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Aaron Ye
Jan 27, 2016
C01
TA: Christina
Yellow Group 3
Lab Partners: Michael, Robert

To Separate and Purify LDH using Various
Techniques as well as Determine the
Isozyme of an Unknown LDH Sample
...
LDH is one enzyme recently
revealed to facilitate the pathway of lactic acid
production by catalyzing a reaction between
pyruvate and lactate, as shown by the reaction
below
...
Presence of many diseases, such
as cancer, elevates LDH in the blood, which
can be detected
...

Multiple purification steps were used to
separate LDH from other proteins found within
a cell
...
Centrifuging will narrow
down the proteins, eliminating proteins from
the nucleus and other proteins residing in
organelles
...
LDH is has a specific
hydrophilicity can be easily precipitated out
using ammonium sulfate precipitation
...
At the end, a
sample of proteins that are of similar solubility
to LDH can be obtained
...
Since LDH has a high
affinity towards NAD+, we can use this
property to purify it
...

Size is also a great factor to further
purify the protein via size exclusion
chromatography
...
6 kDa
...

Isoelectric points of LDH isozymes differ
from one another
...
Other organs have
a different combination of the two subunits, and
this property can help identify where the LDH
came from via isoelectric focusing (Slattery et
al, 1983), since each subunit has a different
isoelectric point
...

The purification and identification of
LDH subunits is important to further
understand metabolism patterns in multicellular
life
...

Materials and Method
It is critical to keep the samples in cold
temperatures during the entire duration of the
experiment
...
This is especially critical before
the sample has been purified
...
This filtered out a clarified
homogenate (CH) after discarding the pellet,
which is a purification intermediate
...
Ammonium sulfate was added until
40% saturation was obtained, which involved
adding 0
...
The sample was then centrifuged at
15,000 xg for 15 minutes and the pellet was
discarded
...
166g of ammonium
sulfate per ml
...

Affinity Purification
The rest of the 65% cut pellet was used
for an affinity assay containing Cibacron Blue,
which has potential binding properties for LDH
...
We collected the eluted
solution in microfuge tubes (1mL each) and
measured them using a spectrophotometer
until the absorbance was a significant low
(~0A)
...
This is the spot test
...
This oxidizes
PMS, which in turn reduces NBT
...

The column fractions that had a
significant blue color were collected together,
and another ammonium sulfate precipitation
was performed using 0
...
The solution was
centrifuged at 15,000 xg for 15 minutes and the
pellet was re-suspended
...
Blue Dextran contained large
molecules, so it eluded first, while Orange G
eluded last
...

20mL of eluded sample in 20 microfuge
tubes was collected
...

Ammonium sulfate precipitation was performed
once more for further purification (0
...
Supernatant
discarded) before the sample was stored as
our size exclusion purified LDH (SE)
...
First, the master reagent was
added, and a recording for the change in
absorbance over time in a spectrophotometer
was performed
...
When NAD+ is
converted to NADH by LDH, absorbance
dramatically increases at 340nm
...

Agarose Gel Electrophoresis
Finally, we conducted two different
types of gel electrophoresis
...
Qualitative
observation revealed the major LDH isozyme
in our unknown sample
...

Results
Centrifugation
and
Ammonium
Sulfate
Precipitation
The first step involved centrifugation of
the cell parts, which is performed to remove
many proteins that reside in organelles, such
as the nucleus, and obtain only materials that
reside within the cytoplasm
...
Afterwards, a large brown pellet
was
present,
which
contained
many
contaminant proteins
...
5mL
...

We first performed a 40% cut by adding in
0
...
The final
volume we added was 20
...
After
centrifuging, the green pellet was discarded
and the green supernatant was kept, which
had a volume of 92mL
...
We needed to add
an additional 0
...
15
...
 Absorbance  for  Af:inity  Chromatography  
4  

Absorbance  (A,  280m)  

3
...
5  

10  

2  
1
...
5  

4  

16   18  
14  

20  

0  
0  

5  

10  

15  

20  

25  

30  

35  

40  

45  

50  

Elusion  Volume  (ml)  

measured out and dissolved
...
The final volume turned out to be 5
...

The results of this part of the experiment
is a sample that has been eliminated of
proteins that are less and more soluble than
LDH
...
Other proteins that do not bind to
Cibacron Blue will elute through first, allowing
us to collect LDH later when we wash it out
with NaCl
...
1
...
98ml
...
295g of ammonium sulfate, and
the dull white pellet was re-suspended in 0
...
579ml
...
The result from this
experiment is further purified based on LDH’s
size
...
1
...
The first peak is the wash
...
Numbers next to the points
indicate fraction numbers
...

20ml was collected in total, and a spot
test revealed that fractions between 1ml and
6ml had bright blue colors and was significant
...
Significant
fractions
are
kept
for
assays
and
electrophoresis analysis
...
852g of
ammonium sulfate and resuspected the pellet
after centrifugation for a final volume of 1
...

Activity Assay
The activity assay is a measurement
method used to determine the rate of the
reaction by measuring absorbance over time
...
An increase in
absorbance shows that a reaction is occurring
...


Clari:ied  Homogenate  1:1    

Absorbance  (A)  at  340nm  

0
...
0012x  +  0
...
99849  

0
...
08  
0
...
04  
0
...
0012  ∆𝐴 60  𝑠𝑒𝑐
=
∗
𝑚𝑖𝑛
𝑠𝑒𝑐
𝑚𝑖𝑛
∆𝐴
= 0
...
072
= 6220𝑀!! 𝑐𝑚!! 1  𝑐𝑚 ∆𝑐
𝑚𝑖𝑛
𝑀
∆𝑐 = 1
...
158 ∗ 10!!  
) ∗ 0
...
158 ∗ 10!!  𝑈
𝑈
= 2
...
002  𝑚𝑙 ∗ 1  𝐷𝐹
𝑚𝑙
𝑈
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒  𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 5
...
73  𝑈
𝑈 = 5
...
2
...
The slope of the line of
best fit indicates the activity
...
This may
be due to a couple of reasons, one being
proteases digesting LDH away in the CH
sample
...

The original expectation was that CH
would have the most relative activity, because
more LDH is present (since some LDH is
filtered out too due to purification)
...


65%  Cut  Pellet  1:40    
Absorbance  (A)  at  340nm  

0
...
0009x  +  0
...
99689  

0
...
06  
0
...
04  
0
...
02  
0
...
68 ∗ 10!!  𝑈
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒  𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 173
...
3
...
The slope of the line of
best fit indicates the activity
...
16  𝑈

The total activity is much higher than
that of CH
...
In
the 65% cut pellet, many contaminant proteins
were removed based on their hydrophobicity
...

The slope appears to be regular, and
nothing out of the ordinary is present
...


Af:inity  Puri:ied  LDH  1:250    
0
...
0008x  +  0
...
99644  

Absorbance  (A)  at  340nm  

0
...
05  
0
...
03  
0
...
01  
0  
0  

10  

20  

30  

40  

50  

60  

70  

Time  (sec)  

Fig
...
The change in absorbance between
15 and 60 seconds after the affinity
purification sample was inserted into the
spectrophometer
...


𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 7
...
63
𝑇𝑜𝑡𝑎𝑙  𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 732
...
The fact that it doesn’t increase further
than the 65% cut pellet suggests that the
proteases that digest LDH have been removed
by the ammonium sulfate precipitation
...

But the total activity dropped slightly, which is
expected since it is impossible to recover
100% of LDH
...
035  

y  =  0
...
0125  
R²  =  0
...
03  
0
...
02  
0
...
01  
0
...
89 ∗ 10!!  𝑈
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒  𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 72
...
7  𝑈

𝑈
𝑚𝑙

Fig
...
The change in absorbance between
15 and 60 seconds after the size exclusion
purificationsample was inserted into the
spectrophometer
...


The sample of the size exclusion had
much less total activity than the previous two
samples
...
The
general sense is that larger proteins travel
faster
...

The main reason that there may be less
total activity is due to our selection of filtration
samples after the spot test
...
In the size exclusion,
however, we only chose 5ml, since we wanted
to be more specific this time
...
This would not, however, explain
the great drop in relative activity
...
061x  +  0
...
98955  

Bradford  Protein  Absorbance  at  595nm  
0
...
6  
0
...
4  
0
...
2  
0
...
Using this, we are able to
estimate the amount of protein we have in our
4 samples
...

Below is a sample calculation for the CH
sample
...
061𝑥 + 0
...
109 = 0
...
0231
𝑥 = 1
...
4082µg of
protein mass per 2µL of sample
...
= 1
...
7041
𝑚𝑙
𝑚𝑔
𝐴𝑐𝑡
...
7041
∗ 10𝐷𝐹
𝑚𝑙
𝑚𝑔
= 7
...
6
...
041
= 612
...
Protein
Concentration
(mg/ml)
7
...
689
108
...
190

Total Protein
(mg)
612
...
2895
82
...
4250

Fig
...
Table containing calculated results
for actual protein concentration and total
protein for all purification intermediates
...
79𝑈 ÷ 612
...
8223
𝑚𝑔

Purification Table

Purification
Step
Clarified
Homogenate
65%
Cut
Pellet
Affinity
Purified LDH
Size
Exclusion
Purified LDH

LDH
Assay
Relative Total
Activity Activity
(U/ml)
Units (U)

Bradford
Assay
Protein
Total
Concentration Protein
(mg/ml)
(mg)

Specific
Activity
(U/mg)

Percent
Yield (%)

Fold
Purification

5
...
73

0
...
567

0
...
60

972
...
7689

262
...
7064

192
...
507

964
...
15

2
...
712

8
...
35

10
...
25

86
...
0238

61
...
4109

17
...
716

Example Calculation for Percent Yield (65%)
𝑃𝑒𝑟𝑐𝑒𝑛𝑡  𝑌𝑖𝑒𝑙𝑑 =

972
...
73

𝑃𝑒𝑟𝑐𝑒𝑛𝑡  𝑌𝑖𝑒𝑙𝑑 = 29
...
7064
0
...
5073

Fig
...
Purification table that contains all our
calculated results
...
9
...


Legend:
2: Heart Standard
5: Purified Sample
8: Muscle Standard

The agarose gel electrophoresis was
done in order to separate the samples by their
isoelectric points, since the subunits heart and
muscle have different subunits each
...

Observation
determined
that
the
unknown sample contains heart subunits, and
is most likely derived from heart tissue
...
10
...

The markers on lane 5 served to
indicate what molecular weight the proteins
were depending on how far they travelled
...

The gel was submerged into a
Coomassie Blue staining solution, which
produces a dark color under the presence of
protein
...


Fig
...
Indicator on what molecular size will
travel what distance down the SDS-PAGE
gel
...
The 65%
ammonium sulfate cut had the greatest specific
activity and, more importantly, the greatest fold
purification
...
5 times
...
2% of the total
protein amount
...
Similarly, the yield also
showed promise
...
In addition, the percent
yield exceeded 100%
...
As a result, no
conclusion can be made about the yield of the
ammonium sulfate precipitation
...
Since
the clarified homogenate has yet to be purified,
in theory it would retain the most amounts of
LDH
...
The clarified homogenate, out of
the four, would be the sample that contains the
greatest amount of contaminant proteins
...
Before purifying
these proteases out, they would act to break
down LDH, decreasing the tested activity when
the master mix was introduced
...

It is difficult to completely control the
clarified homogenate and prevent the digestion
of proteins by proteases
...
Perhaps a
solution would be to measure the LDH activity
and total proteins before completely separating
the cell components into a mush
...
Before destroying cell parts,
proteases would not have access to LDH and
thus would not digest them as easily
...
Then, we would use
this sample, which has not yet been purified,

as the default sample that other purification
steps compare to, instead of the clarified
homogenate
...

A second solution is to add some other
substances that would help denature proteins
...
Of course, this would have to be
controlled as to not irreversibly denature the
LDH as well
...

The affinity purified sample was also
effective at retaining LDH, keeping 75
...
In addition,
the yield was also 75
...
This value is quite
high, and allows us to conclude that the affinity
purified chromatography was successful in
terms of yield
...
4 times)
...
5% and a specific activity of 239% of that of
the ammonium sulfate sample
...

The size exclusion sample was worse
than all the other purification methods in terms
of yield and fold purification
...
8%% of previous LDH
proteins, which is not very impressive
considering 74
...
This failure is evident in its fold
purification, where it is only 15
...
Also considering that the
specific activity dipped to 15
...
It is not much different for
yield, which also produced poor results
...
6%, and the
percent yield was only 17
...
2%
from the previous
...
However, this problem poses a great
threat for the size exclusion chromatography,
since the beads are reliant upon the fact that
no air bubbles are present
...
That way, air bubbles
will not be present within the solution of the
column
...
When it was time
to run the column, many of the beads have run
dry
...
An improvement would be to watch the
level of phosphate buffer at all times and
prevent it from reaching a level below the
beads
...
When leaving it overnight, check
frequently to ensure that the column did not
leak phosphate buffer
...

General improvements for any type of
column purification include preventing any
beads from drying out, and allowing the column
to elude more slowly, or simply increasing the
column length to improve the accuracy of the
purification
...

The
agarose
gel
electrophoresis
revealed that the unknown LDH sample that
we purified has traveled as far as the heart
standard, leaving a trail behind
...
The trail
behind it contains contaminant proteins that
were not 100% filtered out during the
purification steps
...
The clarified homogenate is exactly
what was expected, with a whole set of
proteins
...
The 65% cut pellet, in
comparison to the clarified homogenate, had
much less quantity in terms of protein amount,
but had a thicker band for areas closer to the
stationary phase of the gel
...
Next, the affinity
purified sample did not seem to possess any
proteins at first glance
...
It appears that this chromatography
filtered out all the proteins, but failed to keep
much LDH within the sample
...
Finally,
the size exclusion chromatography sample has
dark band at the same distance as the LDH
standard
...
But compared to
the clarified homogenate, the size exclusion
sample was purified to a successful degree
...
Perhaps the affinity sample
was not loaded properly, or too little volume
was inserted
...

Medical Implications
Cancer is quite unique in its ability to
artificially deflect glucose to undergo lactate
fermentation
rather
than
oxidative
phosphorylation
...
Therefore, targeting LDH is a
method of cancer treatment
...

A second implication is the ability to
detect damaged or destroyed tissue
...
In addition, sickle cell anemia often has
a side effect of elevated LDH levels (Ballas,
2013)
...

The fact that LDH has different subunits
can be useful in identification purposes
...
For
the case of tissue damage detection, this can
theoretically determine the source of the free
LDH
...
As science continues to develop,
LDH may one day be our best friend in terms
of fighting certain illnesses
...
Slattery, W
...
, and A
...
Sinclair
...
" Australian Vet J
Australian Veterinary Journal 60
...

2
...
, and Elizabeth Barto
...
LACTATE
DEHYDROGENASE
ISOZYMES
...

4
...

"Lactate
Dehydrogenase
...
"
Cell
Biochemistry and Function Cell Biochem
...
2
...

5
...
"Lactate
Dehydrogenase 5: An Old Friend and a New
Hope in the War on Cancer
...
1 (2015): 1-7
...
Ballas, S
...
"Lactate Dehydrogenase and
Hemolysis in Sickle Cell Disease
...
1 (2013): 243-44
...
Fiume, Luigi, Marcella Manerba, Marina
Vettraino, and Giuseppina Di Stefano
...
" Future
Medicinal Chemistry 6
...




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