ONE STEP PURIFICATION AND ANALYSIS OF WHEAT GERM ACID PHOSPHATASE

USING GEL FILTRATION CHROMATOGRAPHY

Abstract:
A crude sample of the enzyme wheat germ acid phosphatase (hereby referred to as
WGAP) was purified using a one step purification with gel filtration chromatography. The
sample of WGAP was loaded onto a Sephadex G-75 column and was collected in 30 fractions.
These fractions were tested using a nitrocellulose assay to determine which fractions contained
enzyme specific activity (fractions 7-11). These fractions were then tested using an enzyme assay
to determine which fractions contained the most enzyme, and a protein assay to determine the
protein concentration. The protein and the enzyme assays were used to determine the specific
activity of the fractions with greatest amount of enzyme, fractions 9 and 10, which were 0.674
units/mg and 0.851 units/mg respectively. The specific activities of the fractions were then used
to determine fold purification achieved for WGAP by comparing the specific activities to the
specific activity of the crude (0.465 units/mg). The fold purification of fraction 9 is 1.45, and for
fraction 10 is 1.85. In order to achieve a higher fold purification a multistep purification
technique could be used in the future. In addition to the gel column a 12% SDS PAGE gel. The
SDS PAGE gel was used to determine the number of polypeptides present as well as the
molecular weights of these polypeptides. Bands that appeared darker or more prominent in the
purified fractions as compared to the crude were identified as possible candidates for WGAP;
this occurred for the band located at the 53.7 kDa position on the band

Results:
One-step Purification of WGAP with Gel Filtration Column Chromatography
A Sephadex G75-40 gel filtration column was used to purify a crude extract of wheat
germ acid phosphatase (WGAP). Thirty fractions were collected from the column each
containing 0.43 mL (28 drops) of liquid. Each of the thirty fractions was spotted onto
nitrocellulose paper and underwent a nitrocellulose assay to determine which fractions contained
the most enzyme specific activity. Of these thirty fractions, fractions 7-11 contained the most
enzyme specific activity, so these fractions were used for further analysis. Fractions 7-11 as well
as the previously made dilutions of 1:10 and 1:100 crude underwent an enzyme and a protein
assay. The results of the enzyme and protein assays were used to determine the each of the
fractions specific activity. Fractions 9 and 10 had the highest specific activities, 0.674 and 0.
851 units/mL, respectively (Table 1). The fold purification from the crude enzyme was 1.45 for
fraction 9, and 1.83 for fraction 10. The % yield of these fractions were 58% and 43%,
respectively. However, due to researcher error these fractions were not saved for the next week,
and were discarded. Instead, fractions from another lab group were utilized for SDS PAGE
analysis. The fractions with highest specific activity from this group were fractions 6 and 7,
0.847 and 0.971 units/mL, respectively. The fold purification achieved for that lab group was,
2.19 for fraction 6 and 2.51 for fraction 7.
SDS PAGE Gel
Fractions 6 and 7 from the previous week, along with our 1:10 crude and a BSA standard
were all run through a 12% SDS PAGE gel (Figure 1). The gel was stained with GelCode Blue.
The total length of the gel was 7.2 cm. The total length of all the bands were measured, and then
Rf values were calculated by dividing the distance traveled by the band by the total length of th
gel. These Rf values were then used to determine the molecular weights of the polypeptides

1
using a standard curve. There were 9 bands present in the crude sample of the enzyme, but there
were 11 bands present in both of the fractions. The number of bands indicates the number of
polypeptides present. The most prominent bands on the crude sample were the ones located at
29.5 kDa and 25.1 kDa. The most prominent bands on the both of the purified fractions was the
one located at 53.7 kDa. There were bands found in the purified samples that were not very close
to any bands on the crude sample. These bands had molecular weights of 67.0 kDa and 61.1 kDa.
There was also one band that proved to darker in the purified fractions than in the crude sample.
This band occurs around molecular weight 53.7 kDa. The darkening of this band in the fractions
could indicated better purification of that polypeptide, and could be a potential candidate for
WGAP.

Discussion:
Many different purification techniques can be used to purify a sample of enzyme further
than the the purity of its crude. In this experiment a one step purification process, using a gel
filtration chromatography was used. Gel filtration chromatography is used to separate molecules
on the basis of size. By using a Sephadex G75 a one step purification of wheat germ acid
phosphatase (WGAP) was achieved that yielded a higher purification than the original crude
enzyme. The Sephadex G75 column is used to separate molecules that are less than 80,000 kDA
molecular weight. Substances that are larger than 80,000 kDa are too large to enter the gel beads
of the column and are only located in the fluid between the beads; so these molecules are eluted
quite quickly in the void volume. Since WGAP was found in our later fractions, it is not possible
for the molecular weight of WGAP to be larger than 100,000 kDA, otherwise it would have been
passed through in the void volume. Smaller molecules, such as WGAP are able to enter into the
gel beads and are retarded from exiting the column.
After WGAP had passed through the gel column, and the fractions that contained WGAP
were identified, enzyme and protein assays were run. The results of the enzyme assay were in
units/mL, and the results of the protein assay were mg/mL. These two values were then used to
calculate the specific activity of the fractions and the crude. The specific activities of the
fractions were then compared to that of the crude to compare the degree of enzyme purification
between preparations. The specific activity of crude is 0.465 units/mg, the specific activity of
fractions 9 and 10 are 0.674 units/mg and 0.851, respectively (Table 1). After comparing these
specific activities it was determined that there was 1.83 fold purification (Fraction 10) between
our fractions and our crude. This is significant because this means that our enzyme is purer than
when the experiment started. The WGAP sample was purified approximately a little less than 2
times that of the crude using a one step method of purification. In order to purify the sample
further, a multistep procedure could have been used. In the multistep procedure different types of
columns could be used to separate WGAP based on properties other size, such as charge or
solubility. Another way to achieve a higher specific activity would be to increase, or double the
length of the column. By increasing the size of the column, smaller molecules have more time to
pass through the gel beads of the column. Since the smaller molecules would have spent more
time in the column, they would be able to separate further from molecules with higher molecular
weights. Since the smaller molecules would be separated further, there would be fewer different
types of proteins found in each fraction. Each fraction would contain a greater amount of one
protein, which would yield to a higher purification.
As mentioned previously, a way to achieve a higher purification is to use a multistep
procedure to purify WGAP. Many other researchers have used multistep procedures to purify

2
acid phosphatases. For example, Waymack and Van Etten achieved a very high fold purification
using an 8 step purification procedure (1991). These researchers achieved a purification of
WGAP greater than 7000-fold. In order to achieve this large of a purification, the researchers
began their purification process by running an acetone precipitation, an ammonium sulfate
precipitation and acetone removal, an ammonium sulfate fractionation, and finally a methanol
precipitation. The ammonium sulfate precipitation and fractionation is an example of a
purification technique called salting out. Salting out is used because different proteins precipitate
at different salt concentrations, which can be used to purify the desired protein. All of these
precipitation procedures were used to concentrate their sample of WGAP while simultaneously
removing unwanted substances from their sample. After conducting these purification steps, the
researchers had a 595-fold purification before they even moved into column chromatography.
However, their % recovery or yield was only 16.5% at this point. Waymack and Van Etten cited
that having a higher activity going into the chromatography steps decreases the risk of activity
loss in future steps due to dilution and nonspecific adsorption.
Next the researchers ran a Sephadex G-75 column just as we used in lab. At this point
their fold purification increased from 595-fold to 1980-fold. After this column they used a series
of other chromatography procedures including a SP-Sephadex column, a DEAE-cellulose
column, and a second SP-Sephadex column. A DEAE-cellulose column is positively charged and
separates anionic proteins, while the SP-Sephadex column is a strong cation exchanger column,
which separates out positively charged proteins. After all of these steps the final purification was
a 7030-fold purification with a 1.5% recovery. If we had used any of these purification steps in
conjunction with our Sephadex G75 column, we would have had a higher fold purification of
WGAP.
Another set of researches Igarashi and Hollander also used a multistep procedure to
purify their enzyme (1968). Instead of looking at WGAP these researchers sought to purify rat
liver acid phosphatase (RLAP). First their sample was treated with acid, and the precipitate the
obtained was discarded, but the supernatant was dialyzed. Dialysis procedures are useful in
purification because small molecules and salts can be separated from the larger proteins, located
inside the dialysis bag, via diffusion down their gradient into the dialysis buffer. However this
technique does not separate proteins effectively, so the fold purification was only 5.6-fold at this
point. After dialysis an ammonium sulfate fractionation, similar to the previous paper, was used
ot purify the sample to 6.8-fold. After these two procedures a Sephadex G75 column, like ours,
was used and then a DEAE-cellulose column was used. The DEAE-cellulose column yielded two
peaks of enzyme activity. The first peak was not absorbed into the column, while the other
sample had to be eluted out of the column with NaCl. The first sample was underwent a
chromatography procedure on a hydroxylapatite column. Hydroxylapatite columns are pseudo
ion exchange columns that involve both anion and cation exchange. After this column the sample
was crystalized with ammonium phosphate to concentrate the sample. The fold purification of
this first sample is 890-fold with a % yield of only 2.1%. The second sample obtained from the
DEAE-column underwent chromatography on a Sephadex G200 column instead, which is
another size exclusion column. The fold purification of this sample was only 134-fold with a %
yield of 1.8%. The first procedure for purification was more effective. Again, if any of these
techniques, such as the use of DEAE-cellulose column was used in our experiment, a fold
purification greater than 1.83 could be achieved.
A third set of researchers Aguirre-Garca et al. also used a multistep approach to purify
their acid phosphatase (2000). Aguirre-Garca et al. sought to purify a sample of membrane

3
bound acid phosphatases (MAPs) found in Entamoeba histolytica. They began their purification
with a solubilisation of MAP with Triton X-100. The solubilisation was used to remove the
soluble components of the sample, which led to increase in purity by 12.43-fold, but a decrease
in % yield to 88%. After this a Con A-sepharose column, which is an affinity column, was used
to achieve purification to 16.69-fold and 10.23% yield. The Con A-sepharose column contains
lectins, which is a carbohydrate binding protein, which can bind glyco-groups. Glycoproteins are
bound to the column, while non glycol group proteins are eluted out. After the affinity column a
final DEAE-cellulose column was used to get a final fold purification of 23.05-fold and a yield
of 2.26%.
All of these researchers achieved a higher fold purification by using a multistep
procedure. If we had used even on of these procedures, we could have obtained a larger fold
increase in purity. However, similar to our experiment all the experimenters found a decrease in
% yield as purification increased.
After the one step purification, we ran a 12% SDS PAGE gel to determine the number of
polypeptides present in our sample and their molecular weights. Due to researcher error, the
samples from the previous purification were discarded, so fractions were taken from another lab
group. This lab group identified fractions 6 and 7 as samples that contained the highest specific
activity of WGAP (0.847 and 0.971 units/mL, respectively). These fractions, along with our 1:10
crude, and a BSA standard were loaded on to the gel. The most prominent bands in the crude
were located at 29.5 kDa and 25.1 kDa. The most prominent bands in the fractions were located
at 53.7 kDa, as well as bands located at 30.3 kDA (fraction 6) and 31.0 kDa (fraction 7). The
band located at 25.1 kDa was not found to be very prominent in the fractions. This was because
this protein was probably purified out of the sample during the chromatography process to be as
visible in the gel. Some bands were more prominent in the purified lane as compared to the crude
lane because these polypeptides had an increase in concentration after purification, and now they
show up more prominently in the gel. For example, there were bands that are found in the
purified lanes, at 67.0 kDa and 61.1 kDa, that were not found in the crude lane. These
polypeptides had an increase in concentration after the purification process and now show up
more prominently in the gel.
There was a band that showed up more prominent in the purified lanes as compared to the
crude. This band was found at 53.7 kDa. This band could be considered as a candidate for
WGAP, because this band is more purified as compared to the crude. According to Waymack
and Van Etten in their SDS PAGE gel, they determine the molecular weight of WGAP to be
around 56,000 3000. Our candidate for WGAP is right at the lower range of that finding, so it
could be WGAP. In order to obtain more decisive results, a multistep purification could be used,
just as those used in previously published papers.

4
Table 1: Purification of Wheat Germ Acid Phosphatase (WGAP)
Total Protein Total Specific Activity % Yield Purification
(mg) Activity (units/mg) Level
(units)
Crude 2.15 1 0.465116279 100% 1
Enzyme
Fraction 0.37 0.25 0.674418605 25% 1.45
9
Fraction 0.217 0.18 0.851485149 18% 1.83
10

5
 Fraction Fraction 1:10 Standard
7 6 Crude

250
kDa
150
kDa
100
kDa
75
kDa
53.7 kDa
50
kDa

37
kDa

25
kDa
20
kDa

15
kDa
10
kDa

Figure 1: . SDS-PAGE polyacrylamide gel of fractions that contained the highest WGAP
activity, a 1:10 crude enzyme sample, and a BSA standard. A 12% SDS-PAGE gel was run,
followed by staining with GelCode Blue. Any band that contained at least 50 ng was stained.
Lane 1: molecular weight standards; lane 2: 1:10 crude enzyme dilution, lanes 3-4: purified
fractions after gel filtration chromatography. The band enclosed by dotted lines represents a
possible band that could represent WGAP.

6
 Appendix:
Week 1:

Table 1: Enzyme Assay Absorbance Values

Fraction Absorbance (nm) Absorbance (nm)
5 min 10 min
7 0.019 0.039
8 0.092 0.198
9 0.117 0.268
10 0.107 0.209
11 0.065 0.145
12 0.055 0.112
1:10 Crude 0.254 0.541
Enzyme Dilution
1:100 Crude 0.024 0.048
Enzyme Dilution

Table 2: Enzyme Assay Concentrations1 and Units of Activity Obtained from Fractions and
Dilutions of WGAP
Fraction umol of p- Activity of Kill Activity of Reaction Activity of Enzyme
nitrophenol Tube (units) Tube (units) (units/mL)
7 0.008 0.0008 0.008 0.08
8 0.04 0.004 0.04 0.4
9 0.058 0.0058 0.058 0.58
10 0.043 0.0043 0.043 0.43
11 0.03 0.003 0.03 0.3
12 0.026 0.0026 0.026 0.26
1:10 0.1 0.01 0.1 1
Crude
Enzyme
Dilution
1:100 0.01 0.001 0.01 0.1
Crude
Enzyme
Dilution
1
The 10-minute absorbance readings were used to determine umol of p-nitrophenol using the Enzyme Assay
Standard Curve

7
Table 3: Protein Assay Absorbance and Concentrations
Fraction Absorbance mg/mL

72 0.085

8 0.766 0.73

9 0.845 0.86

10 0.564 0.505

11 0.508 0.405

1:10 Crude Enzyme 1.241 2.15

Dilution
1:100 Crude 0.346 0.215
Enzyme Dilution
2The
absorbance value for fraction 7 is too low to be read on the attached Protein Assay Standard Curve

Table 4: Specific Activities of All Fractions and Crude

Fraction Specific Activity (units/mg)

73
8 0.547945205
9 0.674418605
10 0.851485149
11 0.641975309
Crude Enzyme 0.465116279
3
Due to the inability to calculate the concentration of fraction 7 in the protein assay, no specific activity can be
calculated

Week 2:

NOTE: Due to researcher error, fractions from the previous week were discarded. Instead, the
most reactive fractions for another lab group (Kevin and Priyanka) were used in week 2. The
fractions with highest Specific Activity were fractions 6 and 7.

Table 5: Concentrations and Loading Volumes of Fractions and Crude Enzyme on SDS PAGE
Gel
Fraction Concentrations (ug/uL) Gel Loading Volume (uL)
6 0.51 17.16
7 0.35 25
Crude Enzyme 2.15 4.07
(1:10 dilution)

8
Table 6: Distance traveled by samples on SDS PAGE and Corresponding Rf values
Band Standard Rf values Crude Rf values Fraction Rf values Fraction Rf values
Number (cm) Standard (cm) Crude 6 (cm) Fraction 7 (cm) Fraction
6 7
1 0.95 0.132 1.5 0.208 1.5 0.208 1.5 0.208
2 1.19 0.165 1.9 0.264 1.65 0.229 1.7 0.236
3 1.5 0.208 2.25 0.313 2 0.278 2 0.278
4 1.85 0.257 2.4 0.333 2.15 0.299 2.15 0.299
5 2.52 0.350 2.95 0.410 2.4 0.333 2.4 0.333
6 3.2 0.444 3.6 0.500 3 0.417 2.9 0.403
7 4.35 0.604 3.85 0.535 3.55 0.493 3.5 0.486
8 4.9 0.681 4.25 0.590 3.75 0.521 3.65 0.507
9 6 0.833 5.95 0.826 4.1 0.569 4.1 0.569
10 5.4 0.750 5.4 0.750
11 6 0.833 6 0.833

Table 7: Molecular Weights of Polypeptides

Band Number Standard Crude Fraction 6 Fraction 7
(kDa) (kDA) (kDa) (kDa)
1 250 102.3 102.3 102.3
2 150 72.4 86.3 85.1
3 100 56.2 67.0 67.0
4 75 53.7 61.1 61.1
5 50 39.4 53.7 53.7
6 37 31.3 38.0 42.1
7 25 29.5 32.4 33.1
8 20 25.1 30.3 31.0
9 15 15.1 26.9 26.9
10 20.9 20.9
11 15 15

9
Sample Calculations:

Activity of kill tube = umol p-nitrophenol / 10 minutes

= 0.058 umol / 10 minutes
= 0.0058 umol/min = 0.0058 units

Activity of Reaction Tube = Kill tube * 10

= 0.0058 units * 10
= 0.058 units

Enzyme Activity = Reaction tube * 10

= 0.058 units * 10
= 0.580 units/mL

Purification Table:

Total Protein (Purified Fraction 9) = (Protein concentration of fraction 9) * 0.43 mL

= (0.86 mg/mL) * 0.43 mL
= 0.370 mg
Total Activity (Purified Fraction 9) = (Enzyme activity of fraction 9) * 0.43 mL
= (0.58 units/mL) * 0.43 mL
= 0.250 units
Specific activity (Purified Fraction 9) = Total Activity / Total Protein
= 0.250 units / 0.370 mg
= 0.674 units/mg

Total Protein (Purified Fraction 10) = (Protein concentration of fraction 10) * 0.43 mL
= (0.505 mg/mL) * 0.43 mL
= 0.217 mg
Total Activity (Purified Fraction 10) = (Enzyme activity of fraction 10) * 0.43 mL
= (0.43 units/mL) * 0.43 mL
= 0.185 units
Specific activity (Purified Fraction 10) = Total Activity / Total Protein
= 0.185 units / 0.217 mg
= 0.851 units/mg

Total Protein (Crude) = Protein Concentration (1:10 Crude) *10 * 0.1 mL

= (2.15 mg/mL) *10 * 0.1 mL
= 2.15 mg
Total Activity (Crude) = Enzyme Activity (1:10 Crude) *10 * 0.1 mL
= (1.0 units/mL) * 10 * 0.1 mL
= 1.0 units
Specific activity (Crude) = Total Activity / Total Protein
= 1.0 units / 2.15 mg
= 0.465 units/mg

10
Yield (Purified Fraction 9) = total activity of fraction 9 / total activity of crude
= 0.25 units / 1.0 units
= 0.25 = 25%
Yield (Purified Fraction 10) = total activity of fraction 10 / total activity of crude
= 0.18 units / 1.0 units
= 0.18 = 18.0%

Purification Level (Purified Fraction 9)= specific activity of fraction 9 / specific activity of crude
= 0.674 units/mg / 0.465 units/mg
= 1.45

Purification Level (Purified Fraction 10) = specific activity of fraction 10 / specific activity of
crude
= 0.851 units/mg / 0.465 units/mg
= 1.83

SDS-Page

Rf = Distance traveled / Length of Gel

= 1.5 cm / 7.2 cm
= 0.208

Molecular weight of non-standards = log-1 (log MW)

= log-1(2.01) = 102.01
= 102.3 kDa

11
 1.6

1.4

1.2

1
absorbance

0.8

0.6

0.4

0.2

0
0 0.06 0.12 0.18 0.24 0.3
umol product

Figure 1: Enzyme assay standard curve

12
 1.4

1.2

0.8
Absorbance

0.6

0.4

0.2

0
0.125 0.25 0.5 0.75 1 1.5
Concentration (mg/mL)

Figure 2: Protein assay standard curve

13
 0.9

0.8

0.7

0.6

0.5
Rf Values

0.4

0.3

0.2

0.1

0
1.1760913 1.4260913 1.6760913 1.9260913 2.1760913 2.4260913
logMW

Figure 3: Molecular weight standard curve for SDS PAGE gel

14
 Works Cited

Aguirre-Garca, M. M., J. Cerbn, and P. Talams-Rohana. "Purification and properties

of an acid phosphatase from Entamoeba histolytica HM-1: IMSS." International journal
for parasitology 30.5 (2000): 585-591.

Igarashi, Mitsuo, and Vincent P. Hollander. "Acid phosphatase from rat liver purification,
crystallization, and properties." Journal of Biological Chemistry 243.23 (1968): 6084-
6089.

Waymack, Parvin P., and Robert L. Van Etten. "Isolation and characterization of a homogeneous
isoenzyme of wheat germ acid phosphatase." Archives of biochemistry and biophysics
288.2 (1991): 621-633.

15