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

i i

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.

xvi

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

xix xix

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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