This document describes the characterization and purification of lactate dehydrogenase (LDH) from bovine myocardium tissue. The authors developed quantitative assays to analyze and purify LDH activity extracted from the tissue. Through a series of fractionation and chromatography steps, including ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography, the authors were able to purify LDH from the myocardial extract into near homogeneity. They also determined physical and enzymatic characteristics of the purified LDH through assays and SDS-PAGE gel electrophoresis analysis.

1. Characterization and Purification of Lactate Dehydrogenase from Bos taurus Myocardium
Amber Owens, Brittney Pfeifer, and Joshua Jackson
University of Missouri – St. Louis
Biochemistry 4713, Section 001
Dr. Christopher Wolin
May 13, 2013

2. 2
TABLE OF CONTENTS
INTRODUCTION------------------------------------------------------------------------------------------------------------------------------------3
MATERIALS AND METHODS--------------------------------------------------------------------------------------------------------------------3
PREPARATION OF MYOCARDIAL EXTRACT--------------------------------------------------------------------------------------------------------3
DEVELOPMENT OF AN ENZYMATIC ASSAY FOR LDH-------------------------------------------------------------------------------------4
ABSORBANCE PROFILES FOR NAD+ AND NADH ------------------------------------------------------------------------------------------------4
FEASIBILITY OF LDH ASSAY------------------------------------------------------------------------------------------------------------------------4
ESTABLISHMENTOF OPTIMAL PHFOR LDH ACTIVITY-------------------------------------------------------------------------------------------4
APPROXIMATION OF MICHAELIS CONSTANT,KM,FOR SUBSTRATES ---------------------------------------------------------------------------5
LDH ASSAY OPTIMIZATION AND DETERMINATION OF EXTINCTION COEFFICIENTOF NADH -------------------------------------------------5
PRODUCTION OF A PURIFICATION TABLE---------------------------------------------------------------------------------------------------------6
PURIFICATION OF LDH FROM BOVINE MYOCARDIUM ----------------------------------------------------------------------------------7
AMMONIUM SULFATE FRACTIONATION AND DIALYSIS------------------------------------------------------------------------------------------7
ION EXCHANGE CHROMATOGRAPHY (IEX)-------------------------------------------------------------------------------------------------------7
AFFINITY CHROMATOGRAPHY---------------------------------------------------------------------------------------------------------------------8
CHARACTERIZATION OF PURIFIED LDH ------------------------------------------------------------------------------------------------------8
SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) -------------------------------------------------------8
RESULTS----------------------------------------------------------------------------------------------------------------------------------------------9
ABSORBANCE PROFILES FOR NAD+ AND NADH ------------------------------------------------------------------------------------------------9
FEASIBILITY OF LDH ASSAY------------------------------------------------------------------------------------------------------------------------9
ESTABLISHMENTOF OPTIMAL PHFOR LDH ACTIVITY-----------------------------------------------------------------------------------------10
APPROXIMATION OF MICHAELIS CONSTANT,KM,FOR SUBSTRATES -------------------------------------------------------------------------11
LDH ASSAY OPTIMIZATION AND DETERMINATION OF EXTINCTION COEFFICIENTOF NADH -----------------------------------------------13
PRODUCTION OF A PURIFICATION TABLE-------------------------------------------------------------------------------------------------------16
PURIFICATION OF LDH FROM BOVINE MYOCARDIUM --------------------------------------------------------------------------------17
AMMONIUM SULFATE FRACTIONATION AND DIALYSIS----------------------------------------------------------------------------------------17
ION EXCHANGE CHROMATOGRAPHY (IEX)-----------------------------------------------------------------------------------------------------18
AFFINITY CHROMATOGRAPHY-------------------------------------------------------------------------------------------------------------------19
CHARACTERIZATION OF PURIFIED LDH ----------------------------------------------------------------------------------------------------19
SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) -----------------------------------------------------19
CONCLUSION -------------------------------------------------------------------------------------------------------------------------------------21
APPENDIX -----------------------------------------------------------------------------------------------------------------------------------------23
SAMPLE CALCULATION FOR THE 65%DIALYZED PELLETSHOWN IN TABLES 6 AND 7-------------------------------------------------------23

3. 3
ABSTRACT
Quantitative assays were developed for lactate dehydrogenase (LDH) in order to analyze and purify
the activity of LDH extracted from Bostaurus myocardium.
INTRODUCTION
Lactate dehydrogenase (LDH)plays a criticalrole in the reversible enzymatic conversionof lactate
to pyruvate utilizing an oxidation-reduction reaction between NADH and nicotinamide adenine
dinucleotide (NAD+) as follows: NAD+ + L-Lactate
𝐿𝐷𝐻
⇔ NADH + Pyruvate+ H+. The oxidation of
NAD+ is best accomplished in the presence of L-lactate to yield NADH and pyruvatewith the
addition of bovine myocardial extract. Using an absorbance at 340nm, this reaction can be
monitored because NADH has an absorption peak at 340nm while NAD+ does not.
This enzymatic reaction is important in cellular biochemistry for several reasons. For example, the
reaction is essential to the citric acid cycleto convert pyruvate into ATP for energy in the cells. It is
also important in cellular respiration. LDH converts the body’s sugars into usable energy for bodily
functions. In the medical field, LDH levels in the blood can be measured to determine if there is
tissue damage. If LDH levels are too high in the blood, it suggests that someone is having a heart
attack or has liverdisease. The point of damage can be found by measuring the isoenzymes of LDH
that are found in different concentrations around the body.
By utilizing ammonium sulfate fractionation,ion exchange chromatography and affinity
chromatography, the bovine myocardial extract could be purified. In addition, LDH was purified
from the extract into almost homogeneity and its physical enzymatic characteristics were
determined, such as molecular weight and protein concentrations. An SDS-PAGE analysis was also
done toaid in the analysis of the purified LDH enzyme. This method separated the proteins in
multiple assays, whichhelped to determine how close to pure our sample was. It also gave further
analysis of molecular weight and subunits of the proteins in our enzyme.
MATERIALS AND METHODS
Bostaurus myocardial extract was acquired fromStar Packaging Company in St. Louis, MO.All
assays in the followingexperiments were performed using a Jenway 7315 Spectrophotometer. All
reactants, reagents, and chemicals used in the assays were of reagent grade and acquired from
Sigma Chemical Company in St. Louis, MO.The SilverSNAP Satin Kit II used forsilver staining was
obtained fromThermo Scientific in Rockford,IL.
PreparationofMyocardial Extract
The Bostaurus myocardialextract utilized in these experiments was a gift from Dr. Christopher
Wolin and prepared by him as follows.First, connectiveand epithelial tissue was removed from the
myocardium of a wholebovine heart. The myocardial tissue was then sectioned into 1cm2 pieces
and 300 grams of the tissue was placed into 600ml of icecold 50mM potassium phosphate (KPi),pH
7.4, containing 1mM ethylenediaminotetraacetic acid (EDTA).The mixture was then loaded into a
Model 700 Commercial Blender and homogenized by doing 5 fifteensecond pulses. Next, the
homogenized material was placed in 50ml conical tubes and centrifuged 3 times for10 minutes
using a SorvallGLC-1 desktop centrifuge. Finally, the supernatant was decanted before undergoing
another three rounds of centrifugation using a Sorvall SS-34 rotor in a Sorvall RC2-B floor model

4. 4
centrifuge. This centrifuged homogenate solution is now the myocardial extract to be utilized for
assay development and purification.
Development of an Enzymatic Assay for LDH
Preface
All concentrations given below are the final concentrations found in the reaction (unless otherwise
stated). Furthermore, all reactions measuring the change in absorbance at 340nm took place in a
final reaction volume of 3ml (unless otherwise stated). For NAD+ reduction reactions, reactants
(excluding extract) were loaded into a cuvette and the mixture was then blanked in the
spectrophotometer. The reactions were started with the addition of .1ml of LDH-containing
myocardial extract after blanking. For NADH oxidation reactions, reactants (excluding NADH and
extract) were loaded into a cuvetteand then blanked in the spectrophotometer. The reactions were
started with the addition of NADH and .1ml of LDH-containing myocardial extract. After starting the
reactions, the change in absorbance was measured from 15 to 90 seconds. The rate of absorbance
change per minute, total absorbance change over 90 seconds, Pearson’s coefficient(R2),and shape
of the absorbance profilewere then recorded.
AbsorbanceProfilesforNAD+andNADH
To compare the structure of NAD+ and NADH, absorbance profiles for both were run utilizing the
spectrophotometer. First, the spectrophotometer was blanked withdiH2O in a quartz cuvette.Next,
the reactants (15l of 50M NAD+ and 1785l of diH2O) were loaded into the cuvetteand the
mixture was scanned from 230-400nm. Absorbances, including the maximum absorbances, were
recorded for individual wavelengths in increments of ten. This procedure was repeated, utilizing
50M NADH and 50M ADP (this reaction contained 1770l of diH2O and 30l of ADP).
FeasibilityofLDHAssay
To determine the feasibility and specificity of the assay, the change in absorbance at 340nm was
measured forseveral NADH/NAD+ oxidation-reduction reactions utilizing different substrates. For
NAD+ reduction reactions, 1500l of 100mM Hepes, pH 7.4, 500l of 25mM L-lactate, and 400l of
diH2O were loaded into a plastic cuvetteand blanked in the spectrophotometer. The reaction was
started by adding 500l of 1mM NAD+ after blanking and then assayed from 15-90 seconds before
the desired data was recorded. After the scan, 100µl of diluted bovine myocardial extract was
added to the mixture and the mixture was assayed again. This procedure was repeated two more
times with one starting mixture containing 500µl of 1mM NAD+ (instead of L-lactate) and 900µl of
diH2O, and another mixture containing no NAD+ altogether.
For NADH oxidation, 1500µl 100mM Hepes, 500µl 25mM Pyruvate,and 400µl of diH2O were loaded
into a plastic cuvette.After the cuvettewas blanked, the reaction was started withthe addition of
500µl of .25mM NADH and then assayed. Following the assay, 100µl of extract was added to the
mixture and the mixture was then assayed fora second time. This procedure was repeated again by
adding .25mM NADH and extract simultaneously after blanking withno Pyruvate,and a final time
utilizing 500l of 1mM NADH (instead of .25mM NADH).
EstablishmentofOptimal pH for LDHActivity
The optimal pH for LDH-mediated reactions was determined by measuring the change in
absorbances at 340nm for several NADH/NAD+ oxidation-reduction reactions with pH values
ranging from pH 4 to pH 12 in pH increments of 2. For NAD+ reduction reactions, 500l of 25mM L-

5. 5
lactate, 500µl of .25mM NAD+, 1500µl of varying 100mM buffers (atdiffering pH values), and 400µl
of diH2O were loaded into a plastic cuvette and then blanked. The assay was run from 15-90sec
followingthe addition of 100µl of extract. The buffers used were Acetate, pH 4, Mes, pH 6, Hepes,
pH 7.4, Tris, pH 8, Glycine, pH 10, and KPi, pH 12. For NADH oxidation reactions, 500µl of 25mM
Pyruvate,1500µl of 100mM buffer,and 400µl of diH2O were loaded into a plastic cuvette,blanked,
started with the addition of 500µl of .25mM NADH and 100µl of extract simultaneously, and then
assayed.
Next, pH stability of LDHactivity was determined by preincubating NADH/NAD+ oxidation-
reduction reactions withextract at differing pH values. This procedure was done the same way as
before utilizing the same substrates and buffers as mentioned above except the reactions were
started with the addition of NAD+ or NADH. For example, the NAD+ reduction reactions utilized
500µl of 25mM L-lactate, 1500µl of 100mM buffer, 400µl of diH2O, and 100µl of extract in the
starting mixture. Once it was blanked forone minute, the reaction was started with the addition of
500µl of .25mM NAD+. The NADH oxidation reactions utilized 500µl of 25mM Pyruvate,1500µl of
100mM buffer,400µl of diH2O, and 100µl of extract, and then started with the addition of 500µl
of .25mM NADH.
ApproximationofMichaelisConstant,Km, forSubstrates
The effectof substrate concentration on LDH-mediated NADH/NAD+ oxidation-reduction reactions
was determined by approximating the Michealis constant, Km in the followingways.First, the Km
of L-lactate was determined in the presence of 500µl of 1mM NAD+, 1500µl of 100mM Glycine,pH
10, and 900µl of varying L-lactate samples with concentrations between 1.4 to 45mM. The reactions
were started with the addition of 100µl of 1/20th diluted bovine myocardial extract and assayed
from 15-90seconds at 340nm. Next, the rates of absorbance change/min were recorded and then
graphed on a Lineweaver-Burke plot to determine Km. Based on the Km value for L-lactate(2mM),
the dependence of LDH-mediated NAD+ reduction on NAD+ concentrationcould be determined in
the presence of 200l of 10mM (5 X Km) L-lactate, 1500µl of 100mM Glycine, pH 10, 300l of diH2O,
and 900µl of varying NAD+ samples with concentrations between 56 to 1800M utilizing the same
procedure as before.
The Km of Pyruvatewas also determined using the same procedure from above except NADH was
used to start the reactions instead of extract. To do this, 500l of .25mM NADH, 1500l of 100mM
Glycine,pH 10, and 900l of varyingPyruvatesamples with concentrations between 1.4 to 45mM
were assayed. The data was then graphed to yield a Km value for Pyruvateof 10mM. Next, the
dependence of LDH-mediated NADH oxidation on NADH concentration could be determined in the
presence of 500l of 50mM (5X Km) Pyruvate,1500l of 100mM Glycine,pH 10, and 900l of
varying NADH samples with concentrations between 50 to 500M utilizing the same procedure as
before.
LDHAssayOptimizationand DeterminationofExtinctionCoefficientofNADH
To measure the change in absorbance/minute, different samples of bovine myocardial extract with
varying concentrations from undiluted to 1/160th dilution were used as follows.First, 1500µl of
100mM Glycine,pH 10, 500µl of 1mM NAD+, 700µl of diH2O, and 100l of varyingextract samples
with concentrations fromundiluted to 1/160th dilution were added to a plastic cuvetteand blanked
in the spectrophotometer. The reaction was started with the addition of 200l of 10mM L-lactate
and then scanned from 15-90seconds at 340nm before recording all relevant data. Then, the
changes in absorbance/min vs. the varying concentrations of extract were graphed.

6. 6
The optimized assay used to determine LDHactivity by NAD+ reduction contains: 1500l of
100mM Glycine,pH 10, 500l of 1mM NAD+, 200l of 10mM L-lactate,and 700l of diH2O. The
mixture is blanked in the spectrophotometer and then started with the addition of 100l of extract.
The change in absorbance/min at 340nm is then measured from15-90seconds. This optimized
assay was repeated three times in order to determine its reliability by calculating the percent
deviation.
The specificity of the optimized assay was tested by utilizing 1/80th diluted myocardial extract -
diluted with 50mM K+Phosphate, pH 7.4 as follows.First, 1500l of 100mM Glycine, pH 10 and
1400l of diH2O were loaded into a plastic cuvetteand then blanked in the spectrophotometer. The
reaction was started by adding 100l of the 1/80th diluted extract and then assayed from 15-90
seconds in order to measure the change in absorbance/minute. Next, 1500l of 100mM Glycine,pH
10, 200l of 10mM L-lactate, and 700l of diH2O were loaded into a plastic cuvetteand then
blanked as before. After that, 500µl of 1mM NAD+ was added to the cuvetteto start the reaction and
then the mixture was assayed from 15-90seconds and the proper data was recorded. Then, without
dumping the mixture, 100l of the 1/80th diluted extract was added and the mixture was assayed
an additional time. This procedure was repeated two more times with one starting mixture
containing 500µl of 1mM NAD+ (instead of L-lactate) and 900µl of diH2O, and another mixture
containing no NAD+.
The extinction coefficientand limits of absorbance readings at 340nm for NADH in the optimized
assay forNAD+ reduction was determined by measuring absorbances at 340nm forvarying
concentrations of NADH ranging from 5 to 100M. This was done by loading 1500µl of 100mM
Glycine,pH 10, 500l of 1mM NAD+, 200l of 10mM L-lactate, and 300µl of diH2O into a plastic
cuvetteand then blanking it. After that, 500l of a NADH sample that had been previously diluted
with water to get a concentrationfrom 5 to 100M was added to the cuvetteand scanned using the
single wavelength mode. The concentrations of NADH and the absorbances were then plotted to
yield the extinction coefficient.0039 M-1cm-1 forNADH with absorbance limits between .015–.390.
ProductionofaPurificationTable
To determine the extinction coefficientof bovine serum albumin (BSA),absorbances were
measured at 280nm fordifferent concentrations of BSA ranging from .0781 to 10mg/ml. This was
done by first blanking the spectrophotometer with 50mM K+Phosphate, pH 7.4 in a quartz cuvette.
Then samples containing different concentrations of BSA ranging from .0781 to 10mg/ml that had
previously been diluted with 50mM K+Phosphate, pH 7.4 were loaded into the cuvetteand scanned
using the single wavelength mode. The concentrations of BSA and the absorbances were then
plotted to yield the extinction coefficient.5315 (mg/ml)-1cm-1 forBSA protein withabsorbance
limits between .026–.653.
To determine the concentrationof protein in the undiluted extract as well as the specific activity of
LDH in the myocardial extract, absorbances were measured at 280nm for different concentrations
of bovine myocardial extract ranging from1/160th to 1/5th dilution. This was done by first blanking
the spectrophotometer with 50mM K+Phosphate, pH 7.4 in a quartz cuvette. Next, samples
containing differentconcentrations of extract that had been previously diluted with 50mM
K+Phosphate, pH 7.4 were loaded into the cuvetteand scanned using the single wavelength mode.
The absorbances and previously determined extinction coefficientwere then used to calculate
protein concentrationfor the myocardial extract and specific activity of LDH (See Appendix).

7. 7
Purification of LDH from Bovine Myocardium
AmmoniumSulfateFractionationandDialysis
Purificationof LDH by ammonium sulfate fractionationwas carried out as follows. First, 50ml of
bovine myocardial extract was fractionedto 40% saturation by adding 12.2g of ammonium sulfate.
This was done by pouring the extract into a 100ml beaker along witha stir bar and mixing it on a
stir plate. Next, the ammonium sulfate was added in quarter increments (to avoidclumping) until
all of it was dissolved in the extract. The mixture was then placed on ice for 15 minutes before being
centrifuged in a Sorvell SS-34 centrifuge at 11,000rpm at 4°C for 15 minutes. After 15 minutes, the
supernatant was decanted into a graduated cylinder in order to measure and record the volume of
supernatant (51ml) and pellet (12ml); 1ml of supernatant was saved forfurther analysis and the
pellet was resuspended in 10ml of 50mM K+Phosphate, pH 7.4 and saved in a 50ml conical tube for
further analysis.
A 65% saturation was then carried out by adding 8.4g of ammonium sulfate to the 50ml mixture of
40% supernatant just as before. Once the ammonium sulfate was dissolved, the mixture was placed
on ice for 15 minutes and centrifuged for another 15 minutes. After centrifugation, the 65%
supernatant was decanted into a graduated cylinder to measure the volume of supernatant (51ml)
and pellet (6ml); 1ml of supernatant was saved for further analysis and the pellet was resuspended
in 10ml of ice cold30mM Bicine, pH 8.5 and saved for further analysis.
All of the supernatants and pellets were then assayed for LDH activity (using optimized assay for
LDH activity at 340nm) and protein concentration at 280nm.
Finally, the 65% resuspended pellet containing LDH activity was pipetted into a dialysis tube and
the ends of the tube were clipped shut. The dialysis tube was then placed in a beaker containing
30mM Bicine, pH 8.5 to dialyze overnight at 4°C.
Ion ExchangeChromatography(IEX)
To perform ion exchange chromatography (IEX),the65% dialyzed pellet solution (12ml) was
pipetted into an ion exchange column pre-equilibrated in 30mM Tris-Cl, pH 8.5. The column valve
was then opened and the breakthrough (11.5ml) was collectedin a 50ml beaker until the sample
was just abovethe top of the column matrix; 1ml of breakthrough was saved forfurther analysis.
Next, the column was washed with 30mM Tris-Cl, pH 8.5 and the wash (29.8ml) was also collected
in a clean beaker and then put in a 50ml tube on ice forfurther analysis.
In order to elute LDH from the column, 100ml of 30mM Tris-Cl, pH 8.5 + 0.2M NaCl was added to
the column (to raise salt concentration) and 3ml fractions were collectedinto test tubes as the
sample was eluted from the column. These fractions were then assayed for LDHactivity.All LDH
containing samples were pooled (76ml worth) and stored on ice; 1ml of pooled sample was saved
for further analysis. After that, 50ml of 30mM Tris-Cl, pH 8.5 + 1M NaCl was added to the column
and the eluant (51ml worth) was collected in a clean beaker and stored on ice; 1ml was saved for
further analysis. The column was then washed with50ml of 30mM Tris-Cl, pH 8.5 to re-equilibrate
the column.
After the IEX was completed, 100μl of breakthrough, wash, 0.2M NaCl pooled sample, 1M NaCl
sample, 65% dialyzed pellet, 65% pellet, and extract were all assayed forLDH activity.The total
units of activity were then calculated using the measured volumes in order to determine the
percent recovery of activity.

8. 8
AffinityChromatography
To perform affinity chromatography, the LDH-containing solution that had been previously purified
by IEX was pipetted into a column containing Cibacron Blue matrix pre-equilibrated in 30mM Tris-
Cl, pH 8.5 + 0.2M NaCl. The column valve was then opened and the breakthrough (74ml) was
collectedin a beaker until the sample was just abovethe top of the gel bed. Next, in order to elute
LDH from the column, the columnwas washed with 50ml of 30mM Tris-Cl, pH 8.5 + 0.2M NaCl and
the 0.2M NaCl wash (49.5ml) was also collected in a clean beaker. Finally, the column was washed
with 40ml of 30mM Tris-Cl, pH 8.5 + 1M NaCl (to raise salt concentration) and 5ml fractions were
collectedinto test tubes as the sample was eluted from the column. These fractions were then
assayed forLDH activity and the three fractions containing the highest LDH activity werepooled
(12ml worth)and stored at 4°C for further analysis. The column was then re-equilibrated with
50ml of 30mM Tris-Cl, pH 8.5.
After the affinity chromatography was completed, the IEX sample, Cibacron Blue breakthrough, IEX
pooled 0.2M NaCl sample, and Cibacron Blue pooled 1M NaCl sample were all assayed forLDH
activity and protein concentration.
Characterization of Purified LDH
SodiumDodecyl Sulfate-PolyacrylamideGel Electrophoresis(SDS-PAGE)
To determine how many proteins (and their molecular weights) were in the purified LDH sample,
an SDS-PAGE was run on pure heart-derived LDH, myocardial extract, 65% dialyzed pellet, .2M
NaCl eluted IEX pooled sample, and 1M NaCl eluted Cibacron Blue pooled sample as follows.First,
20l samples were made containing 38μg of protein fromeach solution (all samples were diluted to
20l with diH2O). Next, 80μl of acetone and 4μl of 6X SDS sample buffer [125mM Tris-Cl, pH 6.8,
4%(wt/vol)SDS 20%(vol/vol)Glycerol,.02%(wt/vol)bromophenolblue, and 5% b-
mercaptoethanol] were added to each tube. The tubes were then spun in a microfuge at maximum
speed for a minute to make a protein pellet at the bottom of the tube. After that, the tubes were
heated for5 minutes at 90°C to evaporate any liquid that remained. The tubes were then cooledon
ice fora few minutes before being spun again for a minute. The samples were then put on ice until
they were ready to be utilized in the SDS-PAGE.
The SDS-PAGE was prepared in 1X running buffer. The wells were loaded with 15μl aliquots of
each sample as follows:
1: Molecular Weight Marker
2: Pure Heart-derived LDH
3: 38μg MyocardialExtract solution
4: 38μg 65% DialyzedPellet solution
5: 38μg .2M NaCl eluted IEX pooled solution
6: 38μg 1M NaCl eluted Cibacron Blue pooled solution
The gel was then leftto run fora little over an hour until the dye reached the bottom of the gel.
Once the gel was finished running, it was developed using the SilverSNAP Satin Kit II. After the gel
was stained, band distances were measured and a standard curve was derived from the molecular
weight markers. Using the standard curve,the molecular weight of the most purified band was
determined and compared to the theoretical weight of LDH to ensure that the correctprotein had
been purified.

9. 9
RESULTS
AbsorbanceProfilesforNAD+andNADH
This laboratory experiment revealed that NAD+, NADH, and ADP all have peaks at an absorbance
maximum of 260nm. This is indicative of the adenine functional group present in all of the three
structures. In Figure 1, it is shown that NADH has an additional peak at an absorbance maximum of
340nm, whichmeans that it may be possible to develop an assay at this absorbance. The change in
absorbance at 340nm would indicate the oxidation-reduction reaction of NADH/NAD+. This peak is
also due to the presence of Nicotinamide found exclusively in NADH.
Figure1. NADH and NAD+ share a maximum absorbance at 260nm due to the common adenine
moiety found in each molecule. NADH has an additional maximum absorbance at 340nm due to the
Nicotinamide moiety found exclusively in NADH.
FeasibilityofLDHAssay
This experiment demonstrated that the bovine myocardial extract has some reducing power, but
not a substantial amount to solely complete the reaction. It must be in the presence of L-lactate or
pyruvate to complete the oxidation-reduction reaction of NADH/NAD+ as shown in Figure 2 and
Figure 3. In addition, in the presence and absence of the extract, when NADH is in high
concentrations, it can be an inhibitor forlactate dehydrogenase activity.
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
240 260 280 300 320 340 360 380 400
Absorbance
Wavelength (nm)
Spectra Profile for NADH and NAD+
NADH
NAD+

10. 10
Figure2. The rate of NAD+ reduction depends on various conditions. The complete reaction
mixture contained 1.5ml of 100mM Hepes buffer, pH 7.4, .5ml of 25mM L-lactate, .4ml of diH2O,
.5ml of 1mM NAD+, and .1ml of diluted extract.
Figure3. The rate of NADH oxidation depends on various conditions. The complete reaction
mixture contained 1.5ml of 100mM Hepes buffer, pH 7.4, .5ml of 25mM pyruvate, .4ml of diH2O,
.1ml of diluted extract, and either.5ml of .25mM NADH or 1mM NADH.
EstablishmentofOptimal pH forLDHActivity
This experiment showed that pH 10 was the optimal pH forLDH-mediated activity in the oxidation-
reduction of NADH/NAD+. As shown in Figure 4 and Figure 5, pH 10 had the highest total
absorbance change over 90 seconds forboth NAD+ reduction and NADH oxidation.
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
Complete Rx w/ 1 mM
NAD+
Rx w/o Extract Rx w/o L-lactate Rx w/o 1mM NAD+
A340nm/min
Condition
Rate of NAD+ Reduction under Various
Conditions
0.000
0.020
0.040
0.060
0.080
0.100
0.120
Complete Rx w/ .25
mM NADH
Rx w/o Extract Rx w/o Pyruvate Complete Rx w/ 1 mM
NADH
A340nm/min
Condition
Rate of NADH Oxidation under Various
Conditions

11. 11
Figure4. NAD+ reduction is most optimal at pH 10.
Figure5. NADH oxidation is most optimal at pH 10.
ApproximationofMichaelisConstant,Km, forSubstrates
In order to optimize the assay, a pseudo-first order reaction was created. To do this, Km for the
reduction of NAD+ was determined to be 2mM with a saturating substrate concentration of 10mM.
Km was also determined forthe oxidation of NADH, whichyielded a Km of 6mM. However,the
results did not turn out as expected when graphing them, so this part of the procedure was
repeated because the Km should have been closer to 10mM.
0.000
0.050
0.100
0.150
0.200
0.250
4 6 7.4 8 10 12
TotalAbs340nm/90Sec
pH Value
Rate of NAD+ Reduction under Various
pH Values
w/o Preincubation
w/ Preincubation
0.000
0.100
0.200
0.300
0.400
0.500
4 6 7.4 8 10 12
TotalAbs340nm/90Sec
pH Value
Rate of NADH Oxidation under Various
pH Values
w/o Preincubation
w/ Preincubation

12. 12
Figure6.
Figure7.
y = 18.881x + 10.232
R² = 0.9871
0.0
5.0
10.0
15.0
20.0
25.0
0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800
1/v,(Abs340nm/min)-1
1/[L-lactate] (mM-1)
Lineweaver-Burke Plot for 1mM NAD+-
Dependence on 2mM L-lactate
y = 2,355.3x + 10.8
R² = 1.0
0.0
10.0
20.0
30.0
40.0
50.0
60.0
0.0000 0.0020 0.0040 0.0060 0.0080 0.0100 0.0120 0.0140 0.0160 0.0180 0.0200
1/v,(Abs340nm/min)-1
1/[NAD+] (M-1)
Lineweaver-Burke Plot for 1mM NAD+-
Dependence on 10mM L-lactate

13. 13
Figure8.
Figure9.
LDHAssayOptimizationand Determination ofExtinctionCoefficientofNADH
First, differentconcentrations (from undiluted to 1/160 diluted) of bovinemyocardial extract were
used to find the absorbance change/minute. The changes in absorbance/min vs. the concentration
of extract are graphed in Figure 10. The hyperbolic curveindicates the non-linear nature of the
assay, whichmeans it does not obey the Beer-Lambert law. However,the inner graph is linear and
tells us that the limits of the absorbance values/minute of all future assays must be between 0.015-
0.390.
In testing the feasibility of the LDHassay, it was shown in Figure 2 and Figure 3 that there was an
increase in absorbance when extract and either L-lactateor pyruvate was present at 340nm. This
was retested at the optimal conditions to see whether LDH-dependent absorbances should be
considered when determining enzymatic activity during the purification process. The specificity of
the optimized assay was retested using 1/80th diluted extract under optimal conditions to see
y = 51.8x + 7.8
R² = 1.0
0.0
10.0
20.0
30.0
40.0
50.0
0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800
1/v,(Abs340nm/min)-1
1/[Pyruvate] (mM-1)
Lineweaver-Burke Plot for .25mM NADH-
Dependence on 6mM Pyruvate
y = 9,334.5x - 11.9
R² = 1.0
0.0
20.0
40.0
60.0
80.0
100.0
0.000 0.002 0.004 0.006 0.008 0.010 0.012
1/v,(Abs340nm/min)-1
1/[NADH] (M-1)
Lineweaver-Burke Plot for .25mM NADH-
Dependence on 50 mM Pyruvate

14. 14
whether anything is limited by complete optimization. As shown in Figure 11, the complete reaction
had the highest absorbance, whichindicates that reactions withoutextract wouldresult in very low
or non-present absorbances. Optimization is a limiting factorwhen it comes to non-specificity.
The reliability of the optimized assay was also tested by performing triplicates of the assay
containing 1/80th diluted extract, as shown in Table 1, in order to determine a percent deviation.
The overall percent deviation was 2.23%, with an average value of 0.0403 +/- 0.00087/min. The
percent deviation is acceptable because it is within the 5% error acceptability for reliability.
The relationship between the concentration of NADH and the absorbance at 340nm is shown in
Figure 12. Utilizing the slope of this graph, the extinction coefficient() .0039M-1cm-1, forNADH
was determined.
Figure10.
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.2 0.4 0.6 0.8 1 1.2
Abs340nm/min
[Extract], Fraction of Original
Concentration of Extract vs. The Rate of
Reaction
y = 2.9854x + 0.0076
R² = 0.9996
0
0.02
0.04
0.06
0.08
0.1
0 0.01 0.02 0.03

15. 15
Figure11. The optimized assay was tested for specificity under varying conditions. The complete
optimized assay reaction mixture contained 1.5ml of 100mM Glycine, pH 10, .2ml of 10mM L-
lactate, .7ml of diH2O, .5ml of 1mM NAD+, and .1ml of 1/80th diluted extract.
Table1. This data represents the reproducibility and error tested using 1/80th diluted extract.
The assay was repeated three times in order to make sure the percent and standard deviations
were reasonable.
Trial
Number
Rate of Absorbance
Change A340/min
Total Absorbance
Change over 90 secs.
Pearson's
Coefficient
(R2)
Curve Shape
1 0.041 0.061 0.99 Linear, slight noise
2 0.039 0.059 0.995 Linear, slight noise
3 0.041 0.061 0.99 Linear, slight noise
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
A340nm/min
Testing Optimized Assay for Specificity
y = 0.0039x - 0.0028
R² = 0.9994
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
0.450
0 20 40 60 80 100 120
TotalAbsorbanceat340nm
[NADH], M
Determination of Extinction Coefficient of
NADH in Optimized Assay

16. 16
Figure12. The extinction coefficientforNADH is .0039 M-1cm-1 withabsorbance limits
between .015–.390.
ProductionofaPurificationTable
Determination of protein concentration was done multiple ways. First, absorbances were measured
at 280nm for varying concentrations of Bovine Serum Albumin (BSA) protein. The concentrations
and absorbance readings that did not obey the Beer-Lambert law were excluded from the data. The
relationship between the final concentration of BSA and absorbance at 280nm is shown in Figure
13. As the final concentrationof BSA (mg/ml) increases, so does the absorbance. Utilizing the slope
of this graph, the extinction coefficient(),0.5315(mg/ml)-1cm-1 forBSA was determined.
Next, the concentration of protein in the undiluted extract was determined along with the specific
activity of LDH by measuring absorbances at 280nm fordifferent dilutions of extract, whichis
shown in Figure 14.
Finally, all of the values obtained during the course of the experiment were utilized to complete a
purification table, Table 2.
Figure13. The extinction coefficientforBSA protein is .5315 (mg/ml)-1cm-1 with absorbance limits
between .026–.653.
y = 0.5315x - 0.0081
R² = 0.9996
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Absorbanceat280nm
Final [BSA], (mg/mL)
Determination of Extinction Coefficient of
BSA Protein in 50mM K+Phosphate, pH 7.4

17. 17
Figure14.
Table2. PurificationTable
Sample
Volume
(ml)
U/ml Total U
Protein
concentration
(mg/ml)
Total
Protein
Specific
Activity
(U/mg)
Extract 30 ml 24 720 1.1 33 22
Purification of LDH from Bovine Myocardium
In order to purify the protein, we performed a series of purificationtechniques to obtain the purest
protein possible, and finally compare the experimental sample’s molecular weight and subunits to
the known molecular weight and number of subunits of Lactate Dehydrogenase.
AmmoniumSulfateFractionationandDialysis
The first purifying technique employed was Ammonium Sulfate Fractionation, also knownas
“salting out”.Table 3 shows the Spectrophotometric results for the five samples that were assayed.
Table 3 indicates that the 65% pellet has the highest absorbance and thus, highest LDH activity.
Using the measured absorbances from Table 3 and the extinction coefficientof NADH, the Percent
Recovery of LDH activity was calculated as shown in Table 4.
Dialysis was also performed utilizing the 65% pellet sample because it had a high level of LDH
activity and the highest absorbance. The only reason the total units were not the highest, as shown
in Table 4, was due to the low volumeof sample.
y = 0.5679ln(x) +2.8395
R² = 0.9725
-0.500
0.000
0.500
1.000
1.500
2.000
2.500
0.000 0.050 0.100 0.150 0.200 0.250
Absorbanceat280nm
Fold Dilution of Original Extract
Absorbanceat 280nm for Dilutions of
Extract Ranging from 1/160th to 1/5th
Dilution

18. 18
Table3. Samples collectedby Ammonium Sulfate Fractionation and Spectrophotometric results.
Sample Dilution A340nm/min Total ΔAbs/90sec R2 Curve Shape
Pure Extract 1/20 0.108 0.161 0.997 Linear
40% Supernatant 1/20 0.116 0.174 0.997 Linear
40% Pellet 1/20 0.042 0.062 0.999 Linear
65% Supernatant 1 0.024 0.035 0.989 Linear
65% Pellet 1/80 0.198 0.298 0.994 Linear
Table4.Purificationtable generated by a series of calculations using absorbances from Table 3,
sample volumes, and extinction coefficientof NADH.
Sample U/ml in
Undiluted Sample
Volume
(ml)
Total Units (U) % Recovery
Pure Extract 22 50 1100 100
40% Supernatant 18 51 918 83
40% Pellet 6.6 12 79 7.2
65%Supernatant 0.19 51 9.7 0.88
65% Pellet 120 6 720 65
Ion ExchangeChromatography(IEX)
The second purifying technique employed was Ion Exchange Chromatography (IEX) starting with
the 65% dialyzed pellet and continuing with other samples collected during the course of the
experiment. Table 5 shows the results of the Spectrophotometric analysis and Table 6 is a
purification table utilizing the data from Table 5 and the extinction coefficientof NADH. The results
shown in Table 6 indicate that the 65% pellet has plenty of activity as well as the pure myocardial
extract.
Table5.Spectrophotometric results for samples collected through Ion Exchange Chromatography.
Below the table, are the extinction coefficient,and the limits for the derived coefficient.
Sample Dilution A340nm/min Total ΔAbs/90sec R2
Curve
Shape
Myocardial Extract 1/20 0.108 0.161 0.997 Linear
65% Pellet 1/80 0.198 0.298 0.994 Linear
65% Dialyzed Pellet 1/40 0.136 0.203 0.997 Linear
Breakthrough 1 0.022 0.033 0.963 Log
Column Wash 1 0.016 0.024 0.954 Log (I)
0.2M NaCl Pooled 1/5 0.128 0.191 0.996 Linear
1M NaCl 1 0.034 0.051 0.960 Log (I)
I = Irregular

19. 19
Table6.Purificationtable generated by a series of calculations using absorbance values from Table
5, sample volumes, and extinction coefficientof NADH.
Sample
U/ml in
Undiluted Sample
Volume
(mL)
Total Units
(U)
% Recovery
Myocardial Extract 22 50 1100 100
65% Pellet 120 6 732 67
65% Dialyzed Pellet 42 12 500 45
Column Breakthrough 0.17 11.5 2 0.18
Column Wash 0.12 29.8 4 0.36
0.2M NaCl Pooled 4.9 76 370 34
1M NaCl 0.26 51 13 1.2
AffinityChromatography
The final purifying technique employed was Affinity Chromatography. This form of
chromatography uses a substrate analog, in this case, a dye called Cibacron Blue, to bind to the
enzyme. The Cibacron Blue binds to NAD+, and then NADH is removed before LDH activity is
determined for the samples. After running LDH and protein assays on each sample, the values in
Table 7 couldbe calculated. The results from Table 7 indicate that the 65% Pellet, the Cibacron Blue
1M NaCl Pooledand 0.2M Pooledsamples have the highest fold purifications of Lactate
Dehydrogenase activity.
Table7.Purificationtable forsamples generated from Affinity Chromatography.
Sample
U/ml in
Undiluted
Sample
[Protein]
(mg/ml)
Specific
Activity
(U/mg)
Fold
Purification
% Recovery
Myocardial
Extract
22 72 0.31 1 100
65% Pellet 120 44 2.9 9.4 67
65% Dialyzed
Pellet
42 31 1.4 4.5 45
IEX Pooled 3.1 2.3 1.3 4.2 21
Cibacron
Breakthrough
0.2 0.85 0.24 0.77 1.4
Cibacron
Wash/0.2M NaCl
1.1 0.25 4.4 14 5
Cibacron 1M
NaCl Pooled
4.5 2 2.3 7.4 5
CharacterizationofPurifiedLDH
SodiumDodecyl Sulfate-PolyacrylamideGel Electrophoresis(SDS-PAGE)
In order to characterizethe purified LDH, a Sodium DodecylSulfate PolyacrylamideGel
Electrophoresis (SDS-PAGE)was generated as shown in Figure 15. By utilizing the SDS-PAGE,the
molecular weight of the polypeptide present in the enzymatic activity could be determined.

20. 20
As it turned out, the Cibacron Blue 1M NaCl sample had the most activity and was compared to the
pure heart-derived LDH in order to determine the molecular weight of the purified LDH. This was
done by first measuring the Rf value (distance the sample migrated divided by the length of the gel)
for the Cibacron Blue sample. Next, a standard curve forthe molecular weight marker was graphed
and shown in Figure 16. Then the Rf value for the Cibacron Blue sample was measured and
substituted for x into the linear equation of the line in Figure 16. By taking the antilog of the y-value
that was calculated from the line equation, the molecular weight of the purified LDH could be
calculated as 35kD.
The theoretical molecular weight of pure heart-derived LDH is 36kD, or 36,000 Daltons. The
Cibacron Blue sample was measured to be 35kD or 35,000D, whichgave a percent differenceof 2%.
This shows that the experimental molecular weight of purified LDH is very close to the theoretical
molecular weight of pure LDH according to SDS-PAGE.
Figure15. SDS-PAGE gel after being pulled from the electrophoresis chamber and stained. Reading
right to left(picture was taken from the bottom of the dish), lane 1 contains the Molecular Weight
Marker, lane 2 contains Pure Heart-derived LDH, lane 3 contains, MyocardialExtract, lane 4
contains 65% DialyzedPellet solution, lane 5 contains .2M NaCl eluted IEX pooled solution, and lane
6 contains 1M NaCl eluted Cibacron Blue pooled solution.

21. 21
Figure16. After measuring the distances the polypeptide bands of the molecular weight marker
migrated in the gel shown in Figure 15, a standard curve was graphed. This graph shows the log
values of the molecular weights of the polypeptide bands versus how far the polypeptide bands
migrated from the top of the gel divided by the total buffermovement.
CONCLUSION
The enzyme LDH through rigorous experiments was analyzed and purified as shown in the
protocols. Through analysis of the nicotinamide adenine dinucleotide (NAD+) and L-lactate
reduction, it was noticed that the reaction can be easily monitored using a spectrophotometer
reading at 340 nm over 90 seconds. The change in absorbance shows that the reaction is changing
from NAD+ to NADH and L-lactate to pyruvatedue to the fact that NADH is the only substrate that
absorbs at 340 nm. Itwas foundthat the optimum pH range forLDH activity was from pH 7.4-10,
with the best results seen at an optimal pH of 10. For all further experiments, Glycine, pH 10 was
used as a buffer in the optimized reaction.
An assay was performed forthe Michaelis Constant, Km, and values of 2mM for NAD+ reduction
were determined with a saturating substrate concentration of 10mM. The optimized assay yielding
optimal LDHactivity was determined to be: 1.5ml of 200mM Glycinebuffer, pH10, 0.7ml of dH2O,
0.2 ml of 150mM L-lactate, and 0.5 ml of 6mM NAD+ added to the cuvette and blanked in the
spectrophotometer. The reaction is then started by adding 0.1ml of LDH extract, and the change in
absorbance was measured at 340nm for90 seconds. Under optimal conditions the extinction
coefficientforNADH was determined to be 0.0039M-1cm-1.
Using purificationtechniques previously stated forammonium sulfate fractionation,ion exchange
chromatography, and Cibacron Blue affinity chromatography, LDH from the Bos taurus myocardial
extract was able to be purified. SDS-PAGE analysis showed that the final purification of extract was
the enzyme LDH. The Cibacron Blue band from the polyacrylamide gel was measured at 35kD. The
theoretical molecular weight of pure heart-derived LDH is 36 kD. This confirmed that the purified
y = -1.6407x + 2.5215
R² = 0.9888
0.00
0.50
1.00
1.50
2.00
2.50
0.00 0.20 0.40 0.60 0.80 1.00
Log(Mr)(kD)
Rf = (Distance Moved from Start/ Buffer Movement)
Standard Curve for Log (Mr) vs. Rf

22. 22
Lactate Dehydrogenase is extremely close in molecular weight to the theoretical molecular weight
of pure Lactate Dehydrogenase. The experiment to assay for and purify LDH was a success.

23. 23
APPENDIX
SampleCalculationforthe65% DialyzedPelletshowninTables6and7
(Abs/min)/ (M-1cm-1)(Lcm)=(.136/min)/(.0039M-1cm-1)(1cm) = 35M/min
(M/min)(Rx volume in L)= (35M/min)(.003L) = .11mol/min=.11 U
U/(volumeof extract in ml) = .11 U/.1ml = 1.05U/mL
(U/ml)(DilutionFactor)= (1.05U/ml)(40) = 42U/mL
(U/ml in undiluted extract)(Volume of extract in ml) = (42 U/ml)(12 ml) = 500 U
% Recovery of LDHActivity = (Total Units in Sample/Total Units of Extract) x 100 = 500/1100 =
45%
[Protein] = Abs/((mg/ml)-1cm-1)(Lcm) =.208/(.5315mg/ml-1cm-1)(1cm)(80) = 31mg/ml
Specific Activity = (U/ml in Undiluted Sample)/[Protein] = (42 U/ml)/(31 mg/ml) = 1.4 U/mg
Fold Purification= (Specific Activity of Sample)/(Specific Activity of Extract) = (1.4 U/mg)/(.31
U/mg) = 4.5