FLUORIMETER 6280/6285
Application note: A10-010A
Kinetics of hexokinase measured using the fluorescence
of NADH
+
Introduction
It is almost 100 years since Michaelis and
Menten published their research1 on the kinetic
properties of enzymes, describing how the rate
of an enzyme reaction varies with substrate
concentration. This led them to propose the
formation of an enzyme-substrate complex and
formulate the now famous Michaelis-Menten
equation.
When studying enzyme activity the substrate
must be present in excess so that the amount of
product produced is linear over the reaction
period. By reducing the substrate concentration
to a limiting amount, the rate of enzyme activity
can be reduced and this can be used to
determine the amount of substrate present in a
sample.
Measurement of such a reaction requires the
determination of the amount of product
produced or the disappearance of substrate
consumed. With many assays this cannot
always be done directly, therefore the reaction
may be coupled to a second enzyme that can
convert one of the products into a measurable
substance. One such example is the coupling of
reactions
to
dehydrogenases
that
use
+
nicotinamide adenine dinucleotide (NAD ) or the
reduced form, NADH, as coenzymes. NADH can
be readily measured in a spectrophotometer at
340nm or by fluorimetry with excitation at 340nm
and emission at 460nm.
In this experiment the Glucose (HK) Assay Kit
from Sigma (product code GAHK-20) was used
to investigate the rate of the hexokinase reaction
with varying amounts of glucose. The principle
of the kit is shown below:
Hexokinase
Glucose + ATP
Glucose-6-phosphate + ADP
G6PDH
G6P + NAD+
phosphogluconate in the presence of NAD in a
reaction catalysed by glucose-6-phosphate
dehydrogenase (G6PDH). During this oxidation,
an equimolar amount of NAD+ is reduced to
NADH. The reaction was monitored by
measuring the increase in fluorescence at
460nm with excitation at 340nm.
Methods
The Glucose (HK) Assay Reagent was
reconstituted according to the manufacturers’
2
instructions in 20ml deionised water. 1.5ml of
reagent containing 1.5U of hexokinase was
pipetted into a fluorimeter cuvette and this was
placed in a model 6285 fluorimeter fitted with a
UG1 320-380nm filter (part code 627 126) for
excitation and a 460nm interference filter (part
code 627 167) for emission. The gain was set to
68% which was determined by the auto set gain
function using the final fluorescence of the
100µg glucose sample. The fluorimeter was
connected to a PC running the DataWay data
acquisition software and was set up to take RFU
readings every 3 seconds.
Baseline readings were taken for approximately
1 minute before the addition of glucose as
substrate. Various volumes of standard 1mg/ml
glucose solution were added to the reagent in
separate assays ranging from 5µl (5µg glucose)
to 100µl (100µg glucose) with the volume being
made to 100µl with water in each case. The
substrate was quickly mixed into the reagent in
the cuvette and the fluorescence of the reaction
recorded until it reached a steady rate.
Three drinks with unknown amounts of glucose
were also tested. These were Lucozade Energy
Original (GlaxoSmithKline plc, UK), Pressed
Apple Juice (Wm Morrison Supermarkets plc,
UK) and a sweet white dessert wine. Each drink
was diluted 1 in 50 with deionised water and
25µl aliquots were used in the assay.
6-Phosphogluconate + NADH
Results
Glucose is first phosphorylated by hexokinase in
a reaction with ATP. The product, glucose-6phosphate (G6P), is then oxidised to 6-
The time courses of the reactions with various
amounts of glucose added as substrate are
shown in Figure 1. There was very little
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the values of Vm and Km were calculated to be
1.05 RFU/s and 25.60µg added glucose
respectively. This equates to a Km value of
0.089mM.
difference between the rates of reaction with
75µg and 100µg glucose, indicating that this
amount of substrate was just sufficient not to
limit the reaction.
7
120
y = 24.302x + 0.9494
R2 = 0.9934
6
100
5
80
1/Vi
RFU
4
60
Slope = Km/Vm
3
40
2
-1/Km
20
1/Vm
0
0
-100
1
0
100
200
300
400
-0.05
500
0
0.05
0.1
0.15
0.2
1/[S]
Time (s)
5µg
10µg
15µg
20µg
25µg
50µg
75µg
Figure 3: Lineweaver-Burk double reciprocal
plot to derive Vm and Km.
100µg
The values for Vm and Km can also be derived
from a second type of plot called an EadieHofstee plot5,6. This is a plot of vi versus vi /[S];
the y-intercept is Vm, the slope is – Km and the xintercept is Vm/Km. The data plotted according to
this method is shown in Figure 4.
Figure 1: Time course of the hexokinase assay
initiated with the indicated amounts of glucose at
time 0.
The initial rate or velocity vi of each reaction was
calculated for the period between 30s after
adding glucose to 90s after addition. These are
shown in Figure 2, plotted against glucose
concentration.
1.2
1
Vm
vi
0.8
0.7
y = -26.83x + 1.0878
R2 = 0.9442
0.6
Slope = -Km
0.4
0.6
½ Vm
0.5
vi
Vm
0.8
0.9
Vm/Km
0.2
0.4
0
0
0.3
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
v i /[S]
0.2
Km
0.1
Figure 4: Eadie-Hofstee plot of vi versus vi /[S].
0
0
20
40
60
80
100
120
Glucose
(µg)
Glucose (ug)
3
Figure 2: Michaelis-Menten plot showing the
initial velocity vi as a function of substrate
concentration ([S]), the amount of glucose
added to the assay.
The maximum velocity Vm and the Michaelis
constant Km (the concentration of substrate at
which the reaction is at half maximum velocity)
for the reaction can be determined by plotting
1/vi versus 1/[S]. This double reciprocal plot is
known as the Lineweaver-Burk plot4 and gives a
straight line with a slope of Km/Vm and a yintercept of 1/Vm. Using the equation of the line,
Using the equation of the line from this plot, the
values of Vm and Km were calculated to be 1.09
RFU/s and 26.83µg added glucose respectively
(Km value of 0.093mM), which are slightly higher
but in good agreement with those obtained from
the Lineweaver-Burk plot.
Samples from three different drinks containing
unknown amounts of glucose were also
measured. The glucose content of these
samples can be calculated from standard curves
produced by plotting either the initial rates of
reaction of the known samples against glucose
concentration or by plotting the final
fluorescence values (when the reaction reaches
a steady rate) against glucose concentration.
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Since the relationship of both vi and final
fluorescence are only linear in the range up to
about 25µg glucose added, only these data
points were used for the standard curves. The
unknown samples were diluted to give results
within this range. The two standard curves are
shown in Figure 5.
Conclusions
0.6
A
0.5
y = 0.0187x + 0.0867
R2 = 0.9957
0.4
RFU/s
spectrophotometric assay. The glucose content
of apple juice was expected to be in the range of
10 to 30mg/ml and that for sweet wine, at least
7
18g/l . With the Lucozade Energy drink, the
measured amount of glucose was found to be
about half the total sugars stated on the bottle
label.
0.3
0.2
0.1
0
0
5
10
B. Final RFU
15
20
25
30
ug Glucose
µg
90
B
80
y = 2.4336x + 19.705
R2 = 0.9781
70
Hexokinase catalyses the first step in the
pathway of glucose metabolism known as
glycolysis, the process by which the cell creates
energy in the form of ATP. In mammalian cells
there are four known isozymes of hexokinase
that vary in their subcellular location and
kinetics8. The “low-Km” isozymes (I, II and III)
have a high affinity for glucose (below 1 mM)
and isozymes I and II follow Michaelis-Menten
kinetics at physiological concentrations of
substrates8.
RFU
60
50
40
30
20
10
0
0
5
10
15
20
25
30
ug Glucose
Glucose
µg
Figure 5: A: standard curve using the initial
velocity of reaction. B: standard curve using the
final fluorescence values.
The concentrations of glucose in the three drinks
calculated from the equations of the standard
curves shown in Figure 5 are given in Table 1,
together with the values obtained in a previous
7
experiment using the same assay kit but by
measuring the absorbance at 340nm and
calculating the glucose concentration from the
extinction coefficient of NADH.
Method
Initial rate
Final
fluorescence
Final
absorbance
Glucose (mg/ml)
Lucozade
Apple
Wine
Juice
40.48
15.88
22.35
40.74
15.29
25.70
42.19
15.99
22.15
Table 1: Measured glucose concentrations in
three drinks calculated from the rate of reaction,
final fluorescence and final absorbance values.
The values obtained from the fluorescence
experiments described here are in good
agreement with those obtained from the
The hexokinase reaction with glucose measured
in the experiments described here was followed
by measuring the fluorescence of NADH
produced in the coupled reaction with G6PDH.
The reaction was shown to follow MichaelisMenten kinetics and the Km value of around
0.09mM derived from two types of plot is
consistent with hexokinase I or II.
Both the vi and final fluorescence were used to
determine the glucose concentration of unknown
samples. The Jenway 6280 and 6285
fluorimeters therefore offer a sensitive way to
measure NAD+/NADH coupled enzyme assays
both for quantifying the substrate concentration
of a sample or for following the kinetics of an
enzyme reaction.
References
1. L. Michaelis and M. Menten. Die Kinetik der
Invertinwirkung. Biochem. Z. 49:333 (1913).
2. www.sigmaaldrich.com/etc/medialib/docs/Si
gma/Bulletin/gahk20bul.Par.0001.File/gahk2
0bul.pdf
3. J.F. Robyt and B.J. White. Biochemical
Techniques: Theory and Practice, Chapter
9. Brooks/Cole Publishing Company,
Monterey, California. ISBN 0-534-07944-X
(1987).
4. H. Lineweaver and D. Burk. The
Determination of Enzyme Dissociation
Constants. J. Am. Chem. Soc. 56:658
(1934).
[email protected]
www.jenway.com
Tel: +44 (0)1785 810433
5. G.S. Eadie. The Inhibition of Cholinesterase
by Physostigmine and Prostigmine. J. Biol.
Chem. 146:85 (1942).
6. B.H.J. Hofstee. Non-inverted Versus
Inverted Plots in Enzyme Kinetics. Nature,
184:1296 (1959).
7. Application note: A09-008A. Quantitative
determination of glucose in an energy drink,
apple juice and wine. www.jenway.com.
8. http://en.wikipedia.org/wiki/Hexokinase.
Lucozade is a registered trade mark of the GlaxoSmithKline
group of companies.
[email protected]
www.jenway.com
Tel: +44 (0)1785 810433