Alkaline Phosphatase in Biochemistry Experiment

Biochemistry is used to show the chemical processes that occur in living organisms. In most biochemistry laboratories, researchers dedicated to the study of chemical processes and biological macromolecules. In the lab, many laboratory techniques have been used to isolate, quantitate, measured ammonium salt fractionation and many other fundamental techniques that are used in the laboratory. This paper will display the results of protein purification, isolation, quantitate, and measure the enzymatic parameters of E.coli bacteria Alkaline Phosphatase by using the common biochemistry laboratory techniques. The procedures that are used in this experiment are column chromatography, ammonium sulfate fractions, heat denaturation, and spectrophotometer. During the quantification processes, the concentration of the proteins of alkaline phosphatase needs to be quantified. Four samples were taken and used for the Bradford Protein Assay.

After completion of the isolated and quantification sample of alkaline phosphatase, it was successfully achieved. The samples were placed in a 96-well plate to measure the absorbance of the enzymatic activity. Overall, the purpose of this experiment is to isolate, quantitate and measure the enzymatic parameters of E.coli AP.

Introduction

Alkaline Phosphatase (AP) control in E. coli is based on the environmental control and function. The bacteria, E. coli in which it is expressed in alkaline phosphatase hydrolyzes phosphate monoesters in the high pH environment of the periplasmic space. The periplasmic space is between the inner impermeable membrane and the porous outer membrane to provide the cell with inorganic phosphate. This reaction below is the hydrolyzes phosphate monoesters:

R-O-PO3H- + H2O à R-OH + H2PO4-

Alkaline Phosphatase (AP) is an enzyme that is widely spread in multiple organisms such as prokaryotes and eukaryotes. The physiological feature in the enzyme is that E. coli cleaves to the phosphoryl groups by inorganic phosphate (Pi) which is found in the periplasmic space. The levels in the Alkaline phosphatase are normally low in wild-type E. coli grown in phosphatase rich media, but expression will increase if the cells are grown in phosphate depleted media. The characteristics of E. coli Alkaline Phosphatase is expressed highly in pH > 7.0. E. coli AP is a heat stable homodimer enzyme that has a molecular weight of 86,000 and contains two Zn2+ atoms per dimer. E. coli AP has a pI of 4.5 and maximal activity at pH 8.0.

In this experiment, we used E.coli K-12 mutant AP which is highly expressed and constitutively expressed under all growth conditions. K -12 mutant is the control of phosphatase production is defective. In this experiment, this isolation and purification of E. coli alkaline phosphatase went through a series of steps such as dialysis, salting out, Ion Exchange Column (IEC), and Sigma Fast BCIP. Buffers and reagents played a major role in isolating AP. It is very important that the treatment of E. coli with lysozyme weakens the peptidoglycan in the cell wall which made the outer membrane susceptible to osmotic shock which releases proteins in the periplasmic space without the release of cytoplasmic proteins. However, the EDTA is bound to the periplasmic membrane and has a positive charge ion. EDTA buffer wants to find Ca2+ in the cell wall which can cause the cell wall to become weaker. It is important that EDTA binds to calcium but not to Zinc. If this happens it can cause the cell wall to destroy. In the gram-negative cell, the lysosomes are used to break down the cell wall by hydrolyzing it. MgSO4 is used as a buffer in the bacteria.

The Dialysis plays an important role in AP because it allows the molecules in the solution to separated and the water will move from a highly concentrated area to a low concentrated area. The beaker was filled with buffer and the dialysis bag is placed in the beaker so that the AP can transfer the salts and ions out of the bag. Dialysis process allowed to keep high molecular weight impurities come out in a high concentration to low based on size. The salting out procedure is based on the use of Ammonium sulfate that was added to the protein which isolated the solution. Ammonium sulfate precipice AP proteins. The heat denaturation process was used to further purify the AP. The water bath allows the proteins to denature and which is why ammonium sulfate was added.

The separation of proteins for E.coli is used by the technique Ion exchange column. This technique was used to separate the proteins and examine the charges and structures of the different proteins present using Buffer A and Buffer B. During this procedure the compounds will display positive and negative charges which are caused by the pH change. The proteins are ae applied to the column that allowed the decrease of ionic strength buffer at pH above the isoelectric point of the protein. The proteins can be eluted with a buffer containing a higher concentration of ions. The sampled would be collected and used for elution. BSA was used for quantification of the proteins. A spot test was done to determine how much AP enzyme was present in all stages. Smalls fragments of AP was placed on a parafilm sheet and test. Sigma Fast BCIP was used to place in the small fragment and the enzyme will turn blue which indicated that AP is present.

Lastly, during AP isolation Bradford Method was used to perform an extracted sample of Stage 1, Stage 2, Stage 3, and Stage 4 Enzyme. Bradford is used as a color indicator, and the dye will bind to the protein and the color will begin to change in the 96 well plate. The colors will vary between brown and blue color. Adding Bradford allowed the proteins to bind. The 96 well plate is placed in a spectrometer to calculate the concentration of the proteins present. The numbers that were given after will be used to plot the absorbance trend line and bar graph of the results.

Material and Methods

Alkaline Phosphatase Day One:

Alkaline Phosphatase was isolated from E. Coli K-12 by adding Lysozyme (breaks down the cell wall), DNase(Breaks down present DNA after the cell wall is broken), EDTA(positively charged ion that wants to bind to Ca2+ and Mg 2+), and MgSO4 ( acts as a buffer). Dialysis tube is cut and soaked in a beaker during incubation. After centrifugation, all the reagents were transferred into a dialysis tubing in a beaker with dialysis buffer and was replaced twice.

Alkaline Phosphatase Day Two:

Alkaline Phosphatase was measured from E. Coli K-12 by obtaining the Stage 1 Enzyme and measured the volume (Stage 1 Enzyme decant) After centrifugation, heat denaturation (denature unwanted proteins and make AP heat stable) is next by placing the tube in the water bath. Stage 2 Enzyme is decanting and measured. Enzyme assay is being removed from Stage 2 Enzyme. Ammonium sulfate (salting out, solubility ) is added for Stage 2 Enzyme. After the second centrifugation, Tris HCl is added and MgSO4. The enzyme is transferred into a new dialysis tube submerged in a beaker with fresh dialysis buffer and replaced twice.

Alkaline Phosphatase Day Three:

Alkaline Phosphatase was quantified from E. coli K-12 by retrieving Stage 3 Enzyme and measured the volume. Enzyme assay is removed and placed in an Eppendorf tube. A chromatography column is (separate the charges) with DEAE bed, ten tubes are used for the chromatography column. Stage 3 enzyme is added to the column with buffer A. Buffer A( eluted out of the column)and Buffer B (eluted enzyme from the column). Repeated five times. A spot test (to test to see if any proteins are present in all stages). Parafilm is used to place the small fragments on. Sigma Fast BCIP (will show the AP)is added to each fragment (including column, and subsequent fractions). Highest AP present is used and measured for Stage 4 Enzyme. All stages are needed to be quantified. All stages are placed in the 96 well plate twice, and BSA(proteins show the concentration that needs to be quantified. Bradford Assay (Bind to the proteins, color indicator) is added to each stage. Absorbance is measured with the spectrophotometer. Plot the average for the standard curve to determine the protein.

Results

Figure 2. Protein quantification using BSA Standard. A) The table shows values of volume and concentration of BSA to obtain absorbance at 595 nm. B) The line graph shows the Absorbance at 595nm vs BSA concentration to obtain trendline.

Figure 3. Protein concentration determined by using BSA Standard. A) The table displays values of the protein, volume, and total protein concentration. B) Bar graph displays protein quantification of the four stages based on Protein concentration. C) Bar graph showing total protein concentration in each isolation AP stages.

Alkaline Phosphatase went through a series of steps such as isolation, quantification, separation and enzyme kinetic which the following data shows was collected. For the isolation of alkaline phosphatase, the volumes were different after going through before and after stages of dialysis. The table displays the different measured volumes of before and after the process of dialysis for each stage. Secondly, the heat degeneration is 66.29ml that was measured after the process. The spot test has dark blue in 15 through 18. For the quantification of alkaline phosphatase, the absorbance for each concentration varies: 0.083 and 0.084 is absorbance based on 595 nm of wavelength. The average of absorbances are added together for BSA with a concentration of zero is 0.083+0.084= 0.0835. This will be done of BSA volume: 5ml with a concentration of 20ml absorbance at 595nm of 0.094+0.90=0.092. This will continue until the last volume and concentration. The graph has resulted in concentration vs absorbance at 595 nm with a Y-intercept f y=0.0009x and R^2- 0.0308. The protein quantification is measured based on the numbers from the chart. The protein quantification is also determined by the use of BSA. The four stages absorbances are added together.

Discussion

The purpose of this experiment was to isolate, quantitate, and measure an enzymatic parameter of E.coli Alkaline Phosphatase using the basics of biochemistry laboratory techniques. Based on the results, we were not able to know because we are not sure which in specially for AP. It was imperative that we isolated AP successfully. We went through a series of purification methods while completing this experiment. Heat denaturation, Ammonium sulfate fractionation, Ion exchange chromatography, and spectrophotometry were used to help the purification process. One technique that was important to grasp the concept of was salting in and out. Salting out it was one of the techniques we did for heat denaturation which allowed the AP to become resistant to heat. Overall, the techniques were mastered, and it was well important to understand while completing this experiment.

In figure 1 results are shown that isolation of AP from E.coli K12. Figure 1. A is a table that shows the actual value of each step during the dialysis step and heat denaturation step. Also shows the volume that was recorded during each measured volume. Figure 1.B is a spot test results is an indication of protein that tested to examine if any proteins were present in all the stages. In figure 2 results are shown the protein quantification using BSA standard. Figure 2.A above is given the values of the absorbance that was used based on the spectrophotometer. In each 96-well plate, BSA concentration was pipetted into each as followed: 0 ul, 20ul, 40ul, 60ul,80ul, 100ul, and 200ul. The average of the absorbance is added based on all stages. Figure 2.B a trendline of the absorbance vs BSA. The concentration of the enzyme is showing a slope at low concentration. The numbers that were used in the table and plotted accordingly. The graph represents how well the quantification method concentrated in AP. Figure 3.A. protein concentration is used by determining BSA Standard based on all four stages using 2ul of concentration. The corrected Abs, Protein, Volume and total protein were calculated to get the protein concentration of all stages. Figure 3.B is results are that the concentration of the protein concentration decreased during Stage 2 and Stage 3, but increase slightly in Stage 4. This may be due to incorrect pipetting which caused the St. 4 to be slightly higher in concentration. Figure 3.C shows the total protein that is in all stages. St. 3 was slightly low which is a good indication that about less 0 of protein was still available after isolation and quantification. Bradford Assay(not shown)was used in this experiment as an indicator for the stage enzymes. It allowed users to examine the absorbance. The BSA was added to show the protein concentration, and quantifying this stage it allowed us to see how much was quantified and how much protein is available.

Other Alternative isolation strategies that can be used is to get similar results using a different salt concentration to isolate E.coli protein or Western blot. Using a different salt can get a better salting out method, and allow the ionic strength substance to increase and the high concentration of salt solubility of the AP will drop. Western blot can be used during the separation technique which will separate the proteins an identify AP. The AP proteins will be separated by molecular weight and results are placed onto a membrane producing bands for each of the protein that is present. In AP proteins it will only be visible in proteins.

Overall the experiment was interesting and I learned different techniques on how to isolate AP from E.coli. I also learned how to correctly used the micropipettes, and used them throughout the lab. During this experiment, we were taught how to make a buffer for the dialysis bag. The purpose of this lab was to isolate, quantity, and measure Alkaline Phosphatase from E. Coli 

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