Isolation and Purification of Rabbit Muscle Aldolase

Name: Courtney Horan

Partner’s Name: Allie Socher

3/28/18
Objective

The objective of this lab was to extract, isolate, and purify aldolase from rabbit

muscle in order to then determine its enzyme activity.

Methods

All of the procedures were performed at 4°C and sample aliquots from each fraction

were stored at 20°C for future analysis.

Extraction

Ground rabbit muscle (80g) was hand stirred for 20 minutes with 150 ml of cold

0.03 M KOH and 0.001 M EDTA. The mixture was then centrifuged at 10,000 x g for

10 minutes and then the supernatant solution was decanted. The supernatant was

then filtered through glass wool to remove lipids and the pH of the filtrate was

adjusted to 7.5 with cold 0.1 N KOH. After the volume of the solution was recorded, a

1 ml aliquot of this first solution, Fraction I, was removed for later analysis.

Salt fractionation

An equal volume of 100% saturated ammonium sulfate was then slowly added to

Fraction I to yield a 50% saturated ammonium sulfate solution. After being slowly

stirred for an hour, Fraction I was centrifuged at 10,000 x g for 30 minutes. Its

supernatant was then filtered through glass wool and the volume of this solution,

Fraction II, was recorded. Again, a 1ml aliquot of Fraction II was removed for later

analysis. Ammonium sulfate (65g per liter of Fraction II) was gradually added with
slow magnetic stirring to bring the solution to 60% ammonium sulfate saturation.

The pH of this 60% ammonium sulfate saturated solution was then recorded and

adjusted with 1.0 N H2SO4 with 15 drops to reach a pH of 7.5.

Dialysis

Fraction II was centrifuged at 10,000 x g for 30 minutes and then the supernatant,

Fraction III, was removed to have its volume recorded as well as pH. A 1 ml aliquot

of Fraction III was saved for later analysis. The precipitate from the centrifugation of

Fraction II was dissolved in as little cold equilibrium buffer (10 mM Tris HCl, pH 7.5;

5mM EDTA) as possible. It was then centrifuged at 10,000 x g for 15 minutes. The

volume of the supernatant, Fraction IV, was recorded and a 0.1 ml aliquot diluted

with equilibration buffer to 1 ml was saved for later analysis. Dialysis tubing

(MWCO 14,000Da) that was already soaked in distilled water was filled with

Fraction IV and was dialyzed in a cold room against equilibration buffer.

Affinity purification using phosphocellulose resin

The column (1.5 cm x 11 cm, 20 ml total volume) was set up by packing the

phosphocellulose resin and then equilibrating it using equilibration buffer. After

letting 100 ml of equilibration buffer and obtaining a 1 ml/min effluent flow rate,

Fraction V was added to the column and washed with equilibrium buffer ((2.5 mM

FBP [B-grade]: 50 mM Tris  HCl, pH 7.5; 5 mM EDTA) until the effluent absorbance

at 280 nm fell below 0.1. The liquid following this change in absorbance was labeled

the “wash” which the volume was recorded and a 1 ml aliquot was saved for future
analysis. After the “wash” was eluted, 100ml of substrate elution buffer was used to

elute the aldolase. There were 50 1 ml fractions were collected and assayed by

reading A280. A 0.3 ml aliquot was removed from the fraction tube with the highest

absorbance reading, the “peak” Fraction. Another 0.1 ml aliquot was removed and

diluted to 1 ml with equilibration buffer. Both fractions were saved for future

analysis. Then all of the fractions that were part of the peak were pooled together

and called Fraction VIA. The same was done to the less concentrated fractions that

were part of the “tail” end of the peak and this was called Fraction VIB. The volumes

of both VIA and VIB were recorded and 0.1 ml aliquots from each were diluted to 1

ml with equilibration buffer for later analysis.

Protein determination

A standard curve was created using 7 increments of bovine serum albumin from 1

to 25 μg. 0.15 M NaCl was added to adjust each final volume to 1 ml. The saved

fraction aliquots were each diluted with 0.15 M NaCl to a final volume of 1 ml and 1

ml of Coomasie Brilliant Blue was added. The absorbances were read at 595 nm

after 5 minutes and concentration was recorded.

Aldolase assay

A reaction mixture with a final volume of 3 ml was used for every saved fraction

aliquot to determine absorbance rate when FBP is added. Each reaction mixture

contained dH2O, 87 mM HistidineHCl, 0.13 mM NADH, 6.7 mM FBP, and ~4-5 IU of

αGP (glycerol phosphate) dehydrogenase. To each sample 10 μl of each fraction was

added and then the absorbance was recorded at 340 nm.

Triose phosphate isomerase (TPI) assay

A reaction mixture with a final volume of 3 ml was used for every saved fraction

aliquot to determine absorbance rate when Ga3P is added. Each reaction mixture

contained dH2O, 87 mM HistidineHCl, 0.13 mM NADH, 0.17 M Ga3P, and ~4-5 IU of

αGP dehydrogenase. Each sample also got 10 μl of each fraction and the absorbance

was recorded at 340 nm.

Results

Extraction

After being mixed with 0.03 M KOH and 0.001 M EDTA the resulting supernatant

contained the extracted proteins including aldolase. Fraction I was collected and the

final volume of Fraction I was 159 ml and had a concentration of 28.004 mg/ml.

Salt Fractionation

The following calculation was used to determine the amount of solid ammonium

sulfate to bring Fraction II to a 60% ammonium sulfate saturation:

(310 𝑚𝐿)(0.065𝑔/𝑚𝐿) = 20.15𝑔

The final volume of Fraction II was 311 ml and had a concentration of 1.60134

mg/ml.
Dialysis

Fractions III and IV were collected while preparing for dialysis. The final volume of

Fraction III was 320 ml and the pH was 7.28. The concentration of Fraction III was

6.441 mg/ml. The final volume of Fraction IV was 5.4 ml and the concentration was

72.106 mg/ml.

Affinity purification using phosphocellulose resin

Fraction V, “wash”, “peak”, VIA, and VIA were all collected during the column

chromatography process. The final volume of Fraction V was 9 ml and the

concentration of the saved aliquot was 0.1 M. The final volume of the “wash” was 14

m and the concentration was 0.26536 mg/ml. The final volume of the “peak” was 2

m and the concentration was 14.618 mg/ml. The final volume of both the VIA and

VIB fractions was 10 ml and the concentrations were 8.464 mg/ml and 1.1438

mg/ml respectively.

Protein determination

The Bradford Method was used to determine protein concentration. In order to use

this method a standard curve had to be created. Each fraction then compared its

absorbance to the standard curve to determine protein concentration. Figure 1

shows an increasing absorbance with increasing protein concentration for the

standard curve. This means that absorbance and protein concentration have a

positive correlation.
Figure 1: Standard Curve for Bradford Method

BSA Standard Curve

0.6
y = 0.0219x - 0.054
Absorbance rate (ABS/min)

0.5

0.4

0.3

0.2

0.1

0
0 5 10 15 20 25 30
-0.1
Concentration ( mol/L)

Figure 1. Samples of Bradford Reagent were prepared with known protein

concentration ranging from 1-25 μg and Coomasie Brilliant Blue to have their
absorbance’s read and used to create a standard curve.

The protein content seemed to increase from Fractions I to IV, which made sense

since during the processes that they were collected, the solution was supposed to be

isolated and purified from any other components. The “wash” was found to have the

lowest protein content because it was supposed to be the portion before the

aldolase was eluted out of the column. The “peak” was found to have one of the

highest protein contents because it contained the fraction with the highest

absorbance of aldolase that was eluted out of the column.

Table 2: Protein Content Via Bradford Method

Fraction Final Dilution Bradford Absorbance Protein Concentration

I 4000 0.153 7.001
II 2000 0.175 8.0067
III 400 0.352 16.105
IV 2000 0.788 36.053
V 2000 0.508 23.242
Wash 200 0.029 1.3268
Peak 500 0.639 29.236
VIA 500 0.370 16.928
VIB 200 0.125 5.7190

Aldolase and Triose phosphate isomerase (TPI) assays

Similar assays were set up, one with FBP mirroring the normal aldolase reaction,

and the other with TPI. Absorbance rates were recorded for each reaction in each

assay and then used to calculate enzyme activity, total protein, and specific activity.

Generally both enzyme activity and specific activity both increased with the

fractions.

Table 2: Activity of Aldolase and TPI Assays

Fraction Aldolase Assay TPI Assay

Enzyme Activity Specific Activity Enzyme Activity Specific Activity
(Units) (Units/mg) (Units) (Units/mg)
I 305.28 0.0686 53.424 0.0120
II 5,150.16 10.3413 1,343.52 2.6978
III 1,013.76 0.4918 1,259.52 0.6110
IV 977.184 2.5096 95.904 0.2463
V 1,568.16 3.7484 86.4 0.2065
Wash Volume Was Not Recorded
Peak Volume Was Not Recorded
VIA 576 6.8053 9.6 0.1134
VIB 9.6 0.8393 4.8 0.4197
Activity seemed to relatively increase throughout the fractions although there were

some outliers including Fraction II. Reaction velocity did not seem to have a specific

pattern that was followed.

Table 3: Activity and Reaction Velocity of Aldolase and TPI Assays

Fraction Aldolase Assay TPI Assay

Activity Reaction Velocity Activity Reaction Velocity
(Units) (rate/activity) (rate/activity)
I 305.28 0.00013 53.424 0.00013
II 5,150.16 0.000067 1,343.52 0.000067
III 1,013.76 0.000065 1,259.52 0.000065
IV 977.184 0.00039 95.904 0.00039
V 1,568.16 0.00023 86.4 0.00023
Wash Volume Was Not Recorded
Peak Volume Was Not Recorded
VIA 576 0.00021 9.6 0.00021
VIB 9.6 0.00021 4.8 0.00021

Figure 2: Specific Activity of Aldolase Assays

Specific Activity of Assay II vs TPI

12
Specific Activity (Units/mg)

10

6
Assay II
4 TPI
2

0
I II III IV V Wash Peak VIA VIB
Fraction
 Figure 2. Using both the results from both the Bradford Method and the

Aldolase assays, total enzyme activity and total protein were able to be calculated.

These values were then used to calculate specific activity for each fraction.

Sample Calculations

Protein Concentration (mg/ml) = mg protein in tube x dilution factor

Total Protein (mg) = protein concentration (mg/ml) x total fraction volume

Total Enzyme Activity (Units) = (ΔABS/min x 0.48 x dilution factor x total fraction

volume (ml))/aliquot volume (ml)

Specific Activity (Units/mg) = total enzyme activity/total protein

Discussion

First the aldolase had to be extracted from the rabbit muscle cells. To do this KOH

was used to lyse the cells to release the enzyme. Ammonium sulfate was then used

to decrease the water-protein interactions, which allowed for further purifying by

allowing the aldolase to precipitate out. To then get rid of any remaining salt, a

dialysis tubing of MWCO 14,000Da was used. This was because aldolase molecules

are bigger and therefore the tubing would allow any smaller molecules to be

separated out of the solution. During column chromatography, the aldolase was

bound to the phosphocellulose resin. The equilibrium buffer did not contain any

substrates of aldolase and therefore only washed out all other proteins. The elution

buffer was able to elute the aldolase because it contained FBP in which the aldolase

had a higher affinity to than the resin.

As a general trend, the total amount of protein decreased with each fraction. This

makes sense because the aldolase is supposed to be more purified in each fraction

meaning there should be less background proteins. Fraction III has a higher amount

of protein that doesn’t fit the trend. This fraction was the supernatant collected after

salt fractionation. The aldolase was supposed to be in the precipitate, so the

supernatant was supposed to contain all of the other proteins that did not have

water-protein interactions and therefore were not precipitated out. The total

amount of protein for this fraction could have been higher because of the large

amount of proteins that were separated from the aldolase as well as the solution not

being uniformly mixed and the saved aliquot being more concentrated than the

whole volume.

The affinity chromatography was the most efficient step in increasing specific

activity. In the aldolase assay the VIA fraction had one of the highest specific

activities. This fraction contained the chromatography fractions that surrounded the

peak fraction indicating that this specific activity was due to the affinity

chromatography. Fractions I and III have the lowest specific activity and the wash

should also be expected to have a low specific activity. Both the wash and Fraction

III are expected to contain proteins that were separated from the aldolase so it

makes sense that they would have a low activity in a reaction for aldolase.
The wash step/wash fraction should contain all background proteins that did not

bind to the phosphocellulose resin. The lack of aldolase would cause the specific

activity to be low in the aldolase assay while the presence of many other proteins

would cause the specific activity to be higher in the TPI assay.

In the TPI assay Fraction III also had a relatively high specific activity. This could be

for the same reasoning as in why the wash fraction should have a higher specific

activity. The VIA fraction had one of the lowest specific activities, which makes

sense because it is supposed to be the most purified. The same goes for the peak

fraction.

When comparing this experiment to that of Penhoet and Rutter, they must have

used much larger amounts of aldolase since all of their total protein values are much

larger. Although those values are larger, it seemed as though the specific activities

were smaller. The increase from Fraction I to Fraction II was much larger in this

experiment that that of Penoet and Rutter. This also could have been because of a

calculation error seeing that the values from Fraction II are abnormally high. The

experiments were similar in finding that the phosphocellulose resin column created

the highest specific activity

Purification could have been increased by repeating any of the processes to ensure

that each step was completely successful. There was also an issue with not all of the
total fraction volumes being recorded, which then caused a problem with calculating

enzyme activity, total protein, and specific activity.

References

1. Penhoet, E., & Rutter, W. Detection and isolation of mammalian fructose-

diphosphate aldolases. Retrieved March 29, 2018, from https://www-sciencedirect-

com.ezaccess.libraries.psu.edu/science/article/pii/0076687975421218

2.