The Purification of Aldolase
KyoungMin Kevin Park
Partner: Yuqi Wang
Due: March 26, 2019
Submitted: March 26, 2019
Objective:
The goal for this experiment is to purify, isolate, and figure out the specific activity of
rabbit muscle aldolase through various methods such as extraction, ammonium sulfate
fractionation, dialysis, affinity chromatography, and activity assays.
Materials and Methods:
All steps of the experiment were proceeded under the condition at 4 °C and aliquots were
stored at -20 °C.
Extraction:
Ground rabbit muscle was extracted through suspending 80.1g of rabbit muscle in 150 ml
of 0.03M KOH, 0.001 M EDTA solution while continuously mixing for 20 minutes. Then, the
solution was centrifuged for 10 minutes at 10,000 x g. The supernatant was filtered by glass
wool. The pH of the solution was set up to 7.5 with 0.1 N KOH and then 1 ml aliquot was
replaced separately for the future procedure (Fraction I).
Salt Fractionation:
The supernatant, fraction I, was set up to a 50% saturation of (NH4)2SO4 through adding
an equal volume of (NH4)2SO4 that 100% saturated. The resulting solution was slowly stirred on
ice for 1 hour, then centrifuged for 30 minutes at 10,000 x g. The supernatant was filtered by
glass wool, then a 1ml aliquot was replaced separately (Fraction II). This solution was set up to a
60% saturation of (NH4)2SO4 through adding 65g of solid (NH4)2SO4 per liter of solution that
present. The pH of the solution was set up to 7.5 by 1 N NH4OH. For the continuous experiment,
this solution was saved in a cold room for a week.
Dialysis:
By centrifugation for 30 minutes at 10,000 x g, the precipitate from the above solution
was isolated. Then the pH of the supernatant was recorded and the volume was measured. A 1 ml
aliquot was replaced separately from the supernatant (Fraction III). The remained pellet from the
centrifugation was suspended in 2.5 ml of equilibration buffer (10mM Tris HCL, pH 7.5; 5mM
EDTA). This solution was centrifuged for 15 minutes at 10,000 x g and a 0.1 ml aliquot was
replaced separately from the supernatant, then diluted to 1ml with equilibration buffer (Fraction
IV). A dialysis tubing (MWCO 14,000 Da) was utilized for dialysis of the supernatant against
equilibration buffer at 4°C.
Affinity Column:
About 20 ml of phosphocellulose, the 50/50 slurry in equilibration buffer, was utilized to
isolate aldolase in batch mode. The dialyzed fraction was centrifuged for 15 minutes at 10,000 x
g, and a 0.1 ml aliquot was replaced separately and diluted to 1 ml with equilibration buffer
(Fraction V). Phosphocellulose resin was centrifuged for 5 minutes at 3,000 x g. The outcome
supernatant was replaced separately and then fraction V was added to the resin. This was
prepared to equilibrate for 5 minutes and then was centrifuged for 5 minutes at 3,000 x g. The
supernatant was replaced and the absorbance at 280 nm (A280) was measured by utilizing the
Genesys-5 Spectrophotometer. These steps were continued until the absorbance at 280 nm was
set up to below 0.51. A 1 ml aliquot was replaced from the supernatant and micro-fuged at
maximum velocity for 1 minute (Wash Fraction). The volume of wash fraction was measured as
66ml, and 1 ml of the sample was saved for the future procedure.
Aldolase was eluted from the resin by adding 100 ml of elution buffer (2.5 mM FBP;
50mM Tris, pH 7.5). The resin was suspended, then prepared to equilibrate for 5 minutes. The
outcome was then centrifuged at 3,000 x g for 5 minutes and then the supernatant was gathered.
Bound proteins were eluted with the 1ml/min flow rate, and 40 fractions (1ml for each) were
gathered. A 1 ml aliquot was replaced separately (Fraction VIA) and micro-fuged for 1 minute at
maximum velocity. A 0.1 ml portion from the outcome was then diluted to 1 ml (Highest peak of
VIA). This step was repeated while adding another elution buffer, and centrifuging that
producing Fraction VIB (15ml). Then, the 0.1 aliquots from each Fraction VI were diluted to 1
ml with the equilibration buffer and left for future analysis.
Protein Determination:
A protein standard curve was formed while 1-25 μg of six solutions of bovine serum
albumin and a color reagent (0.0032%(w/v) of Coomassie Brilliant Blue G250, 4.7% of ethanol,
and 8.5% of phosphoric acid were contained) were utilized. Each fraction was properly diluted
and combined with 0.15M NaCl and the color reagent to create the final volume as 1 ml. The
absorbance of the colored solution was set up to 595 nm. This result was used to compare to the
protein standard curve to figure out the concentration of the protein in individual samples.
Assay II for Aldolase Activity.
A 30 ml Master Mix consisting of 86.7mM pH 7.2Histidine HCl buffer, 0.13mM NADH
in His HCl buffer pH 7.2, 6.7mM fructose 1,6-bisphosphate (FBP), 4-5IU/ml αglycerophosphate (α-GP) dehydrogenase was prepared for ten reactions to test aldolase activity;
then, the master mix was shared into 9 cuvettes. Furthermore, 10μl of each fraction were added
into nine cuvettes, and reaction rates of each protein fraction were read at 340nm.
Assay II for Triose Phosphate Isomerase Activity.
A 30ml Master Mix (86.7mM pH7.2 His HCl buffer, 0.13Mm NADH in His HCl buffer
pH7.2, 0.167Mm glyceraldehyde 3-phosphate, and 4-5UI/ml α-GP dehydrogenase) was made to
test the triose phosphate isomerase activity in each protein fraction; the master mix was split into
9 cuvettes, and the reaction rate of each fraction was measured at 340nm after reacting 10μl
protein fraction with the master mix in cuvette.
Results:
Extraction:
To extract muscle aldolase from the rabbit muscle, the osmotic lysis was proceeded. The
supernatant (Fraction I) had pinkish color, and the pH was set up from 7.23 to 7.5 by using 0.1N
KOH. The protein concentration of the Fraction I was recorded as 15mg/ml.
Salt Fractionation:
Ammonium sulfate ((NH4)2SO4) was added into Fraction I to get 50% saturation to
remove the untargeted proteins. The Fraction II’s color was paler than color of Fraction I, and its
pH was set up from 7.3 to 7.5. The recorded concentration of the Fraction II was 5.94mg/ml.
Approximately 20.475g of ammonium sulfate was added to Fraction II to set up 60% saturation
and then was centrifuged. The protein concentration of supernatant (Fraction III) that have
impurities from Fraction II was 3.2932 mg/ml. Then, after dissolving gathered precipitate from
Fraction II and centrifugation, Fraction IV was seemed have no color and its protein
concentration was 130 mg/ml.
Dialysis:
After the Dialysis procedure, the Fraction V was gathered by centrifugation and its
protein concentration was 150 mg/ml.
Affinity Column:
Phosphocellulose resin was utilized by cation exchange chromatography. The wash
sample was gathered first and its protein concentration was recorded as 2.0526mg/ml. In
continue, the aldolase was eluted and gathered in 40 fractions that have 1ml per each fraction.
The fractions displayed diverse absorbance at 280 nm (Figure 1).
Absorbance of Eluted solution at 280nm
3,5
3
Absorbance
2,5
2
1,5
1
0,5
0
0
5
10
15
20
25
30
35
40
Fraction Number
Figure 1) The absorbance of eluted protein at 280nm. The elution was gathered by
phosphocellulose resin at 1ml/min rate flow.
For the 40 fractions, the slope was slowly increased until fraction 10, and it rapidly
increased from 10 to 13 fraction. According to the chart, fraction 13 is the peak of the
absorbance which had 4.85225mg/ml for protein concentration (Figure 1). Moreover, the fraction
VIA had the elution from 11 to 21, which has 2.7875mg/ml for protein concentration. In
addition, the absorbance values of the other fractions tended to decrease. The Fraction VIB that
represent the tail of the peak had 0.5181mg/ml for the protein concentration.
Protein Determination
The Bradford Method was utilized to figure out the protein concentration in each
Fraction. The standard curve was created by bovine serum albumin (BSA) with Brad reagent
before recording the absorbance and concentration of each Fraction (Figure 2). After adding
BSA, the color of the Bradford reagent changed to blue.
Figure 2) Bovine Serum Albumin (BSA) Standard Curve. Diverse amount of BSA (25ug/ml)
that ranged 1-25ug, was combined with adequate amounts of color reagent (combination of
0.032%(w/v) Coomassie Brilliant Blue G250, 4.7% ethanol, and 8.5% phosphoric acid) and
0.15M NaCl. The absorbance of the final solution was set up to 595nm and measured by
Genesys-5 Spectrophotometer.
The Figure 2 above displays an increase in concentration of BSA outcomes that proportional to
the increase in absorbance. The Table 1 contains more detail information below.
Table 1. Protein determination using the Bradford Method
Name of Fraction
Final Dilution
Bradford Absorbance
Protein Concentration (μg/ml)
I
1/4000
0.160
3.7521
II
1/2000
0.134
2.9735
III
1/400
0.298
8.2330
IV
1/5000
0.666
26
V
1/2000
0.278
7.5416
Wash
1/200
0.354
10.263
The Peak
1/500
0.339
9.7045
VIA
1/500
0.218
5.5575
Assay II for Aldolase Activity:
In order to figure out the aldolase activity and specific activity of each fraction, Assay II
for aldolase activity was proceeded. Each fraction contained different reaction rate as displayed
in Table 2. This implies that the reaction of the aldolase in Fraction IV was the highest one
among nine fractions, and there was no aldolase activity detected in Wash Fraction.
Table 2. Absorbance values of each fraction in Assay II for aldolase activity
Name of Fraction
Rate of Aldolase (Abs/min)
Velocity of Aldolase (μmol/min)
I
0.797
0.383
II
0.293
0.141
III
0.053
0.025
IV
1.194
0.573
V
0.213
0.102
Wash
0
0
The Peak
0.135
0.0648
VIA
0.055
0.026
VIB
0.009
0.00432
After combining the data from Table 1 and 2, the protein concentration of each fraction
was figured out as displayed in Table 3. Furthermore, the specific activity of aldolase in nine
fractions were measured based on the total aldolase activities and the protein amount.
Table 3. Data of Enzyme Purification for aldolase
Fraction
Volume
Activity
Total
Protein
Total
Specific
Yield
Fold
Name
(ml)
(Units/ml)
Units
(mg/ml)
Protein(mg)
Activity
(%)
Purification
(units/mg)
I
168
38.3
6434.4
15
2520
2.553
100
1
II
315
14.1
4441.5
5.94
1872
2.3725
69.03
0.93
III
325
2.5
812.5
3.2932
1070.4
0.759
12.63
0.297
IV
6.4
573
3667.2
130
832
4.4077
0.57
1.726
V
15.9
102
1621.8
15.0832
239.822
6.7625
25.2
2.649
Wash
66
0
0
2.0526
135.4716
0
0
0
The Peak
1
64.8
64.8
4.85225
4.85225
13.355
1.007
5.231
VIA
14
26
364
2.7875
39.025
9.327
5.66
3.653
VIB
15
4.32
64.8
0.5181
7.7715
8.338
1.007
3.266
The yield percentage represents the percentage of total units of any Fraction compared to
percentage of Fraction I. Also, the Fold Purification represents the percentage of the specific
activity of any fraction compared to the percentage of Fraction I.
From the Table 3, the yield percentage from fraction I to fraction III, IV, and V were
noticeably decreased, and the activity of aldolase decreased with subsequent procedures except
Fraction III and Wash. During the purification step, total activity had similar trend as the activity
of aldolase. Moreover, total amount of protein tended to decrease as the extraction and
purification procedure goes on. In addition, during the extraction and purification step, the
specific activity of aldolase tended to increase except for the Fraction III and Wash that
contained impurities.
Assay II for Triose Phosphate Isomerase Activity
Assay II utilizing the TPI as the catalyst in main reaction was proceeded to figure out the
contamination of each fraction through Triose Phosphate Isomerase (TPI). The rate of TPI and
the reaction rate of each fraction were recorded in the Table 4 below. Based on the information
of Table 1 and 4, TPI activity, specific activity, and protein concentration were recorded and
explained in the Table 5 below.
Table 4. Absorbance values of each fraction in Assay II for triose phosphate isomerase
activity.
Name of Fraction
Rate of Aldolase (Abs/min)
Velocity of Aldolase (μmol/min)
I
0.144
0.06912
II
0.072
0.03456
III
0.1
0.048
IV
0.051
0.02448
V
0.013
0.00624
Wash
0.027
0.01296
The Peak
0.004
0.00192
VIA
0
0
VIB
0.001
0.00048
Table 5. Data of Enzyme Purification for triose phosphate isomerase
Fraction
Volume
Activity
Total
Protein
Total
Specific
Yield
Fold
Name
(ml)
(Units/ml)
Units
(mg/ml)
Protein(mg)
Activity
(%)
Purification
(units/mg)
I
168
6.912
1161.216
15
2520
0.4608
100
1
II
315
3.456
1088.64
5.94
1872
0.5815
93.75
1.26
III
325
4.8
1560
3.2932
1070.4
1.4573
134.3
3.16
IV
6.4
24.48
156.672
130
832
0.1883
13.5
0.409
V
15.9
6.24
99.216
15.0832
239.822
0.4137
8.54
0.898
Wash
66
1.296
85.536
2.0526
135.4716
0.6314
7.37
1.37
The Peak
1
1.92
1.92
4.85225
4.85225
0.396
0.165
0.859
VIA
14
0
0
2.7875
39.025
0
0
0
Fraction
15
0.48
7.2
0.5181
7.7715
0.926
0.62
2.01
VIB
The yield percentage represents the percentage of total units of any Fraction compared to
percentage of Fraction I. Also, the Fold Purification represents the percentage of the specific
activity of any fraction compared to the percentage of Fraction I.
Based on the information of the Table 5 above, the total activity of TPI decreased with
subsequent procedures of extraction and the purification procedure except the Fraction VIB. The
specific activity of TPI increased until the dialysis procedure that indicated that the TPI in both
Fraction IV and V displayed the lower specific activity than others. During the purification
procedure, the Fraction Wash had the higher specific activity than Fraction V. Since there was no
TPI in the Fraction VIA, the Fraction VIB had larger specific activity than Fraction VIA in the
purification procedure.
The Figure 3 below summarized the information of specific activities of aldolase and
triose phosphate isomerase from Table 3 and 5.
Specific Activites vs. TPI
16
Aldolase
Triose Phosphate Isomerase
Specific Activity
14
12
10
8
6
4
2
0
I
II
III
IV
V
Fraction Name
Wash
Peak
VIA
VIB
Figure 3) Specific Activities of Aldolase and TPI. The activity of each individual enzyme was
determined for each fraction from the aldolase and TPI assays. The protein content was also
determined by utilizing the Bradford Method. Figure 3 indicates that the specific aldolase
activities were relatively higher than that in the TPI except the fraction III.
Sample Calculation:
1. Concentration of protein (mg/ml) = (mg/ml of protein in tube) x (Dilution Factor)
Total protein (mg) = concentration of protein (mg/ml) x (volume of total fraction)
2. Absorbance (340nm) /min = ε x c x l /t
Therefore, Absorbance (340nm) /min x volume / ε x l = U (μmol/min)
ε = molar extinction coefficient (6.2 ml/ (μmol x cm) for NADH at 340nm), l is the path
length (usually 1cm), and c is the solution’s concentration.
Tube Reaction Velocity (μmol/min) = Absorbance (340nm) / min x 0.48 μmol
U/ (volume of sample) x dilution factor = Units/ml
Units/ml x volume of total fraction = Total Units of Activity
3. Specific Activity (Units/mg) = Total Units of Activity/ Total Protein
4. Yield Percentage = Total Units for certain Fraction / Total Units of Fraction I
5. Fold Purification = Specific Activity for certain Fraction / Specific Activity for Fraction I
Discussion:
Throughout the experiment, total four procedures were proceeded to extract and purify
the muscle aldolase from the rabbit muscle, which were extraction/solubilization, stabilization,
concentration/isolation, and the specific assays. For the extraction procedure, 0.03M KOH and
0.001M EDTA were utilized to form a hypotonic condition in osmotic lysis for rabbit muscle.
After the extraction of proteins from muscle cells, the proteins that extracted were stabilized on
ice at 0-4℃ and set up with EDTA. For the enzymes, the low temperature from ice
facilitated to keep enzymes’ activity, and EDTA inhibited proteases that lysed with selected
proteins. Since most of the protease requires metal ions, EDTA were utilized since it tends
to inhibit metalloproteins by chelating metal ions.
After the extraction procedure, the 50-60% saturated (NH4)2SO4 solution was
utilized to precipitate the aldolase. Since the salt tend to compete water with proteins in
the solution, proteins have to be precipitated if there are dissolved salts in water. In
addition, the dialysis procedure was performed since the pores in dialysis membrane only
allowed substances to penetrate. The size of the substances had to be smaller than 14,000
Da (Dalton). Through this procedure, the aldolase and substances that larger than 14,000
Da were filtered by the dialysis bag. In continue, the chromatography was utilized to
concentrate aldolase after the dialysis procedure. The phosphate groups in
phosphocellulose resin bound to arg-148 of aldolase and equilibration buffer was utilized
to washed away the untargeted proteins. Then, the equilibration buffer that consist of FBP,
the natural substrate of aldolase, was utilized to elute the aforementioned bounded
proteins.
After the concentration procedure, Specific assay (Assay II) were utilized to
measure the aldolase and triose phosphate isomerase (TPI) activity for each enzyme. The
rate of disappearance of NADH in coupled reaction was utilized to measure the reaction
rates of two enzymes that tested in experiment at 340 nm. In this procedure,
dihydroxyacetone reaction with NADH was performed to from α-glycerophosphate (α-GP)
with NAD+ by catalyzing the α-GP dehydrogenase. The main reaction for two assays were
different since the aldolase convert fructose to glycerate 3-phospate (Ga3P) and
dihydroxyacetone phosphate (DHAP), and TPI converted Ga3P to DHAP reversibly that
could inhibit the aldolase activity. The main reason for testing TPI activity is because there
are the potentials for contamination through TPI. To determine the concentration of
protein, the Bradford Method was utilized after the Coomassie dye reaction between SO3and NH4+ of proteins while the absorbance of proteins reached to 595nm.
After the isolation step, the total proteins tended to decrease over time except the Fraction
VIA as recorded on the Table 3 above. 15ml of Fraction VIA should contain more proteins than
Fraction Peak since there was only 1ml aliquot for Fraction Peak. In continue, the total aldolase
activity mostly decreased except for Fraction III, Wash, and VIA (Table 3). The Fraction III
contained less aldolase than that in Fraction IV since it was the supernatant that gathered after
the salt precipitation. Also, the Fraction Wash had zero for the aldolase activity since it consisted
of unbounded proteins. The Fraction VIA had relatively higher than Fraction Peak total aldolase
activity since it contained eleven fractions in the dialysis process that represented the peak part.
For the specific aldolase activity, Fraction III and Wash had relatively higher value and Fraction
VIA and VIB had a little bit lower amount of it compare to Fraction Peak (Table 3). The amount
of aldolase activity in Fraction III is very low due to low specific activity. On the other hand, the
Fraction Peak had higher specific activity than Fraction VIA and VIB since the less total protein
and higher total activity were displayed in the Fraction Peak.
Since two assays used the same fraction in the experiment for the enzyme activity test,
the total protein change had the similar trend with that for aldolase which implies that the protein
concentration of each fraction must stay as constant. Except the Fraction VIB, the TPI activities
were tending to decrease with subsequent procedures. Throughout the isolation and purification
procedures, less TPI must display in the later fraction. The total TPI activity in Peak seemed to
be lower slightly due to less volume of Fraction Peak compare to Fraction VIB. Also, during the
salt precipitation, the specific activity of TPI tended to increase since there was more amount of
precipitation of aldolase in 60% saturated (NH4)2SO4 solution. This indicated that TPI was
remaining more in the supernatant for Fraction III. Also, its specific activity tended to decrease
due to more precipitation based on the abundant amount of aldolase were in the Fraction IV.
After the dialysis procedure, the dialysis bag was utilized to filter the proteins that larger than
14,000 Da. As the result of this procedure, the specific activity for TPI was slightly increased.
Throughout the Chromatography procedure, the unbounded proteins were droved out into the
Fraction Wash that lead TPI to be monitored better in Wash. Sine the Fraction Peak, VIA, and
VIB were mainly gathered aldolase throughout the chromatography procedure, the specific
activity of TPI tended to decrease in those fractions.
The most effective procedure in purification to increase the specific aldolase activity was
the chromatography procedure (Figure 3). Based on using the resin to choose aldolase from the
mixture, the large difference in specific activity between Fraction V and Peak was monitored.
Also, the specific TPI activity tended to higher in Fraction Wash than Fraction V. For these
reasons, TPI must be the hydrophilic protein that tend to remain soluble for high concentration.
The wash procedure facilitated TPI to elute since it can’t bind to the phosphate group.
To compare with the experiment from Penhoet and Rutter, they achieved about 71% yield
of aldolase after ammonium sulfate fractionation, and 69% yield of aldolase after
phosphocellulose affinity chromatography.2 On the other hand, our experiment achieved 69%
yield percentage of aldolase after ammonium sulfate fractionation, but only 25.2% of aldolase
after phosphocellulose affinity chromatography was yielded. Though the yield % of aldolase
after ammonium sulfate fractionation were similar between two experiments, there was a huge
gap in between two experiments for phosphocellulose affinity chromatography. The most
predictable reason for this gap is due to the usage of batch mode in this experiment, since the
washes were gone through mixing resin and centrifugation procedures. This might lead to loss of
aldolase throughout the process that result the lower yield percentage in our experiment. In
addition, only two elution went through the resin that indicates some aldolase to stay bounded to
the resin due to the batch mode. Though this alternative method couldn’t bring the close result to
the experiment from Penhoet and Rutter, it still proved that the batch method is able to achieve
the aldolase yield %.
In this experiment, the improvement could be proceeded for reducing the gap among the
fractions for the aldolase activity. The salt might be poured too quickly that lead to premature
precipitation of aldolase in fractions.
To sum up, the experiment performed successfully. The individual subsequent
purification procedure resulted the higher purity of aldolase as shown on the Table 2 and 3
above. The only problem that encountered during the experiment was the relatively lower
aldolase yield percentage compare to the result from Penhoet and Rutter, but it is still not a bad
reason consider for utilizing other method.
References
1. Bradford, M.M., Analytical Biochemistry, 72: 248-254 (1976).
2. Penhoet, E.E. and Rutter, W.J., Methods in Enzymology, 42: 240-249 (1975)