1. Isolation, Purification, and Assay of Wheat Germ Phosphatase
Wilmarie Morales
Natalia Manzano
RISE Program
May 15, 2012
Abstract:
The objective of the experiment was to isolate acid phosphatase from wheat germ and to
determine the protein concentration. The protein was isolated through a centrifuge based
purification technique and later examined through gel electrophoresis. The protein estimation
was done using the BCA method. The results were not what were expected and must be redone
in order to obtain acceptable results.
Introduction Acid phosphatase is a type of enzyme that is
used to free attached phosphate groups from
The wheat plants embryo is known as wheat
other molecules during digestion. The wheat
germ and it is found in the wheat kernel. The
germ embryo uses the phosphatase protein
germ is the most nutritious part of the kernel
in growth and germination. It does this by
containing 28% of protein, which is more
catalyzing and hydrolyzing phosphate
than can be found in most meat products.
groups from macromolecules and smaller
All the proteins found in wheat germ are
molecules that are stored in the wheat seed.
different and serve various essential
purposes for the plant. One of these proteins For this project our goal was to isolate the
is the acid phosphatase enzyme. acid phosphatase enzyme from the wheat
germ through specific methods of isolation,
and to determine the amount of

2. concentration of said enzyme found in our at 2800 x g at 4oC. The supernatant for this
wheat germ samples. centrifugation was denoted as Fraction I (65
ml). An aliquot of 1.0 ml was frozen and
Experimental Procedure
saved for later assay of protein concentration
Materials and enzymatic activity.
Fraction I was then maintained cold and
Commercial wheat germ, sold as
1.26 ml of 1.0 M MnCl2 was added to the
Kretschmer, was used as a convenient
solution. The solution was then centrifuged
source for the phosphatase enzyme. The
and its supernatant (62 ml) and pellet were
reagents used were manganese chloride,
collected. The supernatant was named
ammonium sulfate, and sodium acetate
Fraction II2. The pellet obtained was
buffer-all at 1.0 M concentration. A
suspended in 25 ml of 0.05 M sodium
commercially purchased BCA kit was used
acetate buffer (pH 5.7). When the solution
for the protein estimation. The stacking and
appeared uniform it was centrifuged and the
running polyacrylamide gels were made at
supernatant (25 ml) obtained from this
10% and 4%, respectively.
centrifugation became Fraction III.33.48 ml
Methods
of ammonium sulfate3 were added to
Purification Procedure
Fraction II, bringing it to 35% saturation.
Twenty five grams of wheat germ were
Centrifuge the sample and both the pellet
suspended in 100 ml of deionized water
and supernatant (92 ml) are used for further
o
(4 C). The suspended wheat germ was then
purification. The pellet is suspended in 20
filtered using cheesecloth, the obtained
ml of sodium acetate buffer and centrifuge.
filtrate was titled with a volume of 70 ml.
The supernatant (21 ml) obtained becomes
1
The filtrated was centrifuged for 20 minutes

3. Fraction IV. Add 51 ml of ammonium 2.5µl of sample. The x100 dilutions were
sulfate to Fraction II and raising the solution contained 49.5 µl of 1xPBS for every 0.5 µl
to 57% saturation. Place solution in a hot of sample. 8.28 ml of BCA reagent and
water bath until the temperature reaches 165.6 µl of copper sulfate were added to the
70oC and imediately place in a cold water samples. The samples were then placed in a
bath until the temperature lowers to 30oC. spectrophotometer and the absorbance was
Once the solution has cooled centrifuge and read.
the supernatant (136 ml) obtained is denoted
A standard curve was created in order to
as Fraction V. The pellet obtained from this
compare the concentrations of the rest of the
centrifugation is suspended in deionized
samples.
water and then centrifuged. The obtained
1
All centrifugation was done at 4oC for twenty
supernatant (78 ml) becomes our sixth and minutes at 2800xg
3
The addition of ammonium sulfate had to be done
final fraction (Fraction VI). gradually in order to prevent protein denaturing.
Protein Assay
SDS-PAGE
The technique chosen to undergo the protein
A 10% polyacrylamide gel was prepared.
estimation was the Bicinchonic Acid Assay
All six fractions were placed in the gels and
(BCA). BCA serves the purpose of reacting
the gel ran for two hours.
with complexes between copper ions and
peptide bonds to produce a purple end Results
product (Stoscheck 2000).
Protein Estimation
All six fractions were diluted ten times and
After obtaining the absorbance of all our
then a hundred times. The x10 dilutions
samples we were able to calculate our
were done with 22.5 µl of 1xPBS for every

4. protein concentrations. For the calculations would reduce. Instead, as shown in Figure B,
we eliminated all absorbances that were the concentrations for the second fraction
found to be over 1 and under 0.1(Figure A). were twice as much as the first and then the
concentration for Fraction V were nearly six
times larger than those for Fraction IV.
Figure A- all rows in yellow are the concentrations that
were removed
This measure was taken because the
spectrophotometer reads accurately up to 1.5,
any concentration higher than this causes the
spectrophotometer to become saturated. For
this reason we strain our study to a 0.1 to 1 [Figure B]
SDS
range in order to obtain more accurate
The resulting gel from the SDS-PAGE was
results. Once the concentrations were
unable to show any significant pattern or
calculated the results obtained were not
data. What was expected of the gel was to
promising. The concentrations calculated
show less and less bands as the fractions
had no consistent pattern and no proper
were purified. Instead our gel (Figure C)
deduction could be done from them. The
showed a constant amount of bands for most
expected was that, as the fractions became
gels and then none for others. From this we
more purified the concentration of protein

5. can determine that our protein isolation was added than the absorbance readings could
unsuccessful. have been affected. These are some of the
possible mistakes that could have happened,
but it is impossible to know for sure.
Discussion
The experiment should be redone taking
these things into consideration. If the acid
phosphatase enzyme is properly isolated
than many studies can be conducted
afterwards, such as: classification for the
phosphatase, determining the concentration
of the protein in other specimens and
comparing it with the wheat germ’s
[Figure C]
Our experiment was unsuccessful since the concentration, and also for biomedical
results obtained were not the ones expected. studies. It is believed that acid phosphatase
This can be accountable to different errors can be used for clinical diagnosis, so, if
that could possibly have been made during properly isolated, the relationship between
the procedure. Most likely the mistakes were this enzyme and human disease can be
made during the stage where ammonium further studied (Bull 2002).
sulfate was added to the solution, since this
Acknowledgements
can cause denaturing of proteins. Another
possible mistake could have been during the To Vibha Bansal PhD and her team of
sample dilution process. If the proper students and tecnitians Edmarie Martinez,
volumes of sample and reagents were not

6. Vivian Rodriguez Cruz, and Alexandra Wheat Germ. European J. Biochem.,
Rosado Burgos. 439-44.
To the RISE program and its coordinators
Dr. Eneida Díaz, Dr. Elena González, and
Dr. Robert Ross.
References
Stoscheck ,2000. CM. Quantitation
of Protein. Methods in Enzymology,
50-69.
Bull H., Murray P., Thomas D.,
Nelson P., 2002. Acid phosphatases.
Journal of Clinical Pathology, 65-72.
Ke-Xue Zhu,Hui-Ming Zhou, and
Hai-Feng Qian, 2008. Proteins
Extracted from Defatted Wheat
Germ: Nutritional and Structural
Properties
[http://cerealchemistry.aaccnet.org/d
oi/abs/10.1094/CC-83-0069]
VERJEE Z. H. M., 1969. Isolation of
Three Acid Phosphatases from