Acid Phosphatase Lab

This lab involved several experiments over the span of many weeks. The overall intent of the lab was to be the successful purification of a protein. Specifically, the purification of the enzyme acid phosphatase from wheat germ. Through three major steps we were able to perform this purification. The steps involved were disrupting the source cell, selectively purifying the enzyme from contamination, and finally preserving the original structure of the enzyme and preventing degradation. Aliquots, or small volumes, of the solution were collected from each step in the purification process for enzyme activity and protein content. These calculated values were then used to determine the specific activity, which can be used to analyze the specific

characteristics and analyze the purity of the acid phosphatase. The purification proved to be unsuccessful with a supernatant VI having a total volume of 3.63 mg of protein, 9.3 x 10^-5 of enzyme activity, and 0.34% recovery. However, the SDS-PAGE gel product proved that we did have a small amount of purified protein. Errors occurred in the Sup IV and V, thus will be excluded data and results in this lab report.
Introduction
Cells would not be able to properly carry out the basic functions of life without enzymes.

Enzymes play a role to catalyze numerous reactions and help in the creation of DNA, RNA, and other molecules. This experiment focuses on the enzyme acid phosphatase. Acid phosphatase is a ubiquitous lysosomal enzyme that hydrolyses organic phosphates at an acid pH, which means that they remove phosphate groups under slightly acidic conditions. (Henneberry, 1979). Acid phosphatase has become to be used commonly because of the practical applications such as being used extensively as a serum marker for prostate cancer (Bull, 2005). In this lab a primary focus is to successfully perform an isolation of acid phosphatase and prevent it from degrading while keeping it in its native structure. First, disrupted the cell used as a source via hypotonic shock. Then, through several methods including centrifuging and dialysis, we were able to purify it and protect it from breaking down further. Next, the substrate PNPP was used to follow the extent of which it was purified and characterization. A spectrophotometer was needed to measure the absorbance of the protein solution and compared to a standard curve. Using gel electrophoresis we determined the success that our purifications steps achieved. Absorbance was also measured at 405nm from the hydrolyzed PNPP. The use of these techniques of purification and analysis to determine overall

success.
Materials and Methods
Purification of Acid Phosphatase from Wheat Germ. Beginning by filtering the wheat germ with cold water (40ml of H2O, 10g wheat germ). Then placed the mixture to be filtrated through centrifugation by putting it in a centrifuge tube for 10 minutes at 4,000 rpm at 4 degrees Celsius. This was then decanted into a graduated cylinder and a total volume of 19ml was measured. From this took 0.5ml and stored in a freezer to be used in a later assay. The remainder of our supernatant 1 was then placed into a beaker in an ice bath, stirred with a magnetic bar, and slowly added 11.5 ml of (NH4)2SO4. This was done to prevent precipitation. After stirring for 10-15 minutes it was then again centrifuged for 10 minutes to remove precipitated materials. A total volume of 17ml was measured for supernatant 2 and we again took 0.5ml in an aliquot and stored in a freezer to use in a later assay.
Purification of Acid Phosphatase from Wheat Germ Part 2. The supernatant was placed back into a beaker. 62 ml of (NH4)2SO4 were added per 100ml (13.64ml). This was added slowly while stirring. Afterwards it was placed on a hot plate with a set temperature of 100 degrees Celsius and it was heated until it reached 60 degrees Celsius and held for two minutes. Then the beaker was plunged into an ice bath. The solution was stirred gently until it reached -8 degrees Celsius. It was then centrifuged for 20 minutes at 4,000rpm at 4 degrees Celsius. Once that was completed it was decanted into a graduated cylinder and the volume of supernatant 3 was recorded as 20ml and the pellet was saved. Once again saved 0.5ml and labelled it supernatant 3 and stored in a freezer for use in later assay. The pellet was the completely re-suspended. Used 1/3 of the volume of sup. 3, but no more than 20ml of water. When even suspension was obtained it was transferred to a 40ml centrifuge tube. Centrifuged for 10 minutes at 4,000rmp at 4 degrees. Once centrifuged it was decanted into a graduated cylinder with a noted volume of 15ml. Again saved 0.5ml in an aliquot and labelled it as supernatant 4 and stored in a freezer for use in a later assay. For each ml of sup 4, 12ml of 0.20M EDTA and .05ml of saturated (NH4)2SO4 was added. Then slowly added 1.75ml of cold methanol for each ml of solution of sup 4. Kept solution stirred and on ice for 10 minutes and then stored in a freezer.
Purification of Acid Phosphatase from Wheat Germ Part 3 – Dialysis. A dialysis solution was created by using 0.2M EDTA to make a 0.5L solution of 5M EDTA. While the solution was being created, the sample was unfrozen and centrifuged at 4,000rpm at 4 degrees for 10 minutes. Next, the pellet was re-suspended by using 3ml H2O and centrifuging for 10 minutes at 4 degrees at 4,000rpm. It was separated, and the supernatant was saved. Discarded the pellet and saved a portion in aliquot for assay. A dialysis tubing was wet for 30 minutes in dH2O. A knot was tied in one end of tubing and the combined supernatant was put into the opening. Tied knot in the opening. Then the bag was placed in dialysis tub. Afterwards removed any air bubbles to allow it to properly sink. It was covered in plastic wrapping and left to sit for a week at 4 degrees.
Bradford Assay.

This assay was used to measure the concentration of protein in a solution. Use it to determine the concentration of our saved supernatants 1-6. The total amount of protein is determined and so is the phosphatase activity. By combining the Bradford reagent with our diluted supernatants in a test tube and letting sit for 5 minutes to be read at 595nm we were able to determine the mg/ml of protein. The absorbance was read on a spectrophotometer which measures the light intensity. Using a standard curve we were able to get our readings. The procedure was taking the six supernatants and aligning them to six test tubes. A solution was created with 50x dilution for sup1, 10x for sup 2-3, and no dilution for sup 6. A Bradford reagent was then added to each. Afterwards analyzed using the A595 wavelength. A formula was then used to determine the amount of active protein. Sup 1 yielded 104.31 mg/ml, sup 2 yielded 22.33 mg/ml, sup 3 yielded 1.21 mg/ml, and sup 6 yielded 0.66

mg/ml.
SDS-Page Gel. An SDS-Page gel was ran to determine the levels of purity of enzyme by separating by mass. To start samples of 20ul of supernatants 1-6 were prepared with ul of loading dye. Then it was placed onto a heating block for 5 minutes at 90 degrees. Afterwards loaded 5ul of sample into the SDS page gel after ensuring that the pathways were clear. Added them sequentially until all were loaded. Then the SDS page gel was ran overnight at 75V for gel portion and then at 123V. We then disassembled and rinses stain with Coomassie blue and observed with a molecular weight marker.
Purification Table and Activity Assay. An activity assay was performed to determine the amount of protein present in the supernatants 1-6. It was then used to prepare a protein purification chart that shows total activity of the enzyme. The following table shows the dilutions used. A blank tube was also created.
Sup Dilution Vol H2O
I 1/100 1.5 148.5 L
II 1/100 1.5 148.5 L
III 1/20 7.5 142.5 L
IV 1/100 1.5 148.5 L
V 1/50 3 147 L
VI 1/25 6 144 L
One assay was prepared for each fraction and the control that contained no proteins.
Reagent Volume in mix
1.0 M Sodium acetate 0.4 ml
0.1 M MgCl2 0.4 ml
H2O 1.6 ml
To each of the assay reactions 150ul was added of supernatants respectively to the tubes and h2o to the control. Then began adding PNPP every minute until all 7 were done. Let sit for 10 minutes and then removed one per minute so that each reacted for the same amount of time. The blank was then used to calibrate the spectrophotometer. Each tube was read at A405. Beer’s Law was then used with an assumed value of 18,800 as the molar extinction coefficient.
Results
Purification Table
Supernatant Total Protein Total Enzymatic Activity Specific Activity Percent Recovery Fold Purification
Sup I 1981.9 mg 0.152 mmoles 7.67 x10-5 100 % 1
Sup II 379.6 mg 0.1445 mmoles 3.81 x10-4 95 % 4.97
Sup III 24.2 mg 1.92 x10-3 mmoles 7.91 x10-5 1.26 % 1.03
Sup VI 3.63 mg 5.12 x10-4 mmoles 1.41 x10-4 0.34 % 1.84

The results above show the major loss of protein starting at sup. II through the span of the purification experiment. Our first column represents the total protein in the sample that was calculated through a Bradford assay and multiplied by the volume of the fraction. Total enzymatic activity represents the amount of calculated activity from the activity assay that is multiplied by total volume. Specific activity refers to a direct compare between enzyme activity and its total protein detected. In percent recovery a sup is compared to a sample and observed at any point during progression. And the fold purification is a measure of quality with lower numbers being more pure. Our results we obtained a 2 fold purified product. Error was most likely in pipetting measurements and/or timed assay reactions.
SDS- PAGE Gel

Discussion
Using various techniques including centrifuging, denaturing, dialysis, ion-exchange chromatography and gel chromatography, we were able to determine the purification of wheat germ acid phosphatase. The purified enzyme was examined using several tests such as the Bradford assay, the absorbance’s of activity using PNPP, and gel electrophoresis which gave the molecular mass of the injected samples. After examining the activity of the samples at each step of the ammonium sulfate precipitation, the activity and concentration, we were able to see that our isolation of acid phosphatase was hardly a success. We first disrupted the wheat germ with a hypotonic solution. This step was essential for the release of intracellular components from our cell source. The hypotonic shock let the acid phosphatase leak out of the cell source. The enzyme was then purified through precipitation steps through centrifuging and denaturation while keeping acid phosphatase in form. First there is salting out which is a method that utilizes the reduced solubility of molecules in a solution of high ionic strength. This accomplished purification because it allow the protein to precipitate out of the solution. The protein aggregates to reduce surface area exposure and when enough salt is introduced the protein precipitates. A solvent fractioning was performed by the addition of methanol and was able to accomplish purification through the separation of the acid phosphatase. Through pelleting and resuspension of the acid phosphatase the difference in solubility was used to precipitate out proteins minus acid phosphatase. We used dialysis to accomplish further purification by using EDTA to separate smaller molecules from larger molecules through a semipermeable membrane. Once equilibrium was reached EDTA chelated metal ions to protect the enzyme from denaturation. Although the purification was successful in removing proteins from the supernatant as we can see from sup 6 the purification had a low percent recovery thus an issue must have occurred during the experiment.