The Purification and Isolation of
Rabbit Muscle Aldolase
Hye Soo Lee
Partner: Sohee Min
Date Due: 4/1/14
Date Submitted: 4/1/14
MATERIAL AND METHODS
Note: All procedures were performed at 0-4°C, and aliquots from fractions were stored at
-20°C
EXTRACTION
An 80.2 g of chopped rabbit muscle were mixed with cold (0-4°C) 0.03 M KOH
and 0.001M EDTA. The sample was hand stirred for 20 minutes and centrifuged at
10,000 x g for 10 minutes. The supernatant was filtered by glass wool to remove any
lipids. The pH of filtrate was adjusted to 7.5 using 0.1M KOH. The final volume of the
Fraction I solution was measured to be 157 mL. A 1 mL aliquot was taken, labeled as
Fraction I, and stored.
AMMONIUM SULFATE FRACTIONATION
A 157mL sample of 100% saturated ammonium sulfate was added to Fraction I to
reach 50% saturation and stirred on ice for 60 min. Then the solution was centrifuged to
remove precipitate at 10,000 xg for 30 minutes. The supernatant was filtered using
glass wool and labeled as Fraction II. The final volume of this Fraction II solution was
measured to be 300 mL, and 1 ml of its aliquot was saved. 19.5 g of solid ammonium
sulfate was added at a very slow rate to Fraction II to achieve 60% saturation. The pH
was adjusted to 7.5 using 1.0N NH4OH. The solution and aliquots were stored for a week
in cold room for precipitate formation. Later, Fraction II was centrifuged at 10,000xg for
30 minutes. The supernatant was measured and labeled as Fraction III. 1 ml of its aliquot
was saved, and the pH was measured as 7.39. The precipitate was dissolved in very small
volume of equilibrium buffer (10mM Tris HCl, pH 7.5; 5mM EDTA). The solution was
then centrifuged at 10,000 xg for 15 minutes for removing any insoluble material. The
supernatant was named Fraction IV and its aliquot was diluted to one tenth using
equilibration buffer for later analysis.
DIALYSIS
Fraction IV was dialyzed using tube (MWCO 14,000 Da). Fraction IV was
placed inside the tube, and dialyzed in the cold room against equilibration buffer for a
week. Then the solution was centrifuged at 10,000 xg for 15 minutes and the aliquot from
supernatant was diluted to one-tenth by equilibration buffer. The rest of supernatant was
saved as Fraction V.
AFFINITY CHROMATOGRAPHY
Ion Exchange Chromatography with phosphocellulose column (1.5 cm x 11 cm,
20 m total volume) was set up. The plastic container was filled with 100ml quilibration
buffer (10 mM) Tris. HCl, pH 7.5, 5 mM EDTA). The column was equilibrated at a rate
of approximately 1.5 ml/min. The column was stored in cold room for a week. 20 ml of
equilibration buffer was run through the column, and a 1 ml/min effluent flow rate was
obtained. The "desalted" adolase was then added to the phosphocellulose column. When
the sample was “in” the column, 2 ml of equilibration buffer was add. The headspace was
fiilled with buffer and hooked up the top buffer chamber. The column was washed using
equilibration buffer (flow rate 1.0 ml/min) until the effluent absorbance at 280 nm was
below 0.1. The volume of collected wash was recorded and 1ml was saved for later use.
100 ml of elution buffer (2.5mM FBP [B-grade]: 50mM Tris HCl, pH 7.5; 5mM EDTA)
was run through the column. Forty fractions with approximate volume of 1.0 ml were
collected. The fraction with the highest absorbance was identified as the “peak”, and 0.3
ml was saved. Another 0.1 ml from the “peak” was taken out and diluted to 1 ml with
equilibration buffer. The fractions with highest absorbance were pooled together and
saved as Fraction VIA. The fractions with low absorbance were also pooled together and
saved as Fraction VIB. ALL Fractions including VIA and VIB were saved and stored in 20C freezer for future analysis.
SPECIFIC ACTIVITY DETERMINATION
Protein content determination
A protein standard curve was created from different concentration of bovine
serum albumin (1, 5, 10, 15, 20 and 25 ug/ml). Final volume was adjusted to 1 ml using
0.15 M NaCl, and1 ml of Bradford color reagent was added to each concentrations and
their absorbance was observed at 595 nm. The color reagent used was 0.0032%
Coomasie Brilliant Blue; Ethanol; and 8.5% phosphoric acid. Using Bradford method, the
protein concentrations from all fractions were measured using Genesys-5.
Aldolase Assay I
Each fractions components was mixed with dH2O (0.2ml), Histidine-HCl buffer
(0.2M, pH7.2), NaH2AsO4(120 mM, pH 7.2), NAD+( 25mg/ml ~38 mM), FBP (0.1M,
pH 7.2), Ga3P dehydrogenase (400 IU/ml, ~180ug/ml) and aldolase fractions(10ul of
fraction, 590ul dH2O) in 3ml mixture of final concentrations. The changes in absorbance
of aldolase fractions were measured under 340nm by Genesys 5 spectrophotometer.
Triose Phosphate Isomerase Assay
Each fractions were added to a solution that contains dH2O, Histidine-HCl buffer,
NADH (1.3mM in His-HCl buffer pH 7.2), Ga3P (2.5mM), α-GP dehydrogenase(~40-50
IU/ml) and aldolase fractions(10ul of fraction, 890ul dH2O) in 3ml mixture of final
concentrations. The activity of Triose Phosphate Isomerase was measured at 340 nm
though spectrophotometer
RESULTS
EXTRACTION
From the extraction method using EDTA and KOH, 157ml of Fraction I was
obtained. The pH of Fraction I was 7.49. The protein concentration of Fraction I
calculated to be to be 11.9 mg/ml.
AMMONIUM SULFATE FRACTIONATION
Fraction II was the supernatant that was separated by centrifugation. It had 300
ml of volume and protein concentration of 4 mg/ml. The fractionation by ammonium
sulfate for week resulted in 303 ml of Fraction III and 5.4 ml of Fraction IV. Fraction III
had pH of 7.39. The protein concentration of Fraction III was 4.075 mg/ml and 31.82
mg/ml for Fraction IV.
DIALYSIS
Fraction V was obtained by dialysis had 8.1 ml of volume and contained 37.35
mg/ml of proteins.
AFFINITY CHROMATOGRAPHY
Before chromatography, the column was washed with equilibration buffer. The
58ml of “wash” was collected and had a concentration of 1.04 mg/ml. For elution of
aldolase, forty fractions containing approximately 1.0 ml of each were collected. The
absorbance values for each fractions obtained from column chromatography is provided
by Figure 1. The fraction 12 had the highest absorbance thus selected as “peak” and had
protein concentration of 4.835 mg/ml. The fractions that have relatively high absorbance
at 280nm were pooled together into Fraction VIA (fraction 11-24) which had a protein
concentration 5.515 mg/ml in 14 ml solution. The fractions that have less concentration
of proteins, representing the “tail” of the peak, were pooled together and called Fraction
VIB (fraction 25-37) which had 14 ml of volume and the concentration of 2.143 mg/ml.
Absorbance at 280nm against
eluted fractions
4
3.5
Absorbance
3
2.5
2
1.5
1
0.5
0
3
8
12
16
19
22
25
28
33
Fraction #
Figure 1. Absorbance at 280 nm against eluted fractions: The absorbance values of
fractions that eluted out from column chromatography by elution buffer containing were
measured using spectrophotometer at 280 nm. Total of 30 fractions were obtained, each
having approximate volume of 1.0ml. The fraction 12, which having the highest
absorbance, was identified as peak, fraction 11-24 as Fraction VIA, and fraction 25-37 as
VIB.
SPECIFIC ACTIVITY DETERMINATION
Protein content determination
Different concentration of Bovine Serum Albumin (1, 5, 10, 15, 20 and 25 ug/ml)
and Bradford reagent were used to generate a protein standard curve (Figure 2). Since
absorbance measured for Fraction IV and VIB exceeded the maximum absorbance value
measured from the standard curve, they were diluted to one-tenth and measured again.
Each fraction was diluted before its absorbance at 340 nm was measured. For each
fraction, the dilution factor, absorbance, and associated concentration is recorded in
Table1.
Table 1: The dilution factor, Bradford absorbance, and tube protein concentration for
each Fraction.
Fraction
I
II
III
IV
V
Wash
Peak
VIA
VIB
Final Dilution
4000
4000
400
2000
2000
200
500
Bradford Absorbance
0.101
0.038
0.291
0.407
0.452
0.165
0.279
500
200
0.310
0.303
Tube Protein (ug/tube)
2.976
1.000
10.188
15.911
18.674
5.207
9.670
11.000
10.716
BSA Standard Curve
0.6
Absorbance
0.5
0.4
0.3
0.2
0.1
0
1
5
10
15
20
25
Bovine Serum Albumin Concentration (μg/mL)
Figure 2. Standard Curve from BSA: Protein standard curve was generated using
amounts between 1 and 25ug of bovine serum albumin (BSA; 25ug/ml). The final
volume of each fractions were adjusted to 1ml using 0.15M NaCl, and Coomasie Brilliant
Blue G250 was used to color the proteins. The absorbance of each fractions were read on
Genesys-5 at 595nm.
Aldolase Activity Assay I
The protein concentrations of various fractions obtained by Bradford method were
calculated with the data from Aldolase activity assay I to get the specific activity of
aldolase for each fractions. The Aldolase activity was observed by coupling reaction that
was paired with the main reaction. The main reaction converts FBP into Ga3p and DHAP
by aldolase while the coupling reaction utilizes converted Ga3P to generate NADH. The
appearance of NADH was measured by Genesys-5 at A340nm. The Table 2 illustrates the
total units of aldolase activity, the amount of total protein, and specific activities of
aldolase in each fraction. The data from Fraction I was used to calculate the percentage
yield and fold purification.
Triose Phosphate Isomerase Assay
In this assay, the activity of Triose Phosphate Isomerase was measured. The main
TPI reaction, which converts Ga3P into DHAP, was coupled with the reaction that
utilizes both DHAP and NADH as substrates and the disappearance of NADH was
measured at A340nm using Genesys-5. The same various protein concentrations obtained
through Bradford method was conjugated with the TPI activities to calculate the specific
activity of Triose Phosphate Isomerase. Table 3 was generated with these data that
include total units of activities of TPI as well as its specific activity in different fractions.
The data from Fraction I was used to calculate the percentage yield and fold purification
as aldolase activity assay I. A histogram was created to visualize the trends in specific
activity of each fraction as calculated through the aldolase and TPI assays. The data for
both of these two assays were superimposed together in Figure 3.
Comparison of Specific Activity
between Aldolase and TPI
Specific Activity (units/mg )
10
9
8
7
6
5
"SP for Aldolase Assay I"
4
SP for TPI
3
2
1
0
I
II
III
IV
V
WASH PEAK
VIA
VIB
Fraction #
Figure 3. Comparison of Specific Activity between Aldolase and TPI: The specific
activities for Aldolase Assay I and Triose Phosphate Isomerase Assay were compared.
The absorbance were measured for the appearance or disappearance of NADH using
spectrophotometer at 340nm for both assays. The protein concentration for both assays
were from the same data measured using Bradform method.
SAMPLE CALCULATION (FRACTION II)
Activity in Assay I:
=ΔA340nm/min x reaction vol/6.2 x sv(ml)
=0.106 x 0.48/ 0.01
=5.088 units/ml
Total units in Assay I:
= Activity x volume of stock fraction solution
= 5.088 units/ml x 300 ml
= 1526.4 units
Total protein of Fraction II
= mg protein in tube x DF x volume of stock fraction solution
= 0.001 mg/ml x 4000 x 300 ml
= 1200 mg
Specific Activity in Assay I:
= Total Units of Enzyme Activity (Units) / Total Protein (mg)
= 1526.4 units/ 1200 mg
= 1.272 units/mg
Yield in Assay I:
= Total Units for Fraction II / Total Units for Fraction I x 100%
= 1526.4 Units / 1446.9 Units x 100%
= 105.5 %
Fold Purification in Assay I:
= Specific Activity for Fraction II/ Specific Activity for Fraction I
= 1.272 / 0.774
= 1.64
DISCUSSION
The protein aldolase was isolated and purified from rabbit muscle via various
isolation techniques. These techniques select in favor of aldolase against other proteins
based on the chemical and physical properties of aldolase. The first technique utilized the
osmotic lysis using 0.03 M KOH, 0.001 M EDTA solution. After centrifugation, the
supernatant was passed through glass wool to remove lipids. The cold temperature and
EDTA stabilize the protein such that EDTA inhibits proteases and cold temperatures
protect protein from folding disruption. Next technique utilizes the hydrophobic
properties of aldolase to bind each other and precipitate as ammonium sulfate was added
to compete for water molecules in alsolase solution. This salt fractionation was utilized in
2 steps. The proteins that precipitated at 50% ammonium sulfate saturation were removed
first. Then, ammonium sulfate saturation increased to 60% so that the proteins that salted
out at between 50% and 60% ammonium sulfate saturation would be present in the
precipitate. The physical characteristic of the aldolase was also utilized through dialysis
technique in order to select against the proteins that are smaller than MW of 14,000 Da.
Dialysis is a very efficient method to concentrate a protein since it de-salts the leftover
aldolase and concentrates the certain protein by diffusing any other proteins that are
smaller in size. Finally, chemical characteristic was utilized during ion exchange
chromatography using a column containing phosphoceullulose resin. Since aldolase is a
positive charge at pH 7.5, its Arg-148 residue binds to the phosphate of resin thus
selected against any other protein that are incapable of binding. Later, aldolase was eluted
from the column using elution buffer that contains FBP, the main substrate for aldolase.
The FBP competes with phosphate for binding of aldolase, thus releases aldolase from
the column.
After each subsequent step of isolation procedures, the amount of total protein,
total aldolase activity, and specific activity tend to decrease. The total amount of protein
decreased from 1868.3mg in Fraction I to 77.21 mg in Fraction VIA and 30 mg in
Fraction VIB. This is because the isolation procedures carried out through the experiment
positively selects aldolase in which the total amount of proteins should decrease since the
proteins that are not physically or chemically selected are excluded. Similarly, the total
aldolase activity decreased throughout the experiment from 1446.9 units of activity in
Fraction I to 59.136 units in Fraction VIA and 7.392 units in Fraction VIB. This also
follows the trend in that Fraction I should exhibit the highest total enzyme activity since
it contains all the aldolase yet to be excluded via series of isolation techniques, and
should decrease with each step. Finally, specific activity in units per milligram of protein
also exhibited an overall decrease from 0.774 units/mg in Fraction I to 0.766 units/mg in
Fraction VIA and 0.246 units/mg in Fraction VIB. The experimental value of specific
activity fails to show an overall trend but fluctuate throughout the different fractions.
However, the absence of specific activity in the “wash” represents that aldolase were not
lost during the column chromatography. Also, the high rate of specific activity in the
“peak” represents that aldolase has a higher fraction of the total. Thus, these are good
indicators that the purification was successful. While each of these three trends was
mostly clear, Fraction II consistently didn’t follow this trend in that it increases its total
units of activity to 1526.4 and its specific activity to 1.272. This erroneous data are likely
to be resulted during the process to measure the ΔABS/min. Since the reagents added into
the cuvette were very small in volume, a slight excess, or deficit of one reagent could
have resulted in this erroneous absorbance. Also, it may due to the removal of the
proteins that used to inhibit the function of aldolase. To further determine the exact cause,
further examination for the presence of aldolase inhibitor in rabbit muscle should be
studied.
Selection of “peak” among collection of forty fractions after affinity
chromatography was the most effective purification method in terms of increasing
specific activity. The measured specific activity of 0.774 units/mg of Fraction I increased
by 12-folds during this procedure. The greatest loss of total unit of activity was observed
during the fractionation method via ammonium sulfate. Fraction III, which is the
supernatant isolated from Fraction II by centrifugation, exhibited 305.424 units of total
enzyme activity, which is 84.4% loss of activity compared to the previous fraction, and
78.9% loss of activity compared to Fraction I.
The “wash” of column by equilibration buffer was crucial for the aldolase
isolation. This step is important for the proteins that are not capable of binding to
phosphate within phospho-cellulose resin get washed away leaving the proteins that stay
bound to the resin, such as aldolase. The column was washed until the absorbance of
effluent at 280nm dropped below 0.1 Abs. Since most proteins contain amino acid tyr, trp
and phe which absorb at 280nm, the absorbance value at 280nm below 0.1 is a good
indicator that most proteins are eluted.
To measure the amount of protein contamination at the end of the isolation, the
TPI acticity assay was utilized. The Triose Phosphate Isomerase (TPI) possesses different
characteristic as aldolase and utilizes Ga3p as its substrate. Therefore, to observe the
activity of TPI, the its main reaction, which converts Ga3P into DHAP, was coupled with
–Glycerophosphate dehydrogenase reaction which utilizes DHAP and NADH as its
main substrate and the disappearance of NADH was observed under 340nm. Looking at
Figure 3, it is clear that very high amount of TPI activities were present on Fraction I
through III. After purification via column chromatography, however, the specific activity
of TPI diminished in Fraction VI whereas relatively high activity were detected in the
“wash” suggesting that TPI does not bind to phospho-cellulose resin at pH 7.5 thus most
of TPI are washed away.
The aldolase purification experiment done by Penhoet and Rutter had the aldolase
yield of 67% after ammonium sulfate fractionation and 64% after Phopho-cellulose
chromatography. Our percentage yield for aldoase after salt fractionation, which
corresponds to Fraction III, was 21.1% and after column chromatography, Fraction VIA,
was 4.1%. The fact that our percentage yields were much less means that our methods to
measure the aldolase activity were erroneous and unskilled. The possible reason of less
yield of aldolase is that our column was dried out so that it didn’t bind to alsolase tightly,
or the flow rate may be too high or too low. Also, it is crucial to keep pH at right level
during the experiment since acidic environment can denature aldolase resulting in losing
the activity. Also, pH could change the side chain of aldolase, so that it is unable to bind
the phosphocellulose resin. Since throughout the experiment, the pH level was kept
around 7.5 and significant fluctuation was prevented via use of buffer, the considerable
denaturation of aldolase may not have occurred.
There were few steps that may need to be modified for more efficient isolation in
the future. As state earlier, the major loss of total units of activity occured during the
precipitation method by ammonium sulfate. The significant aldolase activity within
Fraction III indicates the fact that significant amount of aldolase failed to precipitate even
in 50% and 60% saturated ammonium sulfate solution. Therefore for future experiment,
the saturation level may be elevated further, and the period of precipitation should be
lengthened. A longer column could also be used in order to achieve better isolation of
aldolase. The possible error may have occurred during the lab is getting the ΔABS/min
value for any fraction came out to be 0. This is perhaps due to the concentration of
aldolase being too low in that lead to spectrophotometer not correctly measuring the
aldolase activity.
REFERENCE
1) Penhoet, E.E. and Rutter. W.J. (1975) Methods in Enzymol. 42: 240.
2) Chappel, A, Hoogenraad, N.J. and Holmes, R.S. (1978) Biochem. J. 175: 377382.