+

Isolation, Purification, and Assay of Wheat Germ Acid PhosphataseNatalia ManzanoWilmarie MoralesRISE ProgramMay 10th, 2012

+Introduction

Wheat germ is a very small part of a wheat kernel.

Wheat germ is very high in protein.

It contains around 28% protein and has more protein than can be found in most meat products.

The human body needs protein in order to repair tissue damage and to help minerals and nutrients reach our cells.

It contains more potassium and iron than any other food source.

Also has calcium, zinc, magnesium, and vitamins A, B1, B3 and E.

Vitamins B1 and 3 are very important to maintain energy levels and maintain healthy muscles and organs. Vitamin E is a very important antioxidant, prevents heart disease, and is needed to strengthen the body’s immune system.  

+Acid Phosphatase

Acid phosphatase is a type of enzyme used to free attached phosphate groups from other molecules during digestion.

It is basically a phosphomonoesterase, a phosphatase that acts on monoesters.

It is stored in lysosomes and functions when these fuse with endosomes.

Phosphatase enzymes are also used by soil microorganisms to access organically bound phosphate nutrients.

Some plant roots, exude carboxylates that perform acid phosphatase activity, helping to mobilise phosphorus in nutrient-deficient soils.

+Wheat Germ Acid Phosphatase

• Acid phosphatase ocurres in plants and animals, and has a pH optimum below 7.

• Catalyzes the hydrolysis of phosphate groups from macromolecules and smaller molecules that are stored in the wheat seed.

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• The growing wheat embryo uses the freed phosphate in germination and growth.

http://www.google.com.pr/imgres?imgurl=http://nutritioncheckup.com/blog/wp-content/uploads/2010/10/wheat-germ.jpg&imgrefurl=http://nutritioncheckup.com/blog/?p%3D123&h=511&w=535&sz=72&tbnid=3DIxRSBTgElMbM:&tbnh=94&tbnw=98&zoom=1&docid=q_1lvNRjniiFCM&sa=X&ei=xYusT9XhDpKy8AT5ip3TBA&ved=0CJUBEPUBMAQ&dur=112

+Objectives

To purify and isolate acid phosphatase from wheat germ.

Determine protein concentration using Bicinchoninic Acid protein assay.

+Metodology• First we isolated acid phosphatase by suspending

25g of wheat germ in 100 mL prechilled dH20. Filtrated with cheesecloth approximately 70 mL of solution. Centrifuge the filtrate at 4˚C for 20 minutes at 2,800 x g. Collected 65 mL of the supernatant denoted as Fraction I.

• Then purified the acid phosphatase, we transfered Fraction I to a 150 mL beaker, and added 0.396g of 1.0M MnCl2 to 1.0 ml of dH2O. And added 1.26 ml of 1.0 M MnCl2 to Fraction I.

• Collect 62 mL of the other centrifuged supernatant from Fraction I denoted as Fraction II.

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• Suspended the pellet with 4.8 mL of 0.05 M of Solution Acetic Acid and 45.2 mL of 0.05 M Sodium Acetate. The supernatant obtained oof 25 mL was denoted as Fraction III.

• Transferred the remainder of Fraction II to a 400 ml beaker placed in an ice bath. Added 33.48 ml of (NH4)2SO4 to Fraction II. Centrifuged Fraction II and obtained 92 mL of the supernatant.

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• Suspend the centrifuged pellets and added 20 ml of 0.05 M sodium acetate buffer, pH 5.67. The 21 mL of supernatant is denoted as Fraction IV.

• Add 46.92 mL (NH4)2SO4 to Fraction II.

• WILMA YOU DID THIS????• Collected 136 mL of supernatant. This is denoted as

Fraction V• Suspended the pellet obtained in 20 ml of cold, dH2O.

Centrifuge the solution to remove undissolved protein. The78 mL supernatant is denoted as Fraction VI. Record the volume of Fraction VI.

+Materials and Methods

25 g Wheat germ MnCl2, 1.0 MH2O, prechilled to 4˚C

Sodium acetate buffer, 1.0M (pH 5.7)

Cheesecloth (NH4)2SO4,

saturated (pH 5.5)

Ice, crushed Pasteur PipetsBCA Kit Gloves,

disposableBSA standard, 1.0 mg/ml

Weighing traysCentrifuge, high speed

Balance

Ice bucket Spectrophotometer, visible

Magnetic stirrer Microplate

Waterbaths (30˚C and 70˚C)

 

Materials

Methods

Bicinchoninic Acid protein assay (BCA)

SDS PAGE

+Procedure I: Obtaining Fractions

Filter wheat germ with cheese cloth

Centrifuge filtrate (70ml)

Fraction I

Discard Pellet

Add 1.26 ml of MnCl2 1.0MCentrifuge

Supernatant 62 ml

* All centrifugation was done at 2800Xg at 4° for 20min

Fraction II

PelletAdd Sodium Acetate

CentrifugeDiscard Pellet

Supernatant 25 ml

Fraction III

Add 33.48 ml of Ammonium Sulfate (Saturated 35%)

Supernatant 92 mlCentrifuge

Pellet (buffer)

Centrifuge

Discard Pellet

Supernatant 21 ml

Fraction IV

Add 46.92 ml of Ammonium Sulfate

(Saturated 57%)Centrifuge

Supernatant 136 ml

FractionV

Pellet Suspend in

dH2OCentrifuge

Discard PelletFractio

n VI

+

Procedure II: BCA Assay

BCA serves the purpose of reacting with complexes between copper ions and peptide bonds to produce a purple end product.

Estimate visually or measure with a standard spectrophotometer or plate reader (562nm).

+

Add Sample+ dH2O+BCA

+Conc. Of working std (mg/mL)

Stock used

Absorbance

Blank

Abs-Blank

Conc. (mg/ml)

Vol. of sample added

11X

0.162

0.094

0.068 0.200 2.3

1X0.42

40.09

40.330 0.600 6.9

1X0.54

00.09

40.446 1.000 11.5

0.11X/10

0.101

0.094

0.007 0.020 2.3

1X/10

0.126

0.094

0.032 0.060 6.9

1X/10

0.128

0.094

0.034 0.100 11.5

0.011X/100

0.078

0.094

0.000 0.002 2.3

1X/100

0.083

0.094

0.000 0.006 6.9

1X/100

0.094

0.094

0.000 0.010 11.5

Blank Concentration Curve

+Procedure III: SDS-PAGE

Running Gel: 1.88 ml dH2O 1.67 ml separating buffer 2.22 ml Acrylamide 50.0 µl 20% SDS 100 µl 10% Glycerol 1.67 µl TEMED 50 µl APS(100 mg/ml)

Stacking Gel: 2.975 ml dH2O 1.25 ml separating buffer 0.662 ml Acrylamide 225.0 µl 20% SDS 25 µl 10% Glycerol 6.25 µl TEMED 25 µl APS

+Procedure III: SDS

Get your sample obtained from previous purifying technique (Purification)

Set up gel, remove comb

Load Buffer

Load Sample (3:1 sample:buffer)Run Gel

Stain and look at with UV light

Results:Sample Absorbance Blank Abs-Blank Conc. (mg/ml) Vol. of sample added Dilution factor

Fraction I 1X-L 2.511 0.094 2.417 25.466 2.3 1Fraction I 1X-H 3.029 0.094 2.935 6.185 11.5 1

Fraction I X/10-L 0.305 0.094 0.211 22.231 2.3 10Fraction I X/10-H 1.171 0.094 1.077 22.695 11.5 10Fraction I X/100-L 0.13 0.094 0.036 37.930 2.3 100Fraction I X/100-H 0.241 0.094 0.147 30.976 11.5 100

Fraction II 1X-L 3.029 0.094 2.935 30.923 2.3 1Fraction II 1X-H 3.029 0.094 2.935 6.185 11.5 1

Fraction II X/10-L 0.385 0.094 0.291 30.660 2.3 10Fraction II X/10-H 1.636 0.094 1.542 32.493 11.5 10Fraction II X/100-L 0.164 0.094 0.07 73.752 2.3 100Fraction II X/100-H 0.512 0.094 0.418 88.081 11.5 100

Fraction III 1X-L 0.524 0.094 0.43 4.530 2.3 1Fraction III 1X-H 2.035 0.094 1.941 4.090 11.5 1

Fraction III X/10-L 0.143 0.094 0.049 5.163 2.3 10Fraction III X/10-H 0.317 0.094 0.223 4.699 11.5 10Fraction III X/100-L 0.094 0.094 0 0.000 2.3 100Fraction III X/100-H 0.138 0.094 0.044 9.272 11.5 100

Fraction IV 1X-L 0.536 0.094 0.442 4.657 2.3 1Fraction IV 1X-H 1.541 0.094 1.447 3.049 11.5 1

Fraction IV X/10-L 0.149 0.094 0.055 5.795 2.3 10Fraction IV X/10-H 0.303 0.094 0.209 4.404 11.5 10Fraction IV X/100-L 0.102 0.094 0.008 8.429 2.3 100Fraction IV X/100-H 0.163 0.094 0.069 14.540 11.5 100

Fraction V 1X-L 2.373 0.094 2.279 24.012 2.3 1Fraction V 1X-H 3.029 0.094 2.935 6.185 11.5 1

Fraction V X/10-L 0.179 0.094 0.085 8.956 2.3 10Fraction V X/10-H 0.673 0.094 0.579 12.201 11.5 10Fraction V X/100-L 0.151 0.094 0.057 60.055 2.3 100Fraction V X/100-H 0.358 0.094 0.264 55.630 11.5 100

Fraction VI 1X-L 1.443 0.094 1.349 14.213 2.3 1Fraction VI 1X-H 3.029 0.094 2.935 6.185 11.5 1

Fraction VI X/10-L 0.212 0.094 0.118 12.432 2.3 10Fraction VI X/10-H 0.657 0.094 0.563 11.864 11.5 10Fraction VI X/100-L 0.152 0.094 0.058 61.109 2.3 100Fraction VI X/100-H 0.333 0.094 0.239 50.362 11.5 100

Samples Average conc. (mg/mL)

Volumes (mL)

Total protein

(mg)

Fraction I 30.39 65 1974.62

Fraction II 64.16 62 3978.18

Fraction III 5.92 25 147.90

Fraction IV 7.56 21 158.86

Fraction V 34.21 136 4652.61

Fraction VI 33.94 78 2647.45     

+Expected Concentration Curve Fraction Concentration Curve

+Expected Trend Experimental Trend

+250-

150-

100-

75-

50-

37-

25-

15-

Fractio

n V

I

Fractio

n V

Fractio

n V

Fractio

n III

Fractio

n II

Fractio

n I

SDS-PAGE

+Gel Results

Expected Obtained

+Conclusion

Our results were not what was expected.

We were not able to successfully isolate wheat germ acid phosphatase.

This could be accountable to human error (bad pipetting, error in making solutions, etc.).

+Acknowledgements

Vibha Bansal PhD

Edmarie Martinez

Vivian Rodriguez Cruz

Alexandra Rosado Burgos

+References

Stoscheck ,2000. CM. Quantitation of Protein. Methods in Enzymology, 50-69.

Yasuaki Kawarasaki, Hideo Nakano, Tsuneo Yamane, 1999. Purification and some properties of wheat germ acidphosphatases. Elsvier [http://www.sciencedirect.com/science/article/pii/0168945296044779]

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/doi/abs/10.1094/CC-83-0069]