Michelle Grau

Lactate Dehydrogenase Protein Purification and Analysis Laboratory

September 17 2009-October 16 2009- Notebook Pages: 58-96
Introduction:
In this lab investigation, the aim was to purify and analyze Lactate Dehydrogenase (LDH)
enzyme from chicken breast. Protein purification is essential when studying the function, structure,
and interactions of a particular protein. It’s important to determine the concentration of protein if
using it a variety of physical analytic methods such as steady-state kinetics or ligand binding that
rely on molar measurements.
LDH is an enzyme found in most plants and animals and plays a role in glycolysis for the
formation of ATP.1 This enzyme catalyzes the reaction of pyruvate and NADH to form lactate and
NAD+, while at the same time it can catalyze the reverse reaction if there’s a large concentration of
lactate.
If you wanted to purify LDH, or any protein, you must start with a tissue sample containing
the protein. Most proteins are typically found within a cell, so the tissue must be subjected to a
homogenizing process in order to break cell walls and release protein. If the protein is in solution,
you expose it to a selective precipitation. Selective precipitation of proteins can be used as a rough
method to recover a desired protein in a purification. This process depends on the physical or
chemical interaction between the protein and the precipitating agent. In this lab, (NH4)2SO4 is used
to precipitate out LDH.
Dialysis is a process of separating molecules in solution by differences in rates of diffusion
across a semi-permeable membrane. Dialysis can most often remove a large amount of small
impurities in a heterogeneous solution containing your protein. We used dialysis in this lab to
remove the excess, unwanted (NH4)2SO4 and other small impurities while simultaneously
exchanging the extraction buffer with dialysis buffer.
Affinity chromatography is used to obtain a specific substance if it’s mixed in a
heterogeneous solution. Columns used for affinity chromatography are typically composed if inert,
chemically stable polymers, that have specific binding proteins or molecules. You would use a
column that was composed of a specific molecule to which the protein of interest would bind to
with a high affinity. Once the protein solution is applied to the column, all substances and proteins
that do not bind or that bind loosely to the column are removed with buffer washes. Then the
column is washed with a solution to which the desired protein binds strongly to and is isolated. In
this lab we used a Cibacron blue affinity column to purify LDH. This molecule mimics the shape
and charge characteristics of pyridine nucleotides to which dehydrogenase proteins frequently bind
to. To obtain pure LDH from the column, it was washed with an NADH solution because of the
high affinity LDH has for NADH.
Once your protein is purified, there are many techniques to determine the purity and
concentration of your protein. First you typically run an activity assay if the protein has enzymatic
properties, to determine which fractions from the chromatography contain the protein. SDS-PAGE
gel electrophoresis is a good method to use for determining the purity of the protein. This method
separates proteins according their molecular weight and length of polypeptide chain. Proteins all
exhibit the same charge per unit mass due to the binding of SDS resulting in fractionation by size
and mass. Using the gel from SDS-PAGE, you can detect the protein using a specific and visible
antibody against the protein.
Bradford Assays are a routine method for determining protein concentration. A standard
curve made with known protein concentrations is constructed using Coomassie blue dye which
binds to all proteins and absorbs light at 595nm. Based on the absorbance of the solution containing
the protein of interest, using the standard curve you can determine a relatively accurate protein
concentration. The Edelhoch method measures denatured protein concentration, based on the
absorbance at 280nm and the extinction coefficient that is determined by the number of tyrosines,
tryptophans, and cysteins in the polypeptide chain.2 All these methods described were used in the
lab to determine the purity and concentration of LDH protein.
Aims of Experiment:
The purpose of this experiment was to extract and purify LDH enzyme from chicken breast muscle
using a variety of techniques including centrifugation, selective protein precipitation, dialysis and affinity
chromatography. Many different analytical methods were employed to determine the presence, purity and
concentration of LDH such as activity assays, SDS-PAGE, Western Blot, Bradford Assay, Edelhoch, and
QAAA.

Results:

Figure 1. Flowchart Depiction of LDH Protein Purification Procedure and Analysis

Step 1: 50g of Chicken Breast in 75mL

of Extraction Buffer

Extraction Buffer: 10mM TrisHCl pH 8.6, 1mL 2-

mercaptoethanol, 100mM PMSF, 1mM
ethylene diamine

Chicken tissue and extraction buffer homogenized using 4x 30sec

bursts allowing 10 seconds between bursts

Step 2: Centrifugation

27,000 x g, 4°C for 20 min in 250mL conical vials

Supernatant Collected – 52mL (Crude Homogenate)

Step 3: Ammonium Sulfate

Used 20.28g (NH4)2SO4 (0.39g of (NH4)2SO4 per mL of

supernatant)

Added in cold room slowly over 15 minutes and then stirred for an
additional 15 minutes.

Step 4: Centrifugation

Same conditions as above.

Supernatant Collected- 52mL (Ammonium Sulfate Supernatant)

Step 5: Resuspended Pellet in 5mL

of Extraction Buffer

Same buffer as described above.

Suspension contained protein and (NH4)2SO4

Volume of pellet in extraction buffer- 7.2mL

Step 6: Dialysis

Dialyzed suspension two times in 1L of dialysis buffer

Dialysis Buffer: 10mM TrisHCl pH 8.6, 5mM 2-mercaptoethanol

Saved 3 aliquots of dialyzed sample

Step 7: Affinity Chromatography

 Used a Cibacron Blue Affinity Column

Absorbance of all fractions was measured with a UV-Vis

Spectrophotometer at 280nm. Blank was measured with milli-Q
water. Absorbance of PMSF buffer was about 0.004. Absorbance
of each fraction was below 0.1 Abs before moving on to each
consecutive wash.
Flow Through Tris PMSF Wash 10mL of NAD+ wash: Tris PMSF 10mL of NADH wash: Tris PMSF
#1: 10mM TrisHCl pH 8.6, Wash #2: 10mM TrisHCl pH 8.6, Wash #3:
10mM TrisHCl pH 0.5mM 2-mercapto ethanol, 0.5mM 2-mercapto ethanol,
8.6, 0.5mM 2- 1mM Lithium Lactate, 1mM NADH
+ Same solution as Same solution as
mercaptoethanol, 1mM 1mM NAD wash #1 wash #1
PMSF

Fractions: 1-3 Fractions: 4-9 Fractions: 11,12 Fractions: 13-16 Fractions: 17,18 Fractions 19,20

Step 8: Analysis of LDH Purification

Activity Assays of SDS-PAGE Gel Bradford Assay for Edelhoch Analysis

LDH Samples Electrophoresis determination of
LDH concentration Analysis parameters
Analysis parameters located Analysis parameters located in Table 2 and
in Table 1 located in Figure 2 Figure 5

Analysis parameters
located in Table 1

Western Blot LDH mass Bradford Assay for

analysis determination via
BenchMarkTM Protein
Ladder Standard
Analysis parameters
located in Figure 4 Curve
Analysis parameters
located in Figure 3
determination of
LDH concentration
in pooled fractions
Analysis parameters
located in Table 2
 A crude homogenate of chicken breast was obtained and to this, ammonium sulfate was
added to precipitate LDH. This precipitate was treated as described in the flowchart in Figure 1.
Following dialysis, the LDH sample was subjected to a Cibacron blue affinity column. LDH
activity assays were performed to determine which samples and fractions contained a significant
amount of LDH. The results of the LDH activity assays are located in Table 1. The highest
enzyme activity was observed in the crude homogenate. Fraction 17 produced the highest
observed activity from the chromatography fractions. A higher enzyme activity was observed in
the (NH4)2SO4 supernatant than the (NH4)2SO4 dialyzed protein solution.

Bradford Assay Analysis:

Table 1: LDH Concentration, Purity, and Yield Determination via Bradford Assay
Crude (NH4)2SO4 (NH4)2SO4 Fraction Fraction Fraction Fraction Fraction
Sample
Homogenate Supernatant Dialyzed #11 #12 #17 #18 #17 + #186
Dilution 12,500 12,500 125 125 125 125 125 200
Abs 0.141 0.065 0.45 0.347 0.359 0.613 0.396 0.221
LDH Activity
(μmol NADH min- 75.19 13.11 18.95 33.99 38.57 45.86 8.51 27.19
1
mL-1)1
Diluted Protein
Concentration 0.0028 0.0003 0.0130 0.0096 0.0100 0.0184 0.0112 0.0066
(mg/mL)
Undiluted
Protein
34.810 3.353 1.627 1.201 1.250 2.302 1.404 1.362
Concentration
(mg/mL)
Volume (mL)2 52.0 52.0 7.2 5.0 5.5 6.0 6.0 12.5
Total Protein
1810.099 174.338 11.715 6.004 6.877 13.810 8.421 17.025
(mg)
Total LDH
Activity (μmol 3909.88 681.72 136.44 169.95 212.135 275.16 51.06 339.875
NADH min -1)
Specific LDH
Activity (μmol 2.160 3.910 11.647 28.307 30.846 19.924 6.063 19.963
NADH min -1 mg-1)3
Fold Purification4 1.00 1.81 5.39 13.11 14.28 9.22 2.81 9.24
Yield (%)5 100.00 17.44 3.49 4.35 5.43 7.04 1.31 8.69
Table 1: Bradford Assay standard curve was constructed using IgG protein concentrations between 1.25 μg/mL-25.0μg/mL. BioRad Assay Reagent was added
to standards, samples, and fractions that were diluted in 50mM KH2PO4 buffer at pH 8.0. The final volume was 1mL. The blank was measured with 50mM
KH2PO4 buffer at pH 8.0. Absorbance was measured at 595nm using a UV-Vis Spectrophotometer. Path length was 1cm. The equation of the standard curve
determined through the Bradford Assay was: y = 0.0302x + 0.056. Dilutions were made for samples to obtain an absorbance between 0-1. 1LDH Activities-
Activity of 10μL LDH was determined via time course measurements using the UV-Vis Spectrophotometer, monitoring the NADH production by following the
absorbance at 340nm. Duplicates were measured for samples with significant enzyme activity. 2 Volumes- See flowchart. 3 Specific LDH Activity = LDH
Activity/ Undiluted LDH Concentration. 4 Fold Purification = Specific LDH Activity/ Crude Homogenate Specific LDH Activity. 5 % Yield = (Total LDH
Activity/ Crude Homogenate LDH Activity) *100. 6 The absorbance and both diluted and undiluted concentrations for this fraction was determined by the
Bradford Assay as described in Table 2. Absorbance value is an average of two measurements. The enzyme activity was estimated based on the average of the
two fractions. All the subsequent calculations are therefore also estimations.

A Bradford analysis was used to determine the concentration of LDH in a selection of

samples and fractions that had a significant observed enzyme activity. Specific parameters for
the construction of the Bradford Assay standard curve and the results of this analysis are located
in Table 1. The total mass of protein was determined based on the measured volume of each
sample or fraction and its corresponding protein concentration. Total LDH activity was also
calculated based on the total volume of each sample or fraction. The specific LDH activity, fold
purification, and % yield were calculated as described in Table 1. The crude homogenate had the
 largest observed amount of LDH as well as the greatest total LDH activity and % yield while
producing the lowest specific LDH activity and fold purification. Of the column fractions (not
combined), 17 had the largest observed LDH concentration, mass and total LDH activity, while
12 had the largest specific LDH activity, fold purification and % yield. The (NH4)2SO4
supernatant had a larger protein concentration, mass, total LDH activity, and % yield as
compared to the (NH4)2SO4 dialyzed protein solution which had a larger specific LDH activity
and fold purification.
After the SDS-PAGE analysis (Figure 2), fractions 17 and 18 were combined because
they had the largest observed specific LDH activity of the fractions that resulted from the NADH
wash. An enzyme activity was estimated based on the average activity of both fractions. The
absorbance and concentration was determined through a different Bradford Assay as described in
Table 2. The same calculations were made for the combined fractions that were made for all the
samples and individual fractions. This fraction had the largest mass, total LDH activity and %
yield than all the other fractions. The overall yield of the pooled fractions was 17.025mg
LDH/50g chicken breast.

SDS-PAGE Gel Analysis:

Figure 2. SDS-PAGE Gel of LDH Samples and Fractions An SDS-PAGE gel was run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 to determine the purity of
LDH in select samples and 15
14
13
12
fractions that had observed
enzyme activity. An image of
the gel is in Figure 2. An LDH
control was used as a
reference for determining
which band corresponded to
LDH. The lanes with crude
homogenate, dialyzed
(NH4)2SO4 protein, F11, F12,
F13, F17, and F18 all
appeared to have a band that
corresponds to the LDH
control band. Fractions 11, 12,
Figure 2: SDS-PAGE-Each sample was made in SDS-sample buffer: 0.0583M TrisHCl, 5% (v/v)
glycerol, 1.713% (w/v) SDS, 10mM DTT, 0.0017% (w/v) Bromophenol blue. 15μL of each sample and and 17 have the thickest
fraction was added to each well. 5μL of BenchMark Protein standard was added to wells 1 an 14, and bands. There were a lot of
TM

2μL was added to well 15. Lane 1: Standard, Lane 2: Crude Homogenate, Lane 3: Na2SO4
Supernatant, Lane 4: Na2SO4 Dialyzed Protein, Lane 5: Fraction (F)11, Lane 6: F12, Lane 7: F13, observed bands in the crude
Lane 8 F14, Lane 9: F17, Lane 10: F18, Lane 11: F19, Lane 12: F20, Lane 13: Control LDH protein, homogenate lane that didn’t
Lane 14: Standard, Lane 15: Standard. SDS-PAGE was run in running buffer: 0.025M TrisHCl,
0.192M glycine, 0.1% (w/v) SDS. 12% acrylamide gel was used. Proteins were visualized with correspond to LDH. In all the
Coomassie blue staining. fractions, there were other
bands that didn’t correspond to
LDH, however, there were a significantly less number of bands in the fractions as compared to
the crude homogenate as well as the (NH4)2SO4 supernatant and (NH4)2SO4 dialyzed protein.
The SDS-PAGE results were also analyzed for the determination of the molar mass of
LDH. Comparison of the experimental molar mass to the actual molar mass was used for
confirmation that the protein was LDH. Using the largest molecular weight protein band as a
baseline, a standard curve was plotted, located in Figure 3. The bands that corresponded to LDH
in each of the lanes were determined via the Western Blot analysis (Figure 4). In addition, the
LDH band in lane each was predicted based on the corresponding control lane containing LDH.
Figure 3. Standard Curve of BenchMarkTM Protein Ladder from The distance was measured
SDS-PAGE Gel For LDH Mass Determination between the LDH band in lane 17
was measured to and the
baseline. This distance, 3.05cm,
was used in the equation of the
standard curve to calculate for an
experimental molecular weight
of LDH. The distance measured
was 3.05cm. The experimental
molecular mass was determined
to be 36,791g/mol. The percent
difference was <1% between the
experimental mass measurement
Figure 3: Using the SDS-PAGE gel, the distance of each protein benchmark was measured and the actual molecular mass
from the baseline. The baseline was the largest molecular weight benchmark. which is 36514.4g/mol.

Western Blot Analysis:

Figure 4. Results of Western Blot Analysis Using SDS-PAGE gel
Using the second SDS-
PAGE gel, a Western Blot
analysis was accomplished to
confirm the presence of LDH in
each sample and fraction. A 1°
and 2° antibody was used for
detection of the LDH protein and Dialyzed (NH4)2SO4
F17 F18 LDH Control
for visualization purposes. A
visual result of the Western Blot
F11 F12
is located in Figure 3. Antibody Crude Homogenate
treatment resulted in
distinguishable bands on the
membrane, suggesting the
presence of LDH.
Figure 4: SDS-PAGE gel was used for the Western Blot analysis. Gel was transferred to a
nitrocellulose membrane. Membrane was probed with 1° antibody (goat-Anti-rabbit-LDH-antibody) was
used for specific LDH detection on the membrane. Membrane was then probed with a 2° antibody (rabbit-
Anti-goat-IgG-alkine-phosphitase-conjugate) was used for LDH visualization. Spots from left to right:
crude homogenate, dialyzed ammonium sulfate, F11, F12, F17, F18, LDH control. This figure was
enhanced to better visualize the presence of LDH.
 Accurate LDH Concentration Determination via Edelhoch and Bradford Assay Analysis:

Table 2. Accurate Concentrations of Pooled LDH Fractions and Carbonic Fractions 17 and 18
Anhydrase Protein Determined via Bradford Assay and Edelhoch Analysis were combined and
dialyzed against 2
volumes of dialysis buffer
Lactate Dehydrogenase Carbonic Anhydrase
Concentration Concentration
(10mM TrisCl pH 8.6,
0.5mM 2-
Edelhoch Analysis -- 0.69mg/mL
mercaptoethanol) and this
LDH sample was subject
Bradford Assay Analysis 1.3618mg/mL 1.3236mg/mL to a subsequent Bradford
Assay and an Edelhoch
Quantitative Amino
analysis. A sample of
-- 0.691mg/mL Carbonic Anhydrase
Acid Analysis
(CA) was also analyzed
Table 2: Bradford Assay Analysis: A Bradford Assay standard curve was determined using the parameters as in both these techniques
described in Table 1. The concentration of LDH and Carbonic Anhydrase (CA) was determined using the
equation of the standard curve: (y = 0.0288x + 0.0299) The absorbance of LDH and CA was 0.226 and 0.2205 for comparison purposes
respectively. Edelhoch Analysis: The absorbance of LDH and CA samples were obtained in 50mM KPO buffer
4 and to probe the accuracy
pH 7.4 with and without 6M GdnHCl measured at 280nm using a UV-Vis Spectrophotometer. The final volume
was 1mL. The blank was measured with dialysis buffer. The value of ε
Denatured was determined for LDH and CA and limitations of various
2
using the Pace et al. reference . ε
Denatured
-1 -1
= 40,535M cm for LDHA and ε
Denatured
-1 -1
= 43,105M cm for CA. Abs techniques used for
(CA-denat.)= 0.2112131, Abs (CA-nat.)=0.311365. Beer’s Law was used to determine the concentration of CA.
Quantitative Amino Acid Analysis: QAAA was completed on the CA sample to give an accurate concentration determining protein
measurement. concentration.
For the Edelhoch
analysis, absorbance values were measured at 280nm for native and denatured samples of LDH
and CA. 6M Guanidine hydrochloride (GdnHCl) was used to denature the proteins. An
extinction coefficient was determined for both LDH and CA denatured in GdnHCl using the
Pace et. al. reference.2 Using the extinction coefficient for CAdenatured (located in Table 2), a
concentration for CAdenatured was determined and the results are located in Table 2. The values of
Absdenatured and Absnative were measured for CA at 280nm, and knowing the value of εdenatured, the
value of εnative at 280nm was calculated for CA.

Figure 5. Absorbance Scan of Natured LDH Sample

The LDH protein concentration 2.5
could not be determined using the
Edelhoch analysis because a large 2.0
absorbance peak at 260nm completely 1.5
covered the peak at 280nm. A broader Abs
absorbance scan was obtained for the 1.0
natured LDH and is located in Figure 5. 0.5
Absorbance peaks at 260nm and 340nm
suggested the presence of NADH in the 0.0
sample. The absorbance at 340nm for the
native LDH was 0.5020482. Based on
Figure 5: This absorbance scan was of the combined Fraction 17 and 18
sample in dialysis buffer: 10mM TrisCl pH 8.6, 0.5mM 2-mercaptoethanol,
diluted in 50mM KPO4 buffer pH 7.4 . This was one of the Edelhoch analysis
samples. The parameters of the Edelhoch analysis are located in Table 2. ε 340

for NADH = 6220M-1cm-1.

the extinction coefficient for NADH at 340nm, the concentration of NADH was determined to be
0.2675mg/mL

LDH and CA protein concentrations were determined using a Bradford Assay. The
standard curve equation and protein concentrations are located in Table 2. The results of the
Bradford Assay for the combined LDH samples are also located in Table 1.
The sample of CA protein was sent to Texas A&M University Protein Chemistry
Laboratory for quantitative amino acid analysis (QAAA) and the determined concentration is
incorporated into Table 2 as well. It was observed that the Edelhoch method and QAAA
provided a very similar value for the concentration of CA protein. The Bradford Assay
concentration for CA was significantly larger than the other two methods. The only
concentration determined for the combined LDH samples was from the Bradford Assay analysis.

Discussion:
The results of the first Bradford Assay (Table 1), provide concentration data of LDH in
each sample and fraction while also providing information about the purity of LDH. The specific
LDH activity and fold purification are measures of purity; the samples with larger values of
specific LDH activity and fold purification are more pure than samples with lower values. The
chromatography sample with the largest LDH concentration was fraction 17, and the sample with
the highest purity was fraction 12 based on this analytical technique.
The results of the (NH4)2SO4 dialyzed sample in Table 1 provided contradictory
information. The total mass of LDH for this sample was less than the combined mass of LDH
from all the fractions. This wasn’t possible because all the LDH in the fractions came from
(NH4)2SO4 dialyzed sample. It is possible that an alternate dilution was measured and not
correctly accounted for when calculating LDH enzyme activity from the activity assays.
The SDS-PAGE results provided conclusive evidence to evaluate the presence and purity
of LDH in all of the samples and fractions. From the gel in Figure 2, LDH is present in the crude
homogenate, dialyzed (NH4)2SO4, Fractions 11, 12, 13, 17, 18, and possibly in the (NH4)2SO4
supernatant. These samples and fractions all had a band that corresponded to the same distance
of the band in the LDH control lane. Further evidence that these were all LDH bands was
provided through the Western Blot analysis, however it was difficult to visualize the bands for
the (NH4)2SO4 supernatant and fraction 13. From the control LDH lane it was determined that
LDH protein is a single species or produces only one band. This means that LDH is either a
monomer, or an oligomer composed of only one type of monomer.
It seems that fractions 11,12, and 17 contain the most LDH protein based on the thickness
of the band from the SDS-PAGE gel. If I had a chance to change which fractions were
combined, I would probably use fractions 11, 12, and 17 due the high LDH concentration, as
well as the fact that they all had the same level of purity. I assume that the only difference
between the proteins that came out with the NAD+ wash as opposed to the NADH wash was the
confirmation of the protein. I also think that the LDH that came out with NAD+ is just as pure as
the LDH that came out with the NADH. This can be concluded by looking at the different
fractions from each wash on the SDS-PAGE gel. The SDS-PAGE gel however, does not provide
evidence of non-protein impurities.
The presence of impurities in each of the samples was established by the appearance of
other bands than the LDH protein. The crude homogenate had the most significant amount of
impurities. The (NH4)2SO4 supernatant and dialyzed (NH4)2SO4 samples both had impurities, but
in much less concentration than the crude homogenate. All the fractions from the affinity
chromatography had a significantly less amount of impurities as compared to the crude
homogenate. It would appear that all the fractions had relatively the same purity of LDH protein
as the impurity bands in each fraction that had LDH, appeared to be the same intensity. The
impurities are most-likely other proteins found in chicken breast muscle that also have a binding
affinity to NADH, NAD+, and/or molecules containing pyridine groups, as they bound
themselves to the Cibacron blue column that mimics these molecules.
Based on the SDS-PAGE protein BenchMarkTM standard curve located in Figure 3, the
estimated molecular mass was calculated to be 36,791g/mol using the equation of the line. This
estimate is very close to the known mass of LDH, which is 36,514.4g/mol, as determined by
ExPASy Proteomics Service online. The percent difference is <1%. The minor disparities
between the estimate and the actual mass could be attributed to the nature of an SDS-PAGE. In
this analysis, the protein sample is denatured and non-polar regions become covered with SDS
molecules. This adds to the molecular mass of the protein. It is a reasonable explanation as our
estimated mass was slightly higher than the actual mass. In an SDS-PAGE, the protein is
denatured allowing the nonpolar regions of the SDS molecules to associate with the nonpolar
regions of the protein, preventing re-naturation. In this sense, the molecular mass determined by
SDS-PAGE is of the proteins primary structure (plus the mass of the SDS molecules associated
to the protein). If the protein monomer associates with other monomers in its quaternary
structure, you would not be able to determine this when using SDS-PAGE.
From the SDS-PAGE gel it appears that there was LDH protein present in the lane
containing the ammonium sulfate supernatant, however, this lane is rather skewed probably due
to the high concentration of ammonium sulfate. Presence of LDH in this sample provides
evidence that not all of the LDH protein in the crude homogenate precipitated. To precipitate
more of the LDH protein, more ammonium sulfate should have been added
A strange result was observed for the dialyzed ammonium sulfate sample in the SDS-
PAGE gel (Figure 3). It seems that not very much LDH protein was present, rather, a lot of a
protein slightly higher in molecular mass than LDH was present with only a small amount of
LDH. This would not seem correct because the dialyzed ammonium sulfate sample should have
had a large LDH concentration. It’s possible that a sample was mislabeled and incorrectly loaded
into the gel.
From the Edelhoch analysis an accurate CA concentration was determined as it was
almost the exact concentration established from the QAAA. QAAA is a very precise and reliable
method used to determine protein concentrations when the protein is pure. The Edelhoch method
could not be used for the LDH protein because it was determined that a large concentration of
NADH was present in the sample. Both the natured and denatured LDH samples had large
absorbance peaks at 260nm and 340nm, which are two wavelengths for which NADH has a high
absorbance. The peak at 260 completely covered the peak at 280 which was supposed to be used
to determine the LDH protein concentration with the calculated extinction coefficient for
denatured LDH protein in GndHCl at 280nm. The fact that so much NADH was present in
solution stresses how tight LDH binds to NADH. To improve the previously described outcome,
it is possible that the LDH could be dialyzed with pyruvate such that the NADH would be
converted to NAD+, and because LDH has a much lower binding affinity for NAD+, it is possible
that the NAD+ could be eliminated through this dialysis.
The calculation for CA concentration via a subsequent Bradford Analysis appeared rather
large as compared to the concentrations determined through Edelhoch and QAAA. The reason
for this could be that the BioRad Protein Assay Reagent (which turns blue upon protein binding)
binds to all proteins present in solution. If the LDH sample had other protein impurities, which
was observed in fraction 17 and 18 from the SDS-PAGE gel, there would be a larger production
of blue color, increasing the absorbance of the sample. This is a limitation of the Bradford Assay
analysis. If this were the case, than the concentration of the LDH calculated from this Bradford
Analysis may also be higher than the actual concentration. Because the Edelhoch method didn’t
work for LDH and there was no QAAA for LDH, there were no other concentrations of the
combined LDH fractions to use for comparison purposes. QAAA is the most accurate method to
determine protein concentration, because a concentration of each amino acid is determined in the
sample, and based on the number of a particular amino acid present in the protein, an accurate
protein concentration can be deduced. The limitation of this method would be if other protein
impurities were present in the solution, you would obtain an inaccurate concentration calculation.
The Edelhoch method is also very accurate, however, there are a few limitations. For example, if
there’s an impurity in the protein solution that affects the absorbance at 280nm, like we saw with
LDH containing NADH, you will not be able to use this method.

Conclusions:
From the results and analysis it can be concluded that LDH was successfully purified
from chicken breast muscle. There were still some minor impurities following the affinity
chromatography as determined through analysis of the SDS-PAGE, however the protein
appeared to be purified by at least 90% from the crude homogenate. The Western Blot analysis
confirmed that the protein was LDH. The final concentration of the pooled LDH fractions was
1.362mg/mL, as determined by Bradford Assay, with a 8.69% yield. From the CA study, it was
concluded that the Edelhoch method and QAAA are very accurate methods of demining protein
concentration, however QAAA will typically be the most accurate if the protein sample is pure.
 References:

1. Campbell, N.A. and J.B. Reece. 2005. Biology, 7th edition. Benjamin Cummings, San
Francisco.
2. Pace, C. Nick, Felix Vajdos, Lanette Fee, Gerald Grimsley, and Theronica Gray. "How to
Measure and Predict the Molar Absorption Coefficient of a Protein." Protein Science 4
(1995): 2411-423. Print.