1. 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+.
Lactate Dehydrogenase plays a critical role in the
two metabolic pathways; glycolysis and
gluconeogenesis. In glycolysis, LDH converts
Pyruvate to Lactate under anaerobic conditions to
generate a low amount of ATP. Skeletal muscle
commonly undergoes this conversion, generating
Lactic Acid, which is then metabolized by the liver.
The liver then metabolizes the Lactic Acid back to
Pyruvate using LDH again in a process known as
gluconeogenesis. LDH is found in cells of virtually
all of life including eukaryotes and prokaryotes,
and the critical role described above is an
indication why.
Materials and Methods
• Tissue Extraction
Tissue from sources including chicken heart and
breast and bovine heart were collected. Approximately
50 g of tissue was removed and chopped, then
blended in a Tris buffer. The resulting homogenate
was then transferred into centrifuge tubes for
centrifugation.
• Ammonium Sulfate Precipitation
Following initial centrifugation, the supernatant was
recovered for precipitation. Protein in the supernatant
at this point was solubilized by surrounding water
molecules. In order to precipitate out the protein, ionic
strength was increased steadily using Ammonium
Sulfate ions. Ions in solution would displace the
protein molecules at approximately 40% of saturation.
The precipitate was then centrifuged and the pellet
was recovered for dialysis.
• Dialysis
Using 10,000 MW pores in dialysis tubing, the
resuspended pellet was further purified by allowing
impurities and buffer salts to pass through the semi-
permeable membrane. Dialysis was repeated 3 times.
• Ion-Exchange Chromatography
The protein mixture was transferred to a low ionic
strength mobile phase. 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.
• UV-Vis Kinetic Analysis
In order to measure kinetic activity in the Lactate 
Pyruvate direction of the conversion, the production of
NADH was monitored over time at 340 nm. [Lactate]
was increased in successive trials to generate an
enzyme kinetics curve for the LDH isoform.
Title
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XAVIER UNIVERSITY
Isozymes of LDH
Fig 1. Isozymes of LDH tetramers. Isozymes exist in combinations
of H and M subunits. Enzymatic activity was quantified for the M4
and H4 tetramers from various sources (3)
Lactate Kinetics
Fig 4. Chicken breast and heart LDH Lactate  Pyruvate kinetics using
UV-Vis spectrophotometer at 340 nm. Kinetic parameters of interest
include Vmax and Km. Vmax corresponds to the maximal rate of NADH
production and Km refers to the [Lactate] at ½ Vmax.
Table 1. Catalytic efficiency of LDH isozymes. Kcat/Km was used to
measure catalytic efficiency; accounting for both substrate affinity and
turnover number. Chicken heart LDH showed higher catalytic efficiency,
indicating a preference for the Lactate  Pyruvate conversion.
Sources of LDH
Both chicken heart and breast as well as beef
heart were used as sources of Lactate
Dehydrogenase. 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.
Fig 2. Physiological background for LDH. LDH is involved in both
glycolysis and gluconeogenesis, using the interconversion reaction. (2)
Purification and Kinetic Analysis of Lactate Dehydrogenase
Mitchell Cornely, Dr. Stephen Mills (Chemistry)
Acknowledgments
Dr. Stephen Mills
Xavier University Chemistry Department
Stephen Nichols
Aristide Kilundu
References
1. Gibson, C. Michael. "Lactate Dehydrogenase." - Wikidoc. N.p., 12
Feb. 2015. Web. 06 Apr. 2016.
2. Garrett, R., and Charles M. Grisham. Biochemistry. Belmont, CA:
Brooks/Cole, Cengage Learning, 2013. Print.
3. Rajeev, Gandham. "ISOENZYMES & CLINICAL ENZYMOLOGY."
ISOENZYMES & CLINICAL ENZYMOLOGY. N.p., 23 Oct. 2014.
Web. 06 Apr. 2016.
The catalytic efficiency of the breast and heart LDH
indicate a preference for certain directions of the
interconversion based on tissue type. Liver and
skeletal muscle are identical isoforms yet prefer
different conversions. The heart LDH (H4), however,
prefers Lactate  Pyruvate and the breast (M4)
does not based on Kcat/Km. Thus there is an
indication that tetramer differences do impact a
preference for a reaction direction.
The various isozymes of LDH are found
throughout different cell types in the human body.
The isozymes of LDH are tetramers consisting of
H and M subunits in different combinations.
Depending upon cell type and enzymatic
preference (one direction of the interconversion)
the H and M subunits vary. One possible
explanation for the different isoforms is that
different cell types are more prone to one direction
of the interconversion. The expectation, based on
physiology would be that liver and skeletal muscle
tissue would prefer different conversions, but they
are in fact identical isozymes (see Fig. 2). The
interest in this study was to identify whether heart
tissue (H4) would prefer the Lactate  Pyruvate
conversion in order to protect cardiovascular
tissue from damage due to Lactic acid, while
skeletal muscle (M4) would prefer the Pyruvate 
Lactate conversion as it is more commonly
exposed to anaerobic conditions.
Chicken Breast LDH Chicken Heart LDH
kcat (s-1) 945 1.77
KM (mM) 2.7 11
kcat/KM (M-1 s-1) 340,000 160
Conclusions
We measured the kinetic parameters for the
conversion of lactate to pyruvate for both isoforms of
LDH.
Chicken Breast LDH Chicken Heart LDH