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Enzyme are basically Proteins
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BC 367 Experiment 3 Purification and Characterization of the Enzyme Lysozyme Introduction Enzymes are truly remarkable catalysts. For example, catalase can carry out the decomposition of up to 5x10^6 moles of H 2 O 2 per minute per mole of enzyme (the turnover number). Even though the turnover numbers of most other enzymes are substantially lower than this, most of the reactions they catalyze occur at rates at least a million times faster under physiological conditions in the presence of the enzyme than in its absence. In this experiment you will purify an enzyme, using its known activity to monitor the process. The isolation and purification of a specific protein or enzyme is generally a difficult task. First, the enzyme must be liberated from its source tissue in an active form. Fortunately, a wide variety of tissue disruption techniques have been developed. However, the enzyme to be purified is usually only a small percentage of the total protein in the crude extract of the tissue. The object of protein purification is to remove nonprotein contaminants as well as to isolate the protein in question from other proteins. The first objective is relatively easy to obtain, whereas the latter is more difficult. For example, it is not unusual for an enzyme to be 0.1% of the total protein in a crude tissue extract. To purify this enzyme to homogeneity, 99.9% of the protein must be removed, preferably with as little loss as possible of the desired enzymatic activity. This can be a difficult task for two reasons:
Despite the many difficulties associated with enzyme purification, numerous enzymes from many sources have been purified to homogeneity or close to it. The enzyme you will purify in this experiment is lysozyme, one of the first enzymes for which the complete three-dimensional structure was determined by X-ray diffraction. Egg white, human milk, tears, spleen, and many other tissues, including plant sources, contain this enzyme. The systematic name for lysozyme is "mucopeptide N-acetylmuramylhydrolyase." Lysozyme catalyzes the hydrolysis of β-1,4- linkages between N-acetylmuramic acid and 2 - acetamide- 2 - deoxy-D-glucose residues in mucopolysaccharides or mucopeptides of a variety of microorganisms. The reaction catalyzed is illustrated below: O CH 2 OH OH NH 4 Ac O O CH 2 OH NH 4 Ac CH 3 CHC-O- O O O O CH 2 OH OH NH 4 Ac OH HO HO O CH 2 OH NH 4 Ac CH OH CH 3 C-O- O O
Basic Structural Unit of Bacterial Cell Walls Lysozyme N-acetylmuramic acid 2-acetamido-2- deoxy-O-glucose β-1,4 linkage
Lysozyme performs an antibiotic function for the human body. This action is attributed to the ability to destroy invading bacteria by hydrolyzing the mucopolysaccharides of the cell wall. Egg white is particularly rich in lysozyme and thus will be the starting material for your purification. Lysozyme has a lower molecular mass (14.3 kDa from chicken egg white) than most other proteins, and therefore considerable purification can be achieved by simple gel- filtration, which separates proteins on the basis of size. Furthermore, the unique charge characteristics of lysozyme, which has an unusually high pI of 10.5, can also be exploited through ion exchange chromatography. Moreover, lysozyme is a remarkably stable enzyme and retains catalytic activity even after storage for several days at room temperature. These unique characteristics coupled with the fact that a major source of the protein is cheaply obtained at the local grocery store or farm make it an excellent choice for an introduction to enzyme purification techniques.
0.05 M Tris Base, 0.05 M NaCl (pH 8.2)- Tris-NaCl Column Buffer: Calculate the masses of Tris and NaCl needed before coming to lab. Prepare 300 mL of this column buffer to equilibrate and run your column. Adjust the pH with HCl before adjusting the volume to 300 mL. 0.2 M carbonate buffer (pH 10.5): Prepare 50 mL of 0.2 M sodium carbonate (Na 2 CO 3 ) buffer to run your column. Calculate the mass of Na 2 CO 3 needed before lab. Adjust the pH. Note that the pH of this buffer is critical to the success of your experiment. 0.1 M potassium phosphate buffer (pH 7.0): Prepare 100 mL of 0.1 M potassium monobasic phosphate and 100 mL of 0.1 M potassium dibasic phosphate. Use the Henderson-Hasselbach equation to determine approximately how much of the monobasic to add to 100 mL of the dibasic to give a pH of 7.0 (pKa= 6.86). Put 100 mL of the dibasic solution into a beaker and add a few mL less than calculated amount of the monobasic solution. Check the pH with the pH meter and continue to add the monobasic solution until you reach a pH=7.0. Store the remainder of this buffer in a labeled bottle in the cold room when you are done today. C. For those doing ion exchange with CM-Sephadex C50 (the final lab table): CM-Sephadex C50: Start with 0.4 g dry CM-sephadex C50. Swell the dry sephadex in ~200 mL distilled H 2 O by boiling for 1 h, using the “double-boiler” method. After cooling, equilibrate with Tris- NaCl column buffer (see below: 0.05 M Tris, 0.05 M NaCl [pH 8.2]) by decanting H 2 O and replacing it with ~100 mL buffer. Stir gently and allow gel to settle. Decant buffer. Repeat buffer addition, and decant leaving approximately a volume of buffer equal to the volume of the gel slurry. 0.05 M Tris Base, 0.05 M NaCl (pH 8.2)- Tris-NaCl Column Buffer: Calculate the masses of Tris and NaCl needed before coming to lab. Prepare 300 mL of this column buffer to equilibrate and run your column. Adjust the pH with HCl before adjusting the volume to 300 mL. All other solutions should be identical to those prepared in part B above for CM-Sephadex C25. II. Preparation of Columns-Week 1 A. Gel Filtration (to be done by one-third of the groups) Wash your column and then rinse with deionized water. Check to make sure water flows through column. Place a plastic funnel in the top of the column to serve as a packing reservoir and a buffer reservoir. You may wish to secure your funnel with parafilm. Place a plastic stopcock on the tip at the bottom of the column and make sure it is in the closed position. You will use the sephadex G-75 prepared above, which excludes proteins with a molecular weight of about 75 kD. Fill the column about 1/4 full of the Tris-NaCl column buffer. Add enough of the sephadex to fill the column. Turn the stopcock to "on" and allow to drip. Continue to add slurry until the column is filled to about one inch from the top of the column. DO NOT allow the level
IV. Chromatography of the Buffered Egg White Solution- Week 2 IMPORTANT NOTES:
l. Calculation of protein concentration. This comes from the A 280 data used to determine protein concentrations from your lysozyme Warburg-Christian standard curve. Be sure to correct for any dilutions that you made. Sample Calculation: Assume that the absorbance value for a particular fraction corresponds to a protein concentration of 0.5 mg/mL as determined from the standard curve. This fraction was diluted 5- fold for the protein assay. Protein concentration = 0.5 mg x 5 (dilution factor) = 2.5 mg/mL mL
2. Calculation of enzyme activity units and specific activity: You determined the initial activity of the starting material (the buffered egg white) and the column fractions in terms of ΔA 450 /min. The activity of most enzymes is defined in terms of activity units. For lysozyme one unit of activity is defined as being equal to an absorbance decrease at 450 nm of 0.001 per minute at pH 7.0 and 25oC ( - ΔA 450 /min =0.001; note that enzyme activities MUST BE positive numbers). The specific activity is defined as enzyme units per milligram of protein in the assay. The calculation of specific activity is essential for determining the effectiveness of the purification. As the enzyme is purified, the specific activity will increase since inactive protein is removed. Sample Calculations: Suppose, for example, that your buffered egg white had a protein concentration of 12 mg/mL (after correcting for dilution). It was diluted 50-fold for the enzyme activity assay and had an activity of 0.030 ΔA/min. The original, undiluted egg white therefore has the following: Units of activity = ΔA/min = 0.030ΔA/min = 30 units 0.001 ΔA/min/unit 0.001 ΔA/min/unit Correcting for 50-fold dilution: 30 units x 50 = 1500 units Specific activity = Units/mg protein mg protein = 12 mg/mL x 0.1 mL (used in activity assay) = 1.2 mg from standard curve and corrected for dilution already Specific activity = 1500 units = 1250 units 1.2 mg mg protein Similar calculations should be made for each column fraction that shows activity. Column fractions that do not show a gradual and continual change in absorbance during the assay interval do not possess enzyme activity. 3. Calculation of Total Protein, Total Activity, % Recovery, and Fold Purification.
Remember: Total volume of buffered egg white applied to the gel-filtration column was l mL, and the total volume of each gel-filtration fraction was 2 mL. Total volume of buffered egg white applied to the ion-exchange column was 2 mL, and the total volume of each fraction was 2 mL. Total Protein = Total volume x protein concentration (mg) (mL) (mg/mL) Total activity = Specific activity x total protein (units/mg) (mg) % Recovery = Total activity of Fraction x 100 Total activity of egg white Fold Purification = Specific activity of fraction Specific activity of egg white
4. Additional Analysis In your notebook, also include a plot of both protein concentration and total activity versus fraction number (plotted on the same graph). Do not include egg white on the graph. Include this plot in your report. NOTE: you may plot these parameters as "percent of total" (the sum of the fractions) to achieve similar scales for the two graphs. VII. PAGE on the Crude Buffered Egg White and the Purified Lysozyme- Week 3 Polyacrylamide gel electrophoresis (PAGE) is a form of zone electrophoresis used to separate proteins or nucleic acids. The support medium is a polymer of acrylamide cross-linked with N,N dimethyl bis-acrylamide. O C O (^) C O NH 2 NH 2 n -CH 2 -CH-CH 2 -CH Na 2 S 2 O 4 hv or n CH 2 =CH-C-NH 2 PAGE is usually carried out in slabs of the gel thus combining electrophoresis with molecular sieving. By varying the concentration of the gel and its degree of cross-linking, its molecular-sieving properties can be controlled over a wide range, and it can be used very effectively to separate mixtures of proteins. Charged proteins are separated during PAGE into discrete bands. The separated zones of proteins can be made visible with dyes that bind to proteins and scanned photometrically with densitometers that have the capacity to integrate the resulting curves. By this means, a simultaneously qualitative and quantitative analysis of the mobile charged species is obtained upon separation. PAGE gives an almost unparalleled separation of protein components in a sample and is thus a very important analytical tool as a
grease the silicon rubber gasket of the pod lightly with celloseal. The gasket will seal the pod against the alumina (white) plate of the sandwich.
Experimental Procedure While your gel is running, you should obtain electrospray ionization mass spectrometry (ESI MS) data on a sample of pure lysozyme.