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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
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
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
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
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
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

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Rise final research assingment(the one)

  • 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