LDH activity and enzyme kinetics in cow derived
heart tissue
27-2-2024
Biochemie cursusjaar 2023-2024
1
,Abstract
The LDH activity in heart tissue of a cow was measured to get a better understanding on how LDH
works in the heart. LDH plays a crucial role in the production of ATP. The LDH activity was
measured at different steps of purification (40% ammonium sulfate precipitation, 70% ammonium
sulfate precipitation, dialysis and affinity chromatography) to eventually discover at which
purification step the LDH activity has the highest value. To further investigate the structure and
functionality of LDH, a protein electrophoresis was performed to discover the LDH iso-enzymes.
Furthermore, Km and Vmax of the sample with the highest LDH activity were determined out of a
Michaelis-Menten plot to get a better understanding on the enzyme kinetics of LDH. The results
mainly conclude that after the 70% ammonium sulfate precipitation the heart tissue was purified the
most efficient, as in these samples the highest LDH activity was measured. Also, LDH in heart tissue
mostly consists of iso-enzymes LDH1, LDH-2 and LDH-3. This can be explained by the structure of
these iso-enzymes, as they all contain heart-polypeptide chains. The determined Km (0,07 mM) and
Vmax (2,75E-08) reveals that LDH in heart tissue has a low affinity for pyruvate substrate and has a
relatively low maximum reaction rate.
Introduction
Lactate dehydrogenase (LDH) is an enzyme that is present in all animals. The function of LDH is
crucial, as it plays a role in the production of ATP. The following reversible reaction is catalyzed by
the LDH enzyme: L-lactate + NAD+ <-> pyruvate + NADH + H+. When LDH needs to be measured
in experiments, the substrate of pyruvate or L-lactate is used. For the reaction to occur, co-factor
NADH or NAD+ is also necessary. These co-factors are recycled (Labbuddy LDH Literature, z.d.).
At high oxygen levels, large amounts of ATP are accumulated when the pyruvate, that is converted
from glucose during glycolysis, is converted to acetyl coenzyme-A and successively further degraded
in the citric acid cycle and oxidative phosphorylation to CO2 and H2O. Heart muscle cells work under
these high oxygen conditions.
However, sometimes the LDH enzyme works under low oxygen conditions, for example in the muscle
cells. These muscle cells still require significant amounts of ATP, but the citric acid cycle and
oxidative phosphorylation does not work under these low oxygen conditions. This essentially means
that only 2 ATP is formed when glucose is converted to pyruvate. During this reaction NAD+ is
converted to NADH and NAD+ can therefore no longer continue to make ATP. LDH has a solution
for this problem: the reduction of pyruvate to lactate needs a NADH molecule to occur, this yields a
NAD+ molecule. So, now the newly formed NAD+ molecule can yield another 2 ATP when glucose
is degraded to pyruvate (Labbuddy LDH Literature, z.d.).
The LDH enzyme has 5 different forms called iso-enzymes ( LDH-1: H4, LDH-2: H3M1, LDH-3:
H2M2, LDH-4: H1M3 and LDH-5: M4). Each is formed by 4 polypeptide chains. There are H(heart)-
and M(muscle)- type polypeptide chain types. The difference between these types can be found in
their amino acid sequences, this causes a difference in charge. Because of this charge difference, they
can be separated using gel electrophoresis. In muscle the iso-enzyme with 4 identical M chains (M4)
is mostly present, as it converts pyruvate fast to lactate. In the heart iso-enzyme H4 is mostly present,
as the conversion of pyruvate to lactate is much slower and therefore shows an inhibition by pyruvate.
Each LDH iso-enzymes can adapt their function based on the amount of oxygen available and needed
energy (Labbuddy LDH Literature, z.d.).
2
, During this experiment we show a great interest in measuring the LDH activity of the heart of a cow.
Before this can be measured, LDH needs to be purified out of the heart. After this the LDH activity
can be determined by measuring the [NADH] increase or decrease using pyruvate as substrate. This is
the reaction rate and thus the activity of LDH. The measurement at the beginning of the reaction
where no substrate has been converted are going to be the most important to determine the LDH
activity. When we are interested in the speed that it takes to convert substrate into product, we
calculate v (Labbuddy LDH Literature, z.d.).
Materials & Methods
K phosphate buffer
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD-Buffers and
solutions’ (Labbuddy LDH Experiment, z.d.), but with this modification: to get the pH of the buffer
from a pH of 6.67 to a pH of 7.2, 2800 microliters KOH is added.
Isolation
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD-Isolation’
(Labbuddy LDH Experiment, z.d.), but with this modification: 10.03 grams of cow heart is used.
40% Ammonium sulfate precipitation
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- 40%
Ammonium sulfate precipitation’ (Labbuddy LDH Experiment, z.d.), but with this modification: the
centrifugation in ‘step 6’ is performed twice.
70% Ammonium sulfate precipitation
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- 70%
Ammonium sulfate precipitation’ (Labbuddy LDH Experiment, z.d.).
Dialysis
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD-dialysis’
(Labbuddy LDH Experiment, z.d.).
Affinity chromatography
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- Affinity
chromatography’ (Labbuddy LDH Experiment, z.d.), but with this modification: the centrifugation of
‘step 5’ is performed twice.
Reference line protein determination
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- Reference
line protein determination’ (Labbuddy LDH Experiment, z.d.).
Total protein determination
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- Total protein
determination’ (Labbuddy LDH Experiment, z.d.), but with these modifications: in ‘step 1’ sample
70D is diluted 10 times instead of 100 times. The fractions 1-11 are not diluted.
NADH spectrum and extinction coefficient
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD-NADH
spectrum and extinction coefficient’ (Labbuddy LDH Experiment, z.d.).
3
heart tissue
27-2-2024
Biochemie cursusjaar 2023-2024
1
,Abstract
The LDH activity in heart tissue of a cow was measured to get a better understanding on how LDH
works in the heart. LDH plays a crucial role in the production of ATP. The LDH activity was
measured at different steps of purification (40% ammonium sulfate precipitation, 70% ammonium
sulfate precipitation, dialysis and affinity chromatography) to eventually discover at which
purification step the LDH activity has the highest value. To further investigate the structure and
functionality of LDH, a protein electrophoresis was performed to discover the LDH iso-enzymes.
Furthermore, Km and Vmax of the sample with the highest LDH activity were determined out of a
Michaelis-Menten plot to get a better understanding on the enzyme kinetics of LDH. The results
mainly conclude that after the 70% ammonium sulfate precipitation the heart tissue was purified the
most efficient, as in these samples the highest LDH activity was measured. Also, LDH in heart tissue
mostly consists of iso-enzymes LDH1, LDH-2 and LDH-3. This can be explained by the structure of
these iso-enzymes, as they all contain heart-polypeptide chains. The determined Km (0,07 mM) and
Vmax (2,75E-08) reveals that LDH in heart tissue has a low affinity for pyruvate substrate and has a
relatively low maximum reaction rate.
Introduction
Lactate dehydrogenase (LDH) is an enzyme that is present in all animals. The function of LDH is
crucial, as it plays a role in the production of ATP. The following reversible reaction is catalyzed by
the LDH enzyme: L-lactate + NAD+ <-> pyruvate + NADH + H+. When LDH needs to be measured
in experiments, the substrate of pyruvate or L-lactate is used. For the reaction to occur, co-factor
NADH or NAD+ is also necessary. These co-factors are recycled (Labbuddy LDH Literature, z.d.).
At high oxygen levels, large amounts of ATP are accumulated when the pyruvate, that is converted
from glucose during glycolysis, is converted to acetyl coenzyme-A and successively further degraded
in the citric acid cycle and oxidative phosphorylation to CO2 and H2O. Heart muscle cells work under
these high oxygen conditions.
However, sometimes the LDH enzyme works under low oxygen conditions, for example in the muscle
cells. These muscle cells still require significant amounts of ATP, but the citric acid cycle and
oxidative phosphorylation does not work under these low oxygen conditions. This essentially means
that only 2 ATP is formed when glucose is converted to pyruvate. During this reaction NAD+ is
converted to NADH and NAD+ can therefore no longer continue to make ATP. LDH has a solution
for this problem: the reduction of pyruvate to lactate needs a NADH molecule to occur, this yields a
NAD+ molecule. So, now the newly formed NAD+ molecule can yield another 2 ATP when glucose
is degraded to pyruvate (Labbuddy LDH Literature, z.d.).
The LDH enzyme has 5 different forms called iso-enzymes ( LDH-1: H4, LDH-2: H3M1, LDH-3:
H2M2, LDH-4: H1M3 and LDH-5: M4). Each is formed by 4 polypeptide chains. There are H(heart)-
and M(muscle)- type polypeptide chain types. The difference between these types can be found in
their amino acid sequences, this causes a difference in charge. Because of this charge difference, they
can be separated using gel electrophoresis. In muscle the iso-enzyme with 4 identical M chains (M4)
is mostly present, as it converts pyruvate fast to lactate. In the heart iso-enzyme H4 is mostly present,
as the conversion of pyruvate to lactate is much slower and therefore shows an inhibition by pyruvate.
Each LDH iso-enzymes can adapt their function based on the amount of oxygen available and needed
energy (Labbuddy LDH Literature, z.d.).
2
, During this experiment we show a great interest in measuring the LDH activity of the heart of a cow.
Before this can be measured, LDH needs to be purified out of the heart. After this the LDH activity
can be determined by measuring the [NADH] increase or decrease using pyruvate as substrate. This is
the reaction rate and thus the activity of LDH. The measurement at the beginning of the reaction
where no substrate has been converted are going to be the most important to determine the LDH
activity. When we are interested in the speed that it takes to convert substrate into product, we
calculate v (Labbuddy LDH Literature, z.d.).
Materials & Methods
K phosphate buffer
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD-Buffers and
solutions’ (Labbuddy LDH Experiment, z.d.), but with this modification: to get the pH of the buffer
from a pH of 6.67 to a pH of 7.2, 2800 microliters KOH is added.
Isolation
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD-Isolation’
(Labbuddy LDH Experiment, z.d.), but with this modification: 10.03 grams of cow heart is used.
40% Ammonium sulfate precipitation
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- 40%
Ammonium sulfate precipitation’ (Labbuddy LDH Experiment, z.d.), but with this modification: the
centrifugation in ‘step 6’ is performed twice.
70% Ammonium sulfate precipitation
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- 70%
Ammonium sulfate precipitation’ (Labbuddy LDH Experiment, z.d.).
Dialysis
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD-dialysis’
(Labbuddy LDH Experiment, z.d.).
Affinity chromatography
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- Affinity
chromatography’ (Labbuddy LDH Experiment, z.d.), but with this modification: the centrifugation of
‘step 5’ is performed twice.
Reference line protein determination
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- Reference
line protein determination’ (Labbuddy LDH Experiment, z.d.).
Total protein determination
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD- Total protein
determination’ (Labbuddy LDH Experiment, z.d.), but with these modifications: in ‘step 1’ sample
70D is diluted 10 times instead of 100 times. The fractions 1-11 are not diluted.
NADH spectrum and extinction coefficient
According to ‘Labbuddy-Biochemistry- Experiment I: Lactate Dehydrogenase ExperD-NADH
spectrum and extinction coefficient’ (Labbuddy LDH Experiment, z.d.).
3
