of pH. temperature and
substrate concentration
THE SECTIONS BELOW HIGHLIGHTED IN BOLD (in the method section) ARE
THE SECTIONS I NEED ANSWERED WITH THE RESULTS PROVIDED. I ALSO
NEED HELP WITH WHAT I SHOULD INCLUDE IN DISCUTION OF THIS
PRACTICAL, (INCLUDING SOURCES OF ERROR AND IMPROVEMENTS,
LIMITATIONS). Any graphs can be done by hand and also if you could show how you
worked out the VMAX, KM, Q10 and the erzyme activity as ml of HCI at each urea
concentration.
The questions to be answered are highlighted in bold in the method section, there are 3
questions, 1 for EFFECT OF PH, 1 FOR EFFECT OF TEMPERATURE AND 1 FOR
SUBSTRATE CONCENTRATION.

Any help would be greatly appreciated.

The notes below are from handouts from class:

Aims
The aim of this experiment is to determine the optimal pH, temperature and
substrate concentration of the enzyme urease by titra+ion with hydrochloric acid.

Introduction
Urease is found in many seeds and in soil bacteria. This enzyme converts urea into
carbon dioxide and ammonia. The ammonia released can be estimated to give a
measure of urease activity.
HzO + HzN C MHz -»- COz + 2NHs
The effect of pH
The pH of a solution can have several effects of the structure an activity of
enzymes.
For example, pH can have an effect of the state of ionisa+ion of acidic or basic
amino acids. Acidic amino acids have carboxyl functional groups in their side chains.
Basic amino acids have amine functional groups in their side chains. If the state of
ionisation of amino acids in a protein is altered then the ionic bonds that help to
determine the 3-D shape of the protein can be altered. This can lead to altered
protein recognition or an enzyme might become inactive.
Changes in pH may not only affect the shape of an enzyme but it may also change
the shape or charge properties of the substrate so that either the substrate cannot
bind to the active site or it cannot undergo catalysis.
In general enzymes have a pH optimum. However the optimum is not the same for
each enzyme.
(Approximate) Expected Results
Enzyme Activity
There is a limit to the amount of substrate that a single enzyme can process in a
given time. If the concentration of substrate is increased, the rate at which the
product is formed also increases up to a maximum value. The rate of reaction at
which an enzyme is saturated with substrate is known as Vmax.

The Recction for urease obeys simple Michaelis Men+on kinetics.
V = V max [S]
Km + [S]

The effect of temperature

Like most chemical reactions, the rate of an enzyme-catalysed reaction increases
as the temperature is raised. A ten degree Centigrade rise in temperature will
increase the activity of most enzymes by 50 to 100%. Variations in reaction
temperature as small as 1 or 2 degrees may introduce changes of 10 to 207o in the
results. In the case of enzymatic reactions, this is complicated by the fact that
high temperatures adversely affect many enzymes. As shown in Figure 1. the
reaction rate increases with temperature to a maximum level, then abruptly
declines with further increase of temperature. Because most animal enzymes
rapidly become denatured at temperatures above 40°C, most enzyme
determinations are carried out somewhat below that temperature.
METHOD

DETERMINATION of OPTIMUM PH.

LABEL YOUR FLASKS CLEARLY WITH YOUR INITIALS AS WELL AS THE
FLASK NUMBER OR pH VALUE.
1. take 8 clonical flask/beakers. Labal the flasks 1 to 8 and make up the following:

FLASK BUFFER UREA WATER UREASE Mg
No. UREA
0 5ml 0 10ml 1ml 0
1 5ml 0.25ml 9.75ml 1ml 5
2 5ml 0.5ml 9.5ml 1ml 10
3 5ml 0.75ml 9.25ml 1ml 15
4 5ml 1.0ml 9.0ml 1ml 20
5 5ml 1.25ml 8.75ml 1ml 25
6 5ml 1.50ml 8.50ml 1ml 30
\7 5ml 1.75ml 8.25ml 1ml 35

2. Seal with parafilm and incubate at 25°C for 30 minutes.
3. After 30 minutes add 0.2ml of saturated quinhydrone solution (this inhibits the
enzyme by converting -SH groups to -S-S- groups.)
4. Add 6 drops of Methyl Orange to each flask and gently shake.
5. Titrate the ammonia produced against 0.1 M HCI using the methyl orange as an
Indicator as demonstrated (the colour changes from orange to pink).
6. Estimate the enzyme activity as ml of HCI for each pH and plot a graph of your
results.
Urease has an optimum pH, depending on the source of the enzyme, determine this
value from the graph plotted. (THIS IS THE QUESTION I NEED TO ANSWER
USING RESULTS BELOW)

MY RESULTS FOR THE ABOVE EXPERIMENTS WERE AS FOLLOWS:

PH 5.5 6 6.5 7 7.5 8 8.5 9
AVERAGE 0.8 1.5 3.2 6.1 9.4 11.0 2.7 1.0

Determination of effect of substrate concentration
Method:
1 Prepare a series of flasks by labelling 0 to 7, pippette into each 5ml of the
buffer provided at the workstation.
2. Add H2O and urea in the range of 10 to 150mg/flask, as shown below:

FLASK BUFFER UREA WATER UREASE Mg
No. UREA
0 5ml 0 10ml 1ml 0
1 5ml 0.25ml 9.75ml 1ml 5
2 5ml 0.5ml 9.5ml 1ml 10
3 5ml 0.75ml 9.25ml 1ml 15
4 5ml 1.0ml 9.0ml 1ml 20
5 5ml 1.25ml 8.75ml 1ml 25
6 5ml 1.50ml 8.50ml 1ml 30
7 5ml 1.75ml 8.25ml 1ml 35

N.B. Add the Urease last to each flask, and at approximately
the same time.

LABEL YOUR FLASKS CLEARLY WITH YOUR INITIALS AS WELL AS THE
FLASK NUMBER OR Mg UREA.

3. Sec w'th parafilm and incubate at 25°C for 30 minutes.
4. After 30 minutes add 0.2ml of saturated quinhydrone solution (this inhibits the
enzyme by converting -SH groups to -5-S- groups.)
5. Add 6 drops of methyl orange to each flask and gently shake.
6. Titrate the ammonia produced against 0.1 M HCI using methyl orange as an
indicator as demonstrated (the colour changes from orange to pink).

Calculate the erzyme activity as ml of HCI at each urea concentration (substrate)
and plot your results. Calculate the Vmax and Km of this reaction. (THIS IS THE
QUESTION I NEED TO ANSWER BY USING RESULTS BELOW)

MY RESULTS FOR THE ABOVE EXPERIMENTS WERE AS FOLLOWS:

Substrate 0 5 10 15 20 25 30 35
concentration
AVERAGE 0.8 1.5 3.2 6.1 9.4 11.0 2.7 1.0

DETERMINATION OF EFFECT OF TEMPERATURE ON ENZYME
ACTIVITY.
Method
1. You are provided with water baths at the following temperatures -
0°C. 15°C. 30°C. 45°C. 60°C. 75°C. 90°C.
2. Take 7 flasks; label each with one of the above temperatures.
3. Put 5ml of the buffer provided and 5ml of urea solution in each and place into
the relevant water bath for 5 mins.
LABEL YOUR FLASKS CLEARLY WITH YOUR INITIALS AS WELL AS THE
FLASK NUMBER OR TEMPERATURE.
4. Add 1ml of urease to each.
N.B. Add the Urease to each flask at approximately the same
time.
5. Seal with parafilm, and replace the flasks in each of appropriate water baths
and incubate for 30 minutes.
6. After 30 minutes add 0.2ml of saturated quinhydrone solution and titrate as
before.
7. Add 6 drops of Methyl Orange to each flask and gently shake.
8. Titrate the ammonia produced against 0.1 M HCI using methyl orange as an
indicator as demonstrated (the colour changes from orange to pink).
Calculate enzvme activity as ml of HCI at each temperature and plot a graph of
your results ( temperature against enzyme activity).

Calculate the Q10 for the reactions at l5C and 25°C. (THIS IS THE QUESTION I
NEED TO ANSWER BY USING MY RESULTS BELOW)

MY RESULTS FOR THE ABOVE EXPERIMENTS WERE AS FOLLOWS:

Temperature 0 15 30 45 60 75 90
AVERAGE 7 7.9 10.1 11.8 12.2 11.4 10.2