Activity and Inactivation of

Glucose-6-Phosphate Dehydrogenase
by Dr. R. Edwards, edited by Dr. M.E. Fraser
Ø Put the data for the enzyme assays into your lab book.
Ø Make sure the data are well labeled so you can analyze them. For this lab, you will be
writing an introduction and results with references as needed.

Introduction
In this lab you will learn to do enzyme assays and study the activity and inactivation of glucose-
6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides (G6PDH, EC 1.1.1.49).
G6PDH catalyzes the oxidation of glucose-6-phosphate (G6P) and reduction of NADP+ to
produce NADPH in the following reaction:
G6P + NADP+ " 6-phosphogluconolactone + NADPH + H+
NADPH has an absorbance peak at 340 nm (e340 nm = 6.22 mM-1cm-1), but the oxidized form of
this coenzyme (NADP+) does not have absorbance at 340 nm. This makes it easy to measure the
progress of the reaction spectrophotometrically.

The assay used has been modified from an assay from Sigma Aldrich (1) in which the change in
absorbance at 340 nm (DA340 nm) from the NADPH produced is monitored over a period of time.
One unit of enzyme will reduce 1.0 µmole of NADP+ to NADPH per minute at pH 7.4 and 25°C
in the presence of G6P. The enzymatic assay will be used to monitor whether the enzyme is
active or inactive and the stability of G6PDH will be studied under various conditions including
after the addition of stabilizers such as 1,2-propanediol or glucose.

Three methods of inactivation will be tested. Thermal inactivation will be measured by allowing
the enzyme to incubate for 10 minutes at a temperature that will produce about 50% decrease in
activity. The inactivation by freeze-thawing will be measured after three freeze-thaw cycles. The
inactivation from mechanical shear forces will be measured after two minutes of vigorous
stirring.

Although glucose-6-phophate dehydrogenases generally use NADP+, there are reports that the
enzyme from Leuconostoc mesenteroides can utilize NAD+ as well. Thus it will be of interest to
measure kinetic parameters for each of these coenzymes.

Hence, there are two research questions to be tackled in this lab:

a) Are polyol compounds that stabilize this enzyme equally effective with various modes of
denaturation (thermal, freeze-thaw, and mechanical agitation)?
b) What is the coenzyme specificity of this enzyme as measured by the Vmax and Km values for
NADP+ and NAD+?

(1) http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-
glucose-6-phosphate-dehydrogenase.html

MF2-1
Equipment

• UV cuvettes
• Spectrophotometer
• Cuvette washer
• Three heating blocks for thermal denaturation

Reagents

Reagents to measure activity of G6PDH

• Water (all water is distilled water, dH2O)

• 100 mM GlyGly buffer containing 20.0 mM MgCl2, pH 7.4
• 50.0 mM GlyGly buffer containing 10.0 mM MgCl2, pH 7.4
• 10.0 mM glucose-6-phosphate (G6P)
• 1.00 mM sodium NADP+
• 10 mL 1.0 x 10-2 mg/mL G6PDH in 50.0 mM GlyGly, 10.0 mM MgCl2 buffer, pH 7.4 on
ice. Keep all enzyme solutions on ice until the enzyme is added to the assay mixture.
• 2.00 M glucose in 50.0 mM GlyGly, 10.0 mM MgCl2 buffer, pH 7.4
• 2.00 M 1,2-propanediol in 50.0 mM GlyGly, 10.0 mM MgCl2 buffer, pH 7.4
Additional reagents to measure kinetic parameters of G6PDH for NADP+ and NAD+
• 0.75 mL 0.500 mM NADP+ in water (for step 7)
• 0.75 mL 10.0 mM NAD+ in water (for step 7)

Procedure

Measure activity of G6PDH

1. Preparing the assay mixture

Make 70.0 mL of assay mixture from the stock solutions listed above by combining 35.0 mL
100 mM GlyGly buffer containing 20 mM MgCl2, pH 7.4, 8.00 mL 10 mM G6P, 8.00 mL 1
mM sodium NADP+, 19.00 mL water. This is enough for 100 assays. This assay mixture
will be used in steps 2 to 6 and a different assay mixture will be used in step 7.

2. Range finding for the enzyme concentration

Zero the spectrophotometer at 340 nm using 700 µL assay mixture and 100 µL 50 mM
GlyGly, 10 mM MgCl2 buffer, pH 7.4 (mixed) in both the sample cuvette and the reference
cuvette. Leave the reference cuvette in the spectrophotometer.

Using the 50 mM GlyGly, 10 mM MgCl2 buffer, pH 7.4, make 5-fold and 10-fold dilutions
of the stock G6PDH to give ~1 mL of each dilution. Assay the stock enzyme and both
dilutions using the assay procedure below. Select the dilution that gives a DA340/min of
between 0.0100 and 0.1000. Repeat the assay two more times on the dilution that caused a
DA340/min change in the range above. This is done to confirm that you have the right
dilution of the stock enzyme. That dilution will be used for the assays in the remainder
of the experiment. It is referred to as the appropriate dilution.

MF2-2
 Assay procedure:
• Add 700 µL assay mixture at room temperature to a UV plastic cuvette.
• Add 100 µL enzyme solution, mix gently by inversion 3 times, and place in the
spectrophotometer. Read the A340 nm for 90 s. From these measurements, calculate initial
velocity in units of µmol/min and specific activity (U/mg).

Inactivation of G6PDH

3. Thermal inactivation
Using the 50 mM GlyGly, 10 mM MgCl2, pH 7.4 buffer, make 2.00 mL of a two-fold
dilution of the stock G6PDH. Gently mix the solution. Place 400 µL in each of four 2.0 mL
microfuge tubes as shown below. Heat the aliquots as shown below. At the 10-minute mark
remove the tubes from the heating blocks and immediately cool them in an ice bath for 3
minutes. Be sure there is enough water in the ice bath to get good thermal conduction.
Further dilute the enzyme solutions in the tubes to the appropriate dilution. (Think about how
you will make this dilution before you come to the lab.)
#1 for 10 minutes at 25ºC
#2 for 10 minutes at 40°C
#3 for 10 minutes at 45°C
#4 for 10 minutes at 50°C
Assay each aliquot (#1, #2, #3, and then #4). Assay each aliquot again. Assay each aliquot
again. (Note that these assays lead to triplicate assays in a particular order.)

4. Protection from thermal inactivation

Choose which polyol you will use as a protectant (either glucose or 1,2-propanediol). Place
200 µL stock G6PDH into two microfuge tubes and label them. To the first microfuge tube
add 200 µL 50 mM GlyGly, 10 mM MgCl2, pH 7.4 buffer and to the second microfuge tube
add 200 µL 2 M protectant (i.e. add 2 M glucose or 2 M 1,2-propanediol in 50 mM GlyGly,
10 mM MgCl2, pH 7.4 buffer), so that the second tube contains 1 M protectant after the
dilution. Gently mix the solutions. At this point your enzyme has been diluted two-fold.

Using your results from step 3, estimate the temperature required to inactivate ~50% of the
G6PDH (T50%) during a 10-minute incubation. Heat these two solutions to T50% for 10
minutes. At the 10-minute mark remove the tubes from the heating block and cool them in an
ice bath. Add 200 µL 2 M protectant in 50 mM GlyGly, 10 mM MgCl2, pH 7.4 buffer to the
first microfuge tube and 200 µL 50 mM GlyGly, 10 mM MgCl2, pH 7.4 buffer to the second
microfuge tube. At this point your enzyme has been diluted three-fold. After they have
cooled to room temperature (feel cool to the touch), make a further dilution with 50 mM
GlyGly, 10 mM MgCl2, pH 7.4 buffer to produce at least 1.00 mL of each of the enzyme
solutions at the appropriate dilution. Gently mix the solutions. (Think about how you will
make this dilution before you come to the lab if the dilution is 5-fold or 10-fold.) Assay these
solutions in triplicate (alternate the solutions being assayed).

MF2-3
5. Freeze-thaw inactivation
You will be provided with two microfuge tubes containing 400 µL G6PDH that are two-fold
dilutions of the stock enzyme. They have been frozen and thawed. Solution A contains no
additive. Two solutions will contain additive:
B 1 M glucose
C 1 M 1,2-propanediol.
(Note that you will use only one of B or C, in addition to A.) Add 200 µL 2 M additive in 50
mM GlyGly, 10 mM MgCl2, pH 7.4 buffer to tube A and 200 µL 50 mM GlyGly, 10 mM
MgCl2, pH 7.4 buffer to tube B or C. Make dilutions of both enzyme solutions to the
appropriate dilution, gently mix the solutions, and assay them in triplicate (alternate the
solutions being assayed).

6. Mechanical inactivation
Do experiments to find out if mechanical agitation of G6PDH leads to inactivation and
whether the polyol provides protection. Make 400 µL enzyme solution in 2.0 mL microfuge
tubes as shown in the table below. If glucose was used in part 4, use glucose for your
experiment; if 1,2-propanediol was used in part 4, use 1,2-propanediol for your experiment.
Vortex these microfuge tubes for 2 minutes.

Stock G6PDH 50 mM GlyGly, 10 mM 2 M glucose 2 M 1,2-propanediol

(µL) MgCl2, pH 7.4 buffer (µL) (µL) (µL)
X 150 250 - -
Y 150 50 200 -
Z 150 50 - 200

After shaking, add 200 µL of the polyol (2 M glucose or 2 M 1,2-propanediol, depending on

whether you have Y or Z) to the control (X). Then use 50 mM GlyGly, 10 mM MgCl2, pH
7.4 buffer to dilute the solution to the appropriate dilution and gently mix the solution. Use
50 mM GlyGly, 10 mM MgCl2, pH 7.4 buffer to dilute the solution in experimental tube Y or
Z to the appropriate dilution and gently mix the solution. Assay these two tubes in triplicate
(alternate the solutions being assayed).

MF2-4
Measurement of kinetic parameters of G6PDH for NADP+ and NAD+

7. Measuring Vmax and Km

Use the two solutions, 0.75 mL of 0.500 mM NADP+ in water and 0.75 mL of 10.0 mM
NAD+ in water, to determine Vmax and Km.
Prepare 8.0 mL assay mix by combining 4.6 mL 100 mM GlyGly buffer containing 20 mM
MgCl2, pH 7.4, 1.0 mL 10 mM G6P and 2.4 mL water.
Use the stocks and water as listed in the table below to determine Vmax and Km. Mix the
solutions in the cuvette as needed.

ASSAYS Assay mix NADP+ NAD+ Water

without NADP+ (0.5 mM) (10 mM) (µL)
(µL) (µL) (µL)
1 600 5 - 95
2 600 10 - 90
3 600 20 - 80
4 600 40 - 60
5 600 80 - 20
6 600 - 5 95
7 600 - 10 90
8 600 - 20 80
9 600 - 40 60
10 600 - 80 20
Add 100 µL enzyme at the appropriate dilution and record A340 nm for 2 minutes for tube #1,
then for tube #2, etc. Determine initial velocity in units of µmol/min.

Report

Your report for this lab will consist of an Introduction, Results, and References as needed. The
maximum length is three pages (12-point font, double-spaced with tables and figures included).
Introduce why your scientific question is interesting and end the introduction with a key finding
that will lead the reader into the body of your report. The report should be focused on the
objective of your experiments and include tables and graphs as needed. Remember that each
table or graph that you include must make a point that will be in the text of your Results.
Things to think about in writing your report:
• It will be helpful to have sample calculations in an appendix. Sample calculations would
be supplementary materials and not part of the three-page report.
• Your report should have summary table(s) or graph(s) relevant to the question being
studied. If you wish to include raw data, it can be put in the supplementary materials if
there is a compelling reason for the reader to look at the raw data.
• The rubric for grading your reports has five entries: Introduction, Presentation of data,
Results, References, and Overall impression. A maximum of 4 marks can be achieved for
each entry: 4/4 for proficient, 3/4 for competent, 2/4 for approaches competent and 1/4
for unsatisfactory.

MF2-5
 For Technician

Equipment

• UV-plastic cuvettes
• Shimadzu spectrophotometer
• Analytical balance
• Cuvette washer
• Heating blocks for thermal denaturation

Reagents for Activity of G6PDH solutions (all water is dH2O)

• Assay mixture: Each pair of students will make 70.0 mL of assay mixture from the stock
solutions below by combining 35.0 mL 100 mM GlyGly buffer with 20.0 mM Mg2+, pH
7.4, 8.00 mL 10.0 mM G6P, 8.00 mL 1.00 mM NADP+, 19.00 mL water. This is enough
for 100 assays.

o 100 mM glycylglycine buffer with 20.0 mM magnesium chloride. Dissolve 6.60 g

glycylglycine (132.12 g/mol) and 2.04 g MgCl2×6H2O (203.30 g/mol) and in 450 mL
of water and adjust the pH to 7.4 with 1 M NaOH (or 1 M HCl). Bring the total
volume to 500 mL. (This is enough for 14 pairs of students).
o 10.0 mM glucose-6-phosphate (G6P). Dissolve 70.5 mg glucose-6-phosphate
(anhydrous sodium salt, 282.12 g/mol) in 25.0 mL water (3 pairs of students).
o 1.00 mM β-nicotinamide adenine dinucleotide phosphate (NADP+). Dissolve 19.1 mg
NADP+ sodium salt (765.39 g/mol anhydrous base) in 25.0 mL water (3 pairs of
students). Prepare this the same day that it will be used.

• Buffer solution to dilute enzyme: The enzyme will be diluted with 50.0 mM
glycylglycine buffer with 10.0 mM magnesium chloride, pH 7.4. Dissolve 3.30 g
glycylglycine (132.12 g/mol) and 1.02 g MgCl2×6H2O (203.30 g/mol) in 450 mL of water
and adjust the pH to 7.4 with 1 M NaOH (or 1 M HCl). Bring the total volume to 500
mL.

• Enzyme solution: each pair of students will be given 10 mL of 1.0 x 10-2 mg/mL G6PDH
dissolved in 50.0 mM GlyGly, 10.0 mM MgCl2 buffer, pH 7.4. Keep this enzyme
solution (and all enzyme solutions) on ice until the time it is added to the assay mixture.
The concentration of this enzyme solution has been measured by absorbance, assuming
1.31 absorbance units for a 1.00 mg/mL solution. The specific activity was assumed to be
540 U/mL, so a 2.00 U/mL solution would be 3.7 x 10-3 mg/mL. However, in winter 2021
a higher concentration was needed for the students to dilute 5-fold in the range finding.

• 2.00 M glucose (14.40 g glucose (180.16 g/mol) dissolved in 50.0 mM GlyGly, 10.0 mM
MgCl2 buffer, pH 7.4 to 40.0 mL total volume).

• 2.00 M 1,2-propanediol (5.86 mL or 6.08 g (76.09 g/mol, density 1.04 g/mL) dissolved in
50.0 mM GlyGly, 10.0 mM MgCl2 buffer, pH 7.4 to 40.0 mL total volume). 1,2-
propanediol is somewhat hard to pipette, so it would be better to do this by weight.

MF2-6
Additional Reagents for Measurements of Vmax and Km G6PDH solutions (all water is dH2O)

• Make 0.500 mM NADP+ in water. Dissolve 19.1 mg NADP+ sodium salt (765.39 g/mol
anhydrous) in 50.0 mL water (66 pairs of students). Prepare this the same day that it
will be used.

• Make 10.0 mM NAD+ in water. Dissolve 68.5 mg NAD+ sodium salt (685.41 g/mol
anhydrous) in 10.0 mL water (13 pairs of students). Prepare this the same day that it
will be used.

MF2-7