Spence 1
PFK-1 Assay Using Capillary
Electrophoresis to Analyze Enzyme
Inhibition
Lawson Spence
30 November 2021
CHEM 0336L
Dr. Rachael Day
Jeremiah Ellison & Allie Gipson
Abstract:
This lab experiment is utilized for
analysis of the third step of Glycolysis,
which is catalyzed by PFK-1 and converts
F-6-P into Fructose-1,6-Bisphosphate in a
rate limiting committed step. This reaction
can be monitored using Capillary
Electrophoresis. PFK-1 is allosterically
upregulated by ADP and AMP but inhibited
by ATP. This experiment will analyze the
enzyme’s function without inhibitor (ATP)
present, and then compare it with different
levels of inhibitor present. Another section
of these experiments was protein
crystallization. Crystallization is one of the
main techniques used to determine the
structure of a protein. In our experiment
many solutions were created to see which
yielded the best crystals.
Keywords: Enzyme Kinetics, Glycolysis,
Inhibition, PFK-1, allosteric,
Electrophoresis, Protein Crystallization
Introduction:
Phosphofructokinase-1 (PFK1)
catalyzes the irreversible conversion of
Fructose-6-Phosphate (F-6-P) and ATP into
Fructose-1,6-Bisphosphate (F-1,6-BP) and
ADP. Phosphofructokinase is a highly
regulated enzyme and a branching point of
glycolysis. An analysis of PFK is important
in understanding Glycolysis, which is an
important pathway for energy production in
most living organisms.
Mutations in PFK are also important
to examine so that we can better understand
metabolic disorders and other effects that
mutations on this enzyme may have. For
example, a genetic mutation in the PFKM
gene results in Tarui's disease, which is a
glycogen storage disease where the ability of
certain cell types to utilize carbohydrates as
a source of energy is impaired. There are
also important aspects linking PFK’s
function to cancer. One study showed usage
for a PFK inhibition test and its “capacity to
detect malignant neoplasms irrespective of
the organs in which cancer cells start
proliferating.” (Nakamura et. al). PFK-1 is
inhibited by citrate, ATP and ATA via an
allosteric site. Aurintricarboxylic Acid,
(ATA) is a trisodium salt that has a function
as the biological stain 'chrome violet CG.’
ATA has also been found to have antiviral
activity in vitro against coronaviruses such
as SARS, MERS and SARS-CoV-2 (Liu et.
al 2020).
The inhibition from ATA prevalence
prevents the conversion of F-6-P to F-1,6-
BP which in turn, does not consume ATP.
Because of this, this experiment can
visualize the loss of activity of PFK-1 with
various concentrations of inhibitor (ATA)
while keeping the concentration of substrate
and enzyme constant.
This inhibition will be analyzed
utilizing Michaelis-Menten kinetics and a
lineweaver-burk plot to also show the
enzyme’s efficiency in the presence of the
inhibitor. This analysis will examine the
variables associated with Michaelis-Menten
kinetics, for example Vmax and Km, which
show an enzyme's maximum rate and a
pseudo value for the enzyme’s substrate
affinity. The Michaelis constant Km is the
substrate concentration at which the reaction
rate is at half-maximum, and is an inverse
measure of the substrate's affinity for the
enzyme. If there is a small Km, the rate will
approach Vmax more quickly. The value of
Km is dependent on both the enzyme and
the substrate.
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Methods:
This section will be an account of the
procedures that took place in the laboratory.
This experiment was conducted in two
different sessions in the span of two weeks.
The first week consisted of the PFK-1 assay
without any inhibitor present. The following
week conducted the same assay but with our
selected inhibitor (ATA) present in different
concentrations. The results from the second
week and first week were recorded after Tested Group - PFK1 with Inhibitor
completing the Capillary Electrophoresis (ATA)
and saved for comparing the enzyme’s The second week repeated similar
activity with the inhibitor to the control steps to the previous week, starting by
group. preparing a solution. This week, one
Control Group - PFK1 (No Inhibition) solution was made and the components,
During this week, several solutions measured in microliters, are listed in the
were made by various groups participating following table.
in the experiment. The solutions were 10mL
in total and contained varying amounts of F6P SAMPLE ATP TOTAL
enzyme, sample buffer, and ATP. The below BUFFER VOL
values are the components of each solution,
measured in microliters. 2500 5000 2500 10000

F6P SAMPLE ATP TOTAL Afterwards, 1500 microliters of the

BUFFER VOL mixture solution was pipetted into a clean
capillary electrophoresis vial. Following
250 7250 2500 10000 this, varying amounts of inhibitor (ATA)
was added to replicate CE vials. The four
1250 6250 2500 10000 separate ATA volumes were measured as
150 μM, 10 μM, 1 μM, and 0.1 μM. Two
2500 5000 2500 10000
separate lab sections each completed two
different concentrations. The capillary
After the solutions were made, 1500 electrophoresis instrument was again
microliters of each tube was pipetted into a prepared for use by Dr. Day. Results were
capillary electrophoresis vial. The recorded as the components of the solutions
electrophoresis instrument was prepared to began to separate.
run by Dr. Day. The instrument was allowed Capillary Electrophoresis
to run and the charged portions in the Capillary Electrophoresis was
solution were attracted towards the utilized to separate different components of
negatively charged end of the the solutions, so that conclusions about the
electrophoresis. The ATP and ADP began to enzymatic activity could be made. Capillary
split into separate regions, allowing for an electrophoresis was the preferred analytical
analysis. technique because this instrument separates
ions with an applied voltage. The mobility
of different components of the solution is
 Spence 3

dependent upon the charges of the present tables below.

molecules. Other characteristics of the
compounds also affect the rate at which they
are attracted towards the applied voltage.
These include the viscosity, and the atom's
radius.
Neutral species are not affected, and
only ions will move with the electric field. If
two ions are the same size, the one with
greater charge will move the fastest.
UV-Vis is another common
technique for examining traits of a
compound or determining what molecules
may be present in a solution. To study PFK
activity with UV-Vis spectroscopy, the
reaction is typically coupled with further
steps of glycolysis so that NADH is formed,
and monitored at 340 nm. This is typically
the method used during UV-Vis because
most biomolecules will all absorb light in a
similar range of 200-280nm. For example, The solutions were incubated at room
both ATP and ADP will absorb light at temperature and analyzed under microscope
260nm, and would not allow for a the following week.
determination of amounts present. Because
of this, CE (Capillary Electrophoresis) was Results:
chosen as the preferred method of analysis.
Protein Crystallization
Crystallization is one of the main
techniques used to determine the structure of
a protein. The first week the experiments
were set up to obtain crystals of the protein
lysozyme. The second week the resulting
crystals were examined in the respective
crystal trays and observations were recorded
regarding which condition(s) were best for
crystal formation. The solutions containing
lysozyme protein were created following the
This first graph obtained is a graph
of ADP molar ratio vs time. Tuesday's
section lab completed a solution containing
2500 F6P and 2500 ATP. All other solutions
were conducted in other sections or by Dr.
Day. All solution graphs overlaid are present
in the second figure below.
The initial velocity was found by
taking the coefficient logarithmic term of the
 Spence 4

5.79/3.19. The negative inverse was used to

determine a Km = 1.81. Vmax was found by
taking the inverse of the y-intercept value
(5.79). This found Vmax = 0.173. These
values were compared to data given by Dr.
Day, which showed Vi/Vo vs ATA.

best fit line found in each separate graph.

For example, with the 2500 F6P graph, the
value was found to be 0.0916. The initial
velocities for each concentration were found
and plotted against their respective
concentration, forming a Michaelis-Menten
plot. This graph was used to determine
IC50, where inhibition is at 50%. This
occurs when y=0.5, which in this case is at
roughly 2.5 μM.

For further simplification, the

Michaelis-Menten plot was converted into a
Lineweaver-Burk plot by taking the inverse
of each value, (finding 1/S and 1/Vo) and
plotting them against each other.

This plot allowed for a best fit line to

be formed and utilized to estimate the
enzymes Vmax and Km values. The Km
was first found by setting the best fit line
equal to zero.0=3.19 x +5.79 algebra
allowed for x to be determined to be -

Protein Crystallization
Below are the results from the
protein crystallization in the various
solutions. It was found that as sodium
chloride concentration increased, crystal
formation seemed to improve drastically.
Discussion & Conclusion:
These experiments allowed for a
better understanding of a wide array of
topics covered through the first semester of
undergraduate biochemistry. These topics
included enzyme kinetics, the Michaelis-
Menten equation, Lineweaver-Burke plots,
and using capillary electrophoresis for
analysis. The experiment began by using
basic laboratory techniques involving
micropipetting and completing simple
dilutions. The later use of electrophoresis
allowed for a better understanding of the
mechanism of electrophoresis separation.
This occurs where ATP moves at a quicker
rate towards the cathode and allows for
analysis of the varying amounts of
ATP/ADP present in the solution.
The Michaelis-Menten plot allowed
the comparison of ADP formation at
Spence 5
different substrate values (F6P). This
showed that at lower substrate values, the
rate of conversion to ATP was slower. The
data obtained in this experiment stayed
consistent with expected values of higher
rates of product formation at higher
substrate concentrations. Also, when the
inhibitor was present there was a noticeable
decline in the rate of conversion from ATP
to ADP in the presence of PFK1. This
decreased rate stays consistent with what is
expected of an allosteric inhibitor such as
ATA. As was analyzed, Vmax was
decreased in the presence of ATA and Km
was increased, showing a lower affinity
towards the substrate (ATP).
With every experiment, there are
also errors that are likely to give small
margins of error. These errors could stem
from inaccurate measurements/pipetting, or
from losing portions of solution when
transferring liquids or performing a dilution.
Another potential source of error may stem
from the capillary electrophoresis instrument
itself. The best way to counteract all of these
possibilities is to perform the experiment
many more times, and find the average
values of the data that is obtained. Sources
of error could also come from the
calculations made after recording the data,
including any rounding that occurred.
Based on the recorded data, it is
reasonable to conclude and reinforce the fact
that PFK1 activity is inhibited in the
presence of Aurintricarboxylic Acid (ATA).
In future experiments, it would be
interesting to find other sources of inhibition
to compare to the rates found with ATA.
Finding an inhibitor that binds via
competitive inhibition in the active site
would be a useful source of information in
determining which types of inhibitors have
varying rates of inhibition on PFK1’s
enzymatic activity. It would also be useful to
learn more about the inhibitor, and how it
may actually serve as a benefit to introduce
 Spence 6

an inhibitor into the glycolysis pathway in

living organisms.
 Spence 7

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