thesis_mitchell cornely
TRANSCRIPT
A Thesis
Submitted to the Faculty
of
Xavier University
In partial fulfillment of the requirements for the degree of
Bachelor of Science
by
Mitchell J. Cornely
May 14, 2016
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Approved:
________________________________Barbara M. Hopkins, Ph.D.Chair, Department of Chemistry
________________________________Stephen Mills, Ph.D.Thesis Advisor
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PURIFICATION AND KINETIC ANALYSIS OF LACTATE DEHYDROGENASE
BY
MITCHELL J. CORNELY
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ACKNOWLEDGEMENT
The author wishes to thank Dr. Stephen Mills for the openness and guidance which led to the completion of this project, and the Xavier Chemistry department for the use of the laboratory and various supplies. Lastly, but most importantly, the author wishes to acknowledge the inspiration of his parents who have been a tremendous support both in and out of the classroom
-MJC
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CONTENTS
Page
I. INTRODUCTION 1
II. HISTORICAL 2
a. Enzymology of H4 and M4 tetramers 2
b. Pyruvate substrate inhibition 10
c. Quail heart and muscle isozyme differences 12
III. CURRENT RESEARCH 19
a. Materials and Methods 20
i. Tissue extraction 21
ii. Ammonium Sulfate precipitation 22
iii. Dialysis 25
iv. Ion-Exchange chromatography 28
v. UV-Vis kinetics 30
b. Results 36
i. Enzyme purification 36
ii. UV-Vis kinetic assays 39
iii. Ni-Sepharose column separation 40
IV. DISCUSSION AND CONCLUSIONS 42
V. REFERENCES 45
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VI. LIST OF TABLES
Table Page
I Chicken Breast LDH Kinetic Data 19
II Chicken Heart LDH Kinetic Data 27
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LIST OF FIGURES
Figure Page
I Beef Heart and Chicken Breast Ion-Exchange 18
II LDH Kinetics lactate to pyruvate 23
III LDH Kinetics pyruvate to lactate 27
IV Q-sepharose and Ni-sepharose column 3
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ABSTRACT
Lactate Dehydrogenase (LDH) is an important enzyme in the anaerobic metabolism of glucose for the generation of Adenosine Triphosphate. LDH catalyzes the inter-conversion of pyruvate to lactate as NADH is oxidized to form NAD+. LDHhas been shown to be a tetramer comprised of a combination of M and H subunits. The LDH tetramer found in skeletal muscles is comprised of the M-subunit, while the tetramer found in the heart is primarily made up of the H-subunit. It has been suggested that LDH subunits possess different functions and properties, which influence their role in the production of lactate from pyruvate, and vice versa. The aim of this research was to determine whether the LDH found in the heart has different kinetic characteristics than the version found in the skeletal muscle.
Both chicken heart and breast as well as beef heart were used as sources of LDH. Extraction and purification were performed by centrifuging the homogenized tissue and precipitating the sample with ammonium sulfate. The molecules were then separated in dialysis tubing. Ion exchange chromatography (Q-Sepharose ) was also performed to further separate the molecules. A Ni-sepharose ion exchange was also performed in an effort to enhance LDH separation from Hemoglobin and Myoglobin in bovine heart tissue. Finally, kinetic studies utilizing UV-Vis spectroscopy were performed to analyze the enzymatic activity of the LDH isoforms, as well as to investigate whether each isoform favors the conversion of lactate to pyruvate or vice versa.
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Introduction
Lactate Dehydrogenase (LDH) is an important enzyme in the anaerobic
metabolism of glucose for the generation of Adenosine triphosphate. LDH catalyzes the
inter-conversion of pyruvate to lactate as NADH is oxidized to form NAD+. LDH has
been shown to be a tetramer comprised of a combination of M and H subunits depending
upon which tissue the enzyme is from. The LDH tetramer found in skeletal muscles is
mostly of the M-subunit, while the tetramer found in the heart is primarily the H-subunit.
We suggest that the different isoforms of LDH influence the functional properties and
kinetics of the interconversion of lactate to pyruvate and vice versa. The aim of this
research was to determine whether the LDH found in the heart has different kinetic
characteristics than the version found in the skeletal muscle.
LDH was purified from several sources using a combination of chromatographic
methods. Following purification, enzyme kinetics were analyzed using UV-Vis kinetics.
In previous studies the enzymology of beef and chicken LDH was compared,
particularly comparing the H4 and M4 tetramers of bovine and chicken LDH. Data of
particular relevance to the current study are found in table I, the catalytic characteristics
of beef and chicken lactic dehydrogenases. KM and kcat values were reported for the
chicken H4 tetramer as 8.9 x10-5 M and 45,500 s-1 respectively for the pyruvate to lactate
conversion. In the lactate to pyruvate conversion the H4 tetramer had a KM of 7 x 10-3 M.
The M4 tetramer had a KM of 3.2 x 10-3 M and a kcat of 93,400 s-1 in the pyruvate to lactate
conversion. The M4 tetramer had a KM of 4 x 10-2 M in the lactate to pyruvate direction
(1).
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Griffin and Criddle studied the mechanism of substrate-inhibition in excess
pyruvate for LDH. The results suggest that the monomeric subunit of lactate
dehydrogenase combines with the oxidized nucleotide before reacting with pyruvate in a
rate-limiting bimolecular step (2).
In a third study, kinetic characterization of heart and breast LDH from quail was
investigated. The results indicated that there is a difference in the distribution of tissue-
specific LDH isozymes and a difference in energy metabolism in quail because the
distribution of LDH isozymes was correlated with local oxygen tensions, pyruvate
inhibition, and lactate accumulation. In figure 2, comparing effects of concentrated
pyruvate, LDH in the quail heart was inhibited significantly, whereas the breast muscle
was less inhibited. KM values for pyruvate and lactate of heart LDH isozyme
were 0.100 ± 0.04 mM and 7.83 ± 0.52 mM, respectively, whereas for
breast muscle KM values were 0.350 ± 0.035 mM and 23 ± 1.21 mM,
respectively (3).
Following the previous studies, we plan to further study the
kinetic differences between the H4 and M4 tetramers of LDH in an effort
to determine if there is a preference for either direction of the
conversion reaction depending on the tissue source. We expect that
the M4 isoform will preferentially convert pyruvate to lactate due to the
anaerobic conditions skeletal muscle is prone to experiencing. The H4
isoform is predicted to preferentially convert lactate to pyruvate in
order to keep [lactate] lowered in cardiac tissue. The preferences will
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be evident by higher kcat and kcat/KM values for the preferred
conversion.
Materials and Methods
Tissue Extraction
Tissue from various sources including chicken breast and heart and bovine heart
were collected, and approximately 50 g of the tissue was chopped and blended in 75 mL
of 10 mMTris/HCl pH 7.4, 1 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM PMSF
extraction buffer. The resulting homogenate was then centrifuged, and the supernatant
was collected for precipitation.
Ammonium Sulfate Precipitation
The supernatant from the tissue extraction was recovered and transferred to a
beaker for precipitation using ammonium sulfate. Solid ammonium sulfate was ground to
a powder and added to the supernatant while stirring over a period of 30 min. Full
precipitation of the protein occurred at approximately 40% of saturation. The precipitate
was then centrifuged and the pellet was recovered for dialysis.
Dialysis
The pellet from the centrifugation was resuspended in the extraction buffer.
Dialysis using 10,000 MW dialysis tubing (Snakeskin, Pierce) was performed in 500 mL
of 10 mM Tris/HCl pH 8.0 ], 0.5 mM 2-mercaptoethanol dialysis buffer in order to
remove impurities such as ammonium sulfate ions and other small molecules.
Ion-Exchange Chromatography
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Following dialysis, the protein mixture was placed onto an on-exchange column.
The column used for chromatography was a positively charged Q-sepharose column.
Elution was accomplished by gradually increasing the ionic strength of the mobile phase
using an elution buffer of 20 mM Tris/HCl, pH 8.0, with [NaCl] increasing from 0.2 M to
1 M, and 3 mL fractions were collected for further kinetic analysis.
UV-Vis Kinetics
In order to measure the activity of the purified LDH fractions, the production or
consumption of NADH was detected at 340 nm using UV-Vis spectroscopy. The cuvette
contained 3 mM NaHCO3 and 3 mM NAD+ for each assay. Lactate concentration was
varied as follows for each assay: 60 mM, 40 mM, 20 mM and 10 mM. The buffer used to
dilute the lactate was 10 mM Tris/HCl pH 8.6 with 0.5 mM DTT. Kinetic parameters
were derived using the Michaelis-Menten plot of each kinetics assay using Kaleidagraph
for curve fitting.
Results
The protein purification procedure yielded successful isolations of the various
LDH isoforms of the biological sources. The ion-exchange chromatograms were used to
select fractions to be used for the activity assays by determining which fractions had the
highest protein concentration.
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Following the purification, the LDH kinetic activity was analyzed using UV-Vis
spectroscopy. The LDH from chicken breast and heart tissue was studied in the lactate to
pyruvate direction, and then the reverse, in an effort to determine if the catalytic
efficiency varied in a certain conversion for a certain tissue type. [lactate] was varied, and
the rate was measured over a period of 5 min.
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A B
A B
Figure I. Beef Heart and Chicken Breast Ion-Exchange. (A) Beef heart Ion-exchange chromatography print out. Q-Sepharose column with a Tris/HCl buffer mobile phase. The elution buffer increased in NaCl concentration from 0 to1 M to gradually increase ionic strength. (B) Chicken breast Ion-exchange chromatography print out. Q-Sepharose column with a Tris/HCl buffer mobile phase. The elution buffer increased in NaCl concentration from 0 to 1 M to gradually increase ionic strength.
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Kinetic parameters of interest included the maximal rate Vmax, Km and kcat. The
kinetic parameters for chicken heart and breast LDH were compared.
Table I
Kinetic Parameter Chicken Breast LDH lactate to pyruvate
Chicken Breast LDH pyruvate to lactate
Kcat (s-1) 1.77 339.48
Km (mM) 11 0.9336
Kcat/Km (M-1s-1) 160 363600
Kinetic data for chicken breast LDH. Kcat/Km was used to compare catalytic efficiency of chicken breast LDH in the two different conversions.
After comparing the catalytic efficiency of the LDH isoforms from different
tissue types, the next comparison to be made was between the lactate to pyruvate vs.
pyruvate to lactate directions for the chicken heart and breast in order to determine if
there is a preference for either conversion.
The kinetics in the lactate pyruvate direction indicate substrate inhibition as
evident by the slope of the line (peaking and then dropping off). The inhibition could be
impacting the kinetic parameters, but further studies to confirm and determine effects of
inhibition are necessary.
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A B
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Again, following kinetic analysis, the parameters were compared in order to
determine a potential preference.
Table II
Kinetic Parameter Chicken Heart LDH lactate to pyruvate
Chicken Heart LDH pyruvate to lactate
Kcat (s-1) 945 311.55
Km (mM) 2.7 1.63
Kcat/Km (M-1s-1) 340,000 1,903,000
Kinetic data for chicken heart LDH. Kcat/Km was used to compare catalytic efficiency of the chicken heart LDH in the two different conversions.
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Figure III. LDH Kinetics pyruvate to lactate. (A) Chicken breast LDH kinetics converting pyruvate to lactate. Chicken heart LDH converting pyruvate to lactate. Measured using UV-Vis spectrophotometer at 340 nm.
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After the kinetic assays were performed, beef heart was then purified using two
different ion-exchange methods in order to improve purity and lessen contamination by
hemoglobin and myoglobin. One method utilized the standard Q-sepharose resin as a
stationary phase and the other used a nickel-sepharose (Ni-sepharose) resin. The fractions
were analyzed using UV-Vis spectroscopy in order to determine which contained the
highest LDH activity.
Discussion
Kinetic parameters from the Michaelis-Menten plot were used to analyze the H4
and M4 tetramers of LDH from various sources. kcat represented the rate of conversion
from lactate to pyruvate in the presence of NAD+. kcat/KM represented the catalytic
efficiency of the LDH, determined by enzyme affinity and rate of the conversion. The
goal of the study was to determine whether the tetramers of LDH consisting of only one
subunit (H4 or M4) would prefer a certain direction of the interconversion reaction. The
basis for which is that the cell types in which the tetramers are found would likely be
conditioned to use one direction of the conversion over the other. In the case of skeletal
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Q 11 Q 12 Q 16 Q 17 Ni Ft Ni 10
Ni11 Ni 12
Ni 13
0.00E+00
1.00E-07
2.00E-07
3.00E-07
4.00E-07
5.00E-07
6.00E-07
7.00E-07
Assay
Rat
e (M
/s)
Figure IV. Q-sepharose and Ni-sepharose column. Stationary phase comparison using UV-Vis activity of LDH.
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muscle LDH, which is more commonly exposed to anaerobic conditions, the pyruvate to
lactate conversion should be preferred. The heart LDH should prefer the opposite
conversion in order to protect the heart from damaging lactic acid concentrations in
critical cardiac tissues. Thus, the catalytic efficiency of the chicken breast LDH in the
pyruvate to lactate direction should be higher than in the lactate to pyruvate direction. We
observed kcat/KM for the chicken breast LDH in the pyruvate to lactate direction was
363,600 M-1s-1 and 160 M-1s-1 in the lactate to pyruvate direction. The catalytic efficiency
for the chicken heart LDH should be higher in the lactate to pyruvate direction than the
pyruvate to lactate direction, but the data suggests otherwise (340,000 M-1s-1 vs.
1,903,000 M-1s-1).
The heart LDH (H4) proceeded at a faster rate (kcat) than the breast muscle LDH
(M4) in the lactate to pyruvate direction. Additionally, the catalytic efficiency (kcat/KM) of
the heart LDH in the lactate to pyruvate direction was more than 2000x higher than that
of the breast muscle LDH. Although the heart LDH was more efficient in the pyruvate to
lactate direction, just as the breast LDH was, it is important to note that the H4 tetramer is
capable of a more efficient conversion of lactate to pyruvate, suggesting the heart LDH
has a physiological need for a more efficient conversion of lactate to pyruvate than does
the breast LDH. It is also of interest to note that the breast muscle did have a much higher
efficiency in the pyruvate to lactate direction, which suggests a possible preference.
Physiologically, skeletal muscle is more exposed to anaerobic conditions than heart tissue
would be, thus confirming the breast muscle’s preference for the pyruvate to lactate
conversion.
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Finally, the Ni-sepharose purification proved to be a more effective method than
Q-sepharose for bovine heart tissue. The high hemoglobin and myoglobin content in the
homogenate affected the purity of the LDH, but the nickel column successfully bound the
impurities and the LDH flowed through the column as indicated by the high activity of
the Ni-sepharose flow through.
Conclusions
The purpose of the study was to further analyze the kinetic differences
between the H4 and M4 tetramers of LDH in an effort to determine if
there was a preference for either direction of the conversion reaction
depending on the tissue source. We expected that the M4 isoform
would preferentially convert pyruvate to lactate due to the anaerobic
conditions skeletal muscle is prone to experiencing. The H4 isoform
was predicted to preferentially convert lactate to pyruvate in order to
keep lactate concentration lowered in cardiac tissue. The preferences
would be evident by higher kcat and kcat/KM values for the preferred
conversion. This prediction was confirmed for the chicken breast (M4)
isoform, as the catalytic efficiency was more than 2000x higher in the
pyruvate to lactate direction than lactate to pyruvate. The catalytic
efficiency of the H4 isoform was 2000x higher in the lactate to pyruvate
direction, suggesting a more efficient ability to convert lactate to
pyruvate than skeletal muscle. Further investigation will be performed
to test other LDH sources.
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REFERENCES
1. Pesce, A; McKay, R. H. The Comparative Enzymology of Lactic
Dehydrogenases. Biochemistry. 1964
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2. Griffin, J. H.; Criddle, R. S. Substrate-Inhibited Lactate Dehydrogenase. Reaction
Mechanism and Essential Role of Dissociated Subunits. Biochemistry. 1970, 9,
1195–1205.
3. Singh, R.; Sastry, K.; Pandey, N.; Shit, N.; Agrawal, R.; Singh, K.; Mohan, J.;
Saxena, V.; Moudgal, R. Characterization Of Lactate Dehydrogenase Enzyme in
Seminal Plasma of Japanese Quail (Coturnix Coturnix Japonica). Theriogenology.
2011, 75, 555–562.
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