Lab3-Protein Purification

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Lab 3-1 LAB 3 PEROXIDASE EXTRACTION AND PURIFICATION FROM HORSERADISH ( Armoracia rusticana ) ROOTS Name:_____________________________________________ Date_____________________ Dear Student: If you’re typing directly into this document, please use BLUE font color for anything you type so I can tell it apart from what I have written. INTRODUCTION Protein purification from biological tissue is a critically important skill that any lab biologist should be familiar with. In this lab exercise, we will learn about how biologists purify proteins for future use. Among the future uses could be chemical analysis, medical uses and others. We will try to purify a specific protein called peroxidase. The enzyme we use in this lab will be used by us in lab 3 and we will also investigate this enzyme in our IRPs, so this is a very important enzyme to learn about. We will purify this protein from roots of horseradish. The structure of this enzyme is shown below as both a surface model and a ribbon model. As you can see, there are many alpha helix structures in this protein. In addition, the active site of the enzyme is dominated by an iron heme group. The protein is slightly negative at cellular pH. Plant peroxidases chemically reduce harmful H 2 O 2 and other similar reactive molecules to water by oxidizing some organic substrate. H 2 O 2 and other similar reactive molecules are very harmful to biological tissues because they oxidize important biological molecules (they cause oxidative stress). So peroxidase is an important stress enzyme that plants use to cope with the oxidative stress caused by H 2 O 2. Without this enzyme, plants (and even animals) would have an abnormal buildup of H 2 O 2 , which could lead to some damaging results for the cells. One way to see this reaction is to react H 2 O 2 with a synthetic chemical called guaiacol. The product, called tetraguaiacol is an amber color and can easily be detected using a spectrophotometer:

Lab 3-2 Knowing the reaction above will be very useful toward understanding complete the procedures of this lab. The goal of this lab is to familiarize you with the techniques of protein purification and analysis. This lab will be conducted over a three-week period. First, we will start with a crude protein extraction and purification to separate proteins from other macromolecules. In the second lab meeting, we will specifically purify peroxidase from other proteins. Finally, in the third lab, we’ll analyze how well we did. 3A. CRUDE TOTAL PROTEIN EXTRACTION AND PARTIAL PURIFICATION BY SALTING OUT In 3A, you will extract and partially purify all proteins from root tissue of horseradish plant. The goal is to extract proteins from the cells and tissues of the root and to separate proteins from other macromolecules, such as carbohydrates and nucleic acids. With most proteins, purifications must be done under cold conditions to prevent denaturation. The advantage of working with peroxidase is that it is heat-stable, so we can work at room temperature for long periods, but should be stored in a frozen state. PROCEDURE Reagents and Materials: 1. Horseradish roots 2. Homogenization buffer  0.1 M phosphate buffer, pH 7.0. This buffer helps control the pH 3. 25 mM phosphate buffer, pH 7.5 4. Ammonium sulfate  salt used to precipitate proteins 5. Cheesecloth  used to filter homogenized chicken to remove large unhomogenized chunks and to remove lipids 6. 50 ml centrifuge tubes pre chilled 7. Blender  for homogenization 8. Desalting column 9. Microfuge tubes 10. Disposable pipets 11. Pipet tips 12. Weigh boats 13. 100 ml beaker (2/grp) pre chilled 14. 250 ml beaker (2/grp) pre chilled 15. 125 ml e flask (1/grp) pre chilled 16. Small petri dish (1/grp) pre chilled 17. Stir plates (1/grp) 18. Stir bars (1/grp) 19. Ice 20. Sharpies 21. 50 ml graduated cylinder (1/grp) 22. Ultracentrifuge at 5 o C 23. Ring stand with clamps (1/grp)

Lab 3-3 Homogenization, Filtration and Centrifugation 1. WEAR GLOVES THROUGHOUT THE WHOLE PROCEDURE. Horseradish can be irritating to skin and some of the chemicals we use may be skin and eye irritants. 2. Tissue Preparation — Obtain about 50 g horseradish root and peel/discard the skin 3. Chop up the root into small pieces with a knife. Record the actual weight of the chopped up you used after removal of extraneous tissues: _______g 4. Soluble Protein Extraction — Place the minced tissue and 75 ml of cold 0.1 M phosphate buffer in a blender and put the top on the blender. Homogenize the tissue 4x in 30 second bursts (or maybe more-ask your instructor). 5. Filtration — Obtain 4 layers of cheesecloth and place them over a 250 ml beaker. Pour homogenization buffer through the cheesecloth to pre-wet it and then squeeze out the excess buffer from the cheesecloth. Discard the buffer in the beaker down the sink. Now pour the homogenized tissue onto the cheesecloth in two or three batches. In between batches, push the chunks to extract as much liquid as possible through the cheesecloth using a spatula or the bulb end of a transfer pipette. Squeeze out the cheesecloth to get all remaining filtrate through the cheesecloth. Once all the homogenate has been filtered, you are ready for the next step. The cheesecloth will remove unhomogenized chunks of tissue as well as lipid from the solution. 6. Centrifugation — Put 25 ml of the filtered sample into a pre-chilled 50 ml centrifuge tube (a clear round bottom tube with a screw-on cap). Label the tube by writing on it with a sharpie pen (don’t use label tape). Balance the tubes following the directions of your lab instructor. Centrifuge the balanced tubes (opposite each other in the centrifuge) at 3000 x g for 20 min. 7. Save two 1 ml aliquots of the supernatant (aka crude homogenate) into microfuge tubes and label as “C. H., Grp #, & Lab section”. Keep these samples in an ice bath at your station until the end of lab. The instructor will freeze these at the end of class for later analysis 8. Measure and record the volume of the remaining supernatant in a graduated cylinder. Volume of the supernatant ____________ ml 9. Discard the pellet that is at the bottom of your centrifuge tube. Salting-out Proteins 10. Ammonium Sulfate Precipitation — Ammonium sulfate is a salt that, when added to a protein solution, will cause the proteins to come out of solution by disrupting the hydration sphere of the

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Lab 3-4 dissolved proteins. This allows us to separate proteins from carbohydrates and nucleic acids, which stay in solution. Doing so will also let us concentrate the proteins by first precipitating them out of a large volume and then later re-dissolving them in a smaller volume. To do this, we will add solid (NH 4 ) 2 SO 4 to our centrifuged supernatant slowly until we reach a salt saturation level of 80% (0.57 g (NH 4 ) 2 SO 4 per ml of filtrate). a. Knowing the volume of the supernatant, and knowing that you need 0.57 g of (NH 4 ) 2 SO 4 per ml of filtrate, obtain the total amount of (NH 4 ) 2 SO 4 that you’ll need and write down this amount: Amt. of (NH 4 ) 2 SO 4 needed: ________________________g b. Slowly (over a period of 15 min) add 0.57 grams of ammonium sulfate per 1 ml of your centrifuged protein solution. It is best to perform this step in a chilled beaker on a magnetic stirrer. Place your sample into a beaker. Use a small magnetic stir bar to keep things mixed up. c. Avoid stirring too violently because this could shear (tear up) the proteins. If you see too many large bubbles forming then you are shearing your proteins. d. Stir for an additional 15 min after adding the ammonium sulfate to give the salt a chance to dissolve. 11. Gently pour the salted-out solution into a clean centrifuge tube. Avoid pouring out the stir bar and also avoid the undissolved salt crystals that may be at the bottom of the beaker. 12. Centrifugation — Centrifuge the sample as before in a pre-chilled balanced centrifuge. At the end, gently pipet the supernatant into a separate 50-ml blue-capped sample tube labeled “SUPER” + GRP# and section…etc. Give this tube to your instructor for freezing. 13. Keep the pellet, consisting of precipitated proteins, in the centrifuge tube. QUESTIONS 1. Explain the purpose of why we salted out the proteins. Why was it done and how does it work? 2. Why do we need to remove the (NH 4 ) 2 SO 4 salt before we go on to future steps? Briefly explain how the salt is removed from the protein.

Lab 3-5 3B. PEROXIDASE PURIFICATION USING GEL FILTRATION AND ION EXCHANGE CHROMATOGRAPHY Next, we will use ion exchange chromatography to separate proteins based on their charge. However, we must first remove the ammonium, sulfate salt that was used during the last lab period. Desalting the Protein Sample We must remove the ammonium sulfate salt from the protein pellet. High concentrations of salt, such as ammonium sulfate, can interfere with subsequent protein purification steps so removing this salt is necessary. To remove the salt, we will use a chromatography technique called gel filtration chromatography. Gel filtration chromatography separates different chemicals by their different sizes. The process of gel filtration chromatography employs a column (see figure below) that contains a buffer called the mobile phase and a semi solid (usually) material called the stationary phase. The sample to be desalted is placed in the column sample reservoir and gravity is used to pull the sample through the first frit (or filter) and into the column. Since the stationary phase consists of beads with small pores in them, the salt, which is a small molecule compared to the proteins, enters the pores and therefore spends more time in the beads. The larger proteins do not pass through the pores and simply go around the beads. Therefore, the larger proteins move faster through the column than the smaller salt ions. At the bottom of the column, the proteins come out (elute) first followed by the salt. This results in separation of the proteins from the salt. Once the sample has been desalted, the proteins can now be subjected to another kind of chromatography called ion exchange chromatography, which we’ll begin on the second day of the procedure.

Lab 3-6 Procedure for Gel Filtration Chromatography 1. Designate a small container for buffer waste and label it. Also, get a large plastic container and fill it with ice to use as ice storage. 2. Resuspend ammonium sulfate pellet — add 2 ml of 25 mM phosphate buffer to the ammonium sulfate pellet. Gently mix the buffer and the solid material until the pellet dissolves. Keep on ice as much as possible during the procedure. 3. Be sure that no buffer remains above the top frit of the column. If there’s buffer above the top frit, then use a disposable transfer pipet to remove it and discard into your waste container. 4. Load the desalting column — load 3 ml of the mixture on the desalting column. Allow the liquid to drain to the frit (the plastic cover on the column resin). The column will only drain if there is pressure by fluid above the frit. When you load your sample into the sample reservoir this creates pressure on the fluid (buffer) inside the column. The result is that the buffer elutes out of the column. Discard the flow through, which is mostly buffer. 5. Elute the sample from the desalting column — add 5 ml 25 mM phosphate buffer to the column and collect the flow through in a 15 ml blue-capped tubed labeled “Desalted” as well as your group number and lab section. The flow through contains the peroxidase and all other proteins minus the salt. 6. Remove the desalting column from the ring stand and record the volume of the flow-through in the tube ____________ ml 7. Save two 1 ml aliquots of the desalted material in two microfuge tubes and label as desalted fraction with your group number and your lab day (two labels better than one: one label on side and one on cap). Keep these as well as the larger 15ml tube on ice. Now that the ammonium sulfate has been mostly removed, we can now use ion exchange chromatography to separate proteins based on their charge. Different proteins have different total charges at cellular pH. Some are weakly or strongly negative (anions) and others are weakly or strongly positive (cations). Protein total charge comes from the types and number of charged amino acids they contain. (Remember, some amino acids are negative while others are positive. If a protein has lots of negative amino acids on its surface, then that protein will be strongly negative.)

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Lab 3-7 Ion exchange chromatography allows the separation of proteins based on their charge. The resin within the column can have either positive groups attached to it (these are called anion exchangers because they capture anionic proteins) or negative group (called cation exchanges). If a mixture of proteins is added to an anion exchange column, negatively charged proteins will bind to it, with highly negative proteins binding most strongly and weakly negative proteins binding more weakly. Positively charged proteins don’t bind to the column at all. The positive proteins can be eluted simply by passing a neutral buffer through the column. Once these are removed, an increasing gradient of salt concentrations can be passed through the column to dislodge first the weakly negative protein and then, as the salt concentration gets higher, the more strongly negative proteins. The idea here is that the salt ions will compete with the protein for binding to the cationic resin, eventually dislodging the proteins that wash out of the column. At cellular pH, peroxidase is a weakly anionic protein (weakly negative) and so we should use an anion exchange column (positive). The column we’ll use is DEAE-sephacel: positive DEAE (diethylaminoethyl) covalently attached to cellulose resin (see figure to right). Negative proteins in our desalted sample (including peroxidase) will bind to the DEAE-sephacel and we can then separate these from positive proteins. The negative proteins will then be eluted in a gradient of KCl salt (0-300 mM). As the buffers are passed through the column, we will collect 5 ml fractions in test tubes. This ensures that we separate different proteins from each other. To know that an eluent (the stuff coming out of the column) is free of proteins, we will measure the absorbance of each fraction at 280 nm, a wavelength at which ALL proteins absorb light.

Lab 3-8 Procedure for Ion Exchange Chromatography 1. You are now ready to further purify your sample through ion-exchange chromatography. Obtain an ion exchange chromatography column and secure it to the ring stand. What you want to do is put the thawed and mixed desalted sample on the column and then subsequently remove: a. All the proteins that do not bind to the column b. And then all the proteins that bind weakly to the column c. And finally, all the proteins that bind strongly to the column 2. Label about 15-20 blue-capped plastic test tubes using label tape. Place the following information on the labels: sequential numbers (from 1 to 15), your group number, and your lab day 3. Let the storage buffer that is above the column frit flow out into a waste container and then discard it. Your instructor may have already done this for all the groups in the class in order to save time. 4. Place tube # 1 in a plastic beaker containing ice below the column. 5. Next, Load the complete desalted protein solution onto the sephacel column. Begin to collect a 5 ml fraction in tube #1. It is likely that tube #1 will not reach 5 ml with only your desalted sample. Therefore, once ALL of the desalted sample has flowed through past the top frit you can begin adding 10 ml of 0 mM KCl buffer wash to the column. 6. Once tube # 1 reaches the 5 ml mark, remove it from beneath the column and replace it with tube # 2. Keep collecting 5 ml fractions in tube # 2 and all subsequent tubes. While collecting the fractions, one member of the team can invert tube #1 to mix it and measure the absorbance of tube # 1 at 280 nm. Since proteins, absorb at 280 nm you can determine if proteins are present in the flow-through by measuring the absorbance of the fractions. 7. Once the absorbance of tube #1 (F1) is measured, pour the contents of the cuvette back into the F1 blue-capped tube and store this tube in a large plastic beaker filled with ice. 8. It is likely that the absorbance of F1 will be high because all un-bound proteins have flowed into it. The 0 mM KCl buffer will push out all these neutral or positively charged proteins. 9. Keep collecting 5 ml fractions and keep washing with 5 ml of buffer in more and more fractions until the absorbance of a fraction reaches below 0.1, indicating that no more proteins are flowing out of the column. 10. Once the absorbance of a fraction goes below 0.1 and once there is no liquid above the top frit (everything has drain through the column), you can add 10 ml of the first KCl salt solution, the 50 mM KCl. Continue collecting 5 m l fractions below the column. You must indicate (in the table on the next page) which fraction was in place when you first started adding the 50 mM KCl . KCl will dislodge weakly-binding proteins which will elute from the column. This salt concentration may be sufficient to dislodge the peroxidase from the column. You should start to see the A280 values go up because proteins are now eluting from the column.

Lab 3-9 11. If the A280 of the last fraction is still high, add another 5 ml of 50 mM KCl. Keep doing this until the A280 is less than 0.1 12. Repeat step 11 with all remaining KCl solutions (150 mM and 250 mM). Keep collection 5 ml fraction. Once the A280 of the last fraction from the 250 mM wash goes below 0.1, you are done with the ion-exchange procedure. Remember to indicate on the table which fraction first received the 150 mM KCl and the 250 mM KCl. 13. At the end, remove all the fraction tubes from the ice bath in which you are storing them, place them in blue wire rack and give this rack to your instructor to freeze. 14. Record the results in the table below and also prepare a bar graph of your results with fraction number on the X-axis and absorbance at 280 nm on the Y-axis. Mark which fraction corresponds to which step in the procedure (50, 150, 250 mM KCl) Fraction Number Abs. Fraction Number Abs. Fraction Number Abs. Fraction Number Abs. 1 7 13 19 2 8 14 20 3 9 15 21 4 10 16 (if needed) 22 5 11 17 23 6 12 18 24

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Lab 3-10

Lab 3-11 Questions 1. List the fractions (by fraction number) that showed up as absorbance peaks from the previous graph and carefully explain the causes of the high absorbance values for each peak (you must describe what class of protein makes up each peak and what elution buffer is responsible for eluting that class of protein). 2. If the procedure of this lab was done perfectly and all the results are as expected, which one sample (out of all samples) do you think will contain the highest overall protein concentration ? Fully explain why you think these will contain the highest protein concentration? 3. If the procedure of this lab was done perfectly and all the results are as expected, which sample do you think will have the highest peroxidase purity ? Fully explain .

Lab 3-12 3C-TOTAL PROTEIN QUANTIFICATION AND PEROXIDASE SPECIFIC ACTIVITY DETERMINATION Protein Concentration of the Samples and Fractions In order to determine how well we purified peroxidase, we need to know something about the amount of peroxidase relative to all proteins in each sample. A very pure sample will contain a high concentration of peroxidase compared to other non-peroxidase proteins. To determine a sample’s “purity” we have to determine that sample’s specific activity of that protein. Specific activity is the activity of your target protein divided by the total protein concentration of a sample. Thus, it is clear we need to measure two things for each sample that we obtained: (1) the total protein concentration and (2) the activity of peroxidase. In the next procedure, you will determine the total protein concentration of all the samples that you obtained. Next week we will determine the activity of the proteins in each sample. The method that we will use for determining protein concentrations is called the Bradford Assay, which you learned about in lab 1. Remember, the Bradford reagent binds to any and all proteins indiscriminately. When the Bradford reagent binds to a protein, the resulting product’s color is blue and its absorbance has a maximum at 595 nm. The absorbance level depends on the protein concentration. Protein + Bradford Reagent  PB Complex (clear) (brown) (blue) To measure the amount of protein in your unknown samples, you first need to calibrate the change in Bradford reagent absorbance induced by different amounts of protein. You already did this in lab 1 when you determined the extinction coefficient of BSA bound to the Bradford Reagent (remember??). Question In your own words, briefly explain the theory behind how we will determine the total protein concentration in our samples.

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Lab 3-13 Procedure for Determining the Protein Concentrations of Samples 1. Thaw the fractions collected from ion exchange chromatography. Once thawed, invert the tubes several times to mix the contents of the tubes. Keep the fractions on ice. 2. Obtain 4-5 cuvettes and use these to perform the Bradford Assay. (A used cuvette can be reused by discarding the Bradford Reagent/protein mixture into the waste container in the hood, rinsing with isopropyl alcohol and then rinsing with water.) 3. Make a blank cuvette by adding 0.25 ml D.I. water to the cuvette and adding 1 ml of Bradford Reagent. Cover the cuvette with parafilm and invert. Wait 5 minutes and then invert the cuvette again invert to mix and then blank the spectrophotometer at 595 nm (the absorbance maximum of the Bradford-Protein complex) with this sample. 4. To a cuvette add 250 µl of a sample (for example, the CRUDE HOMOGENATE). Next, add 1000 µl of Bradford Reagent. As you make each cuvette, cover it with parafilm and invert three times to mix. Let the cuvette sit at room temperature for at least 5 minutes, invert again to mix and take an absorbance reading at 595 nm. (If there is a need, your instructor may ask that you dilute your protein samples by 1/10 th and use these diluted samples in the Bradford assay. The samples that might require a dilution are as follows: CH, DS, Super. Ask your instructor. If you do dilute your samples, you will need to correct for this dilution by multiplying your measured absorbance values by 10. However, if you do not need to dilute the sample then DO NOT multiply by 10.) 5. Repeat this procedure with each sample and fraction that you have (SUPERNATANT, DESALTED AND F1-F?). You must do this without delay to ensure you don’t run out of time. As soon as a fraction comes off the IEC column and its absorbance at 280 has been taken (previous procedure) then you must perform the Bradford Assay on it. 6. Record absorbance values in the table below and use the extinction coefficient you determined from lab 1 (BSA/Bradford standard curve) in the Beer-Lambert equation to determine the corrected protein concentration. I would like all members of the same group to use the same extinction coefficient. Compare all your coefficients from lab 1 and if they are all similar, use an average for your whole group. In this way, I can compare group member answers to each other.

Lab 3-14 Sample Abs. Protein Conc. ( μ g/ml) Sample Abs. Protein Conc. ( μ g/ml) Crude Homogenate (CH) F10 Supernatant F11 Desalted (DS) F12 F1 F13 F2 F14 F3 F15 F4 F16 F5 F17 F6 F18 F7 F19 F8 F20 F9 7. Prepare a bar graph of your results with the sample identifier on the X-axis and the protein concentration (as μ g/ml) on the Y-axis (It’s OK to connect these data points with lines because there is no mathematical relationship between the X and Y variables).

Lab 3-15

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Lab 3-16 Questions 1. Which sample contained the highest protein concentration? ______________________________ 2. Does this result agree with what you may have predicted back on page 11? Fully explain .

Lab 3-17 Specific Activity of Peroxidase in Samples and Fractions When conducting protein or enzyme purification studies it is necessary to have some way of determining how pure your purified sample is. This is normally done by determining the concentration of the protein or enzyme that you are trying to purify compared to the concentration of total protein. Equation 1 The denominator in the equation above is easy to determine and you have already done so (see section 2B). However, there are two problems with this method: (a) This method does not give any indication of the structural and functional integrity of the protein that you are trying to purify. (b) The numerator in the above equation is hard to determine. It is hard to determine the concentration of one specific protein that is mixed in a solution with many other proteins! Instead of measuring purity using the technique of equation 1, we can instead use another indicator of purity called the specific activity which is given in the equation below: Equation 2 The activity of an enzyme can be estimated by determining the amount of product that the enzyme catalyzes over time. Equation 3 One unit of enzyme activity is the amount of enzyme that is necessary to produce 1 μmole of p roduct in 1 minute, Equation 4 So, instead of measuring the concentration of enzyme in our samples, we measure what those enzyme molecules are doing, their activity . Activity can be used as a substitute for concentration because the two terms are somewhat equivalent. If a sample is very active, then you can infer that the sample probably has a high concentration of the enzyme causing the activity. Purity = conc. of protein of interest conc. of total protein Specific Activity = Activity of the Enzyme to be Purified g Total Protein µ Activity (U) = Amount of Product Time 1 unit of activity = 1 mole of product min = 1U µ

Lab 3-18 Activity can be determined by measuring how much product is forming over a given time period. If the product is colored or absorbs strongly at some wavelength then one can measure the rate of product formation by monitoring the absorbance of the solution over time. This measure of activity (increase in absorbance over time) can then be converted to the increase in product amount over time by using the extinction coefficient for the product. Once activity is determined, we can then determine a relative activity by dividing activity by the volume of enzyme used in the experiment (measured U/ml). Equation 5 Therefore (using the preceding definition of units): if we add 0.01 ml of an enzyme preparation, and find that the reaction proceeds at 10 μmol/min, the relative activity of the enzyme preparation is: Equation 6 Ultimately, the most important measure of purity is the specific activity . The activity and relative activity tell you how concentrated the enzyme is, but NOT how pure it is. What we want is a high activity of the enzyme without a lot of other proteins around. To estimate this we use the concept of SPECIFIC ACTIVITY : Equation 7 which is the activity in units divided by the number of micrograms of total protein in the sample. The number of milligrams of protein is a measure of total protein, active or inactive, enzyme or non-enzyme, which is what you determined last week. As an example: Let’s say you have three enzyme fractions. We take 200 μl of each and we assay for activity. We also take 0.5 ml of each and assay for total protein content. The results are as follows: Fraction Units (μmol/min) Protein Concentration (μg/ml) C.H 100 3000 1 10 10 2 20 10 3 25 30 The specific activity of each fraction is calculated as follows: Relative Activity = Activity Volume of Enzyme Used = U ml 10 mol / min 0.01 ml = 1000 mol / min ml = 1000U ml µ µ Specific Activity = Relative Activity Total Protein Concentration = U / ml g protein / ml U g protein µ µ =

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Lab 3-19 For fraction 1, we have 10 units that came from 200 μl or 0.2 ml, so we have 10U/0.2 ml = 50U/ml. Specific activity = U/ μg = U.ml -1 /μg.ml -1 = 50/10 = 5 U/μg For Fraction 2, the specific activity is 10 U/ μg , for Fraction 3 it is 4.2 U/ μg and for the C.H. the specific activity is 0.17 U/ μg . As you can see, even though the C.H. had the highest activity, it was not the purest sample. In fact, it was the least pure sample because it had the highest concentration of total protein. The purest fraction is Fraction 2. A useful thing to know is how pure your samples are compared to the unpurified state as found in the C.H. This is called FOLD PURIFICATION and is easily calculated by simply dividing the specific activity of a sample by the specific activity of the C.H. So, the fold purification of Fraction 2 is 10 𝑈𝑈 . μg −1 0 . 17 𝑈𝑈 . μg −1 = 60 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑓𝑓𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑓𝑓𝑝𝑝 Today you will measure the activity of the purification samples that you got in lab 2B. Next, you will calculate the specific activity of these samples as a way of gauging how well your purification was. To measure the activity of peroxidase in the samples we must incubate a small subsample of each sample with the substrates H 2 O 2 and guaiacol. Question Based on your understanding of the procedure, predict which sample(s) you think will have the highest peroxidase activity . Which samples will have the highest peroxidase specific activity ? Will they be the same? Explain your reasoning . You will first add reaction mixture to spectrophotometry cuvettes and then you will add a small subsample of your purification samples to these and measure the absorbances over time in your spectrophotometer. Details are below: 1. Prepare a reaction mixture by mixing the following into a 25 ml glass flask: 4.5 ml guaiacol + 5.5 ml H 2 O 2 + 10 ml sodium phosphate buffer. Cover the flask with parafilm and invert to mix. 2. Obtain as many small-volume spectrophotometry cuvettes as the number of samples you have. The samples you will analyze are the same samples for which you determined the total protein concentration in lab 2B. Label these tubes with tape using the appropriate name for the sample to be analyzed. Be careful not to place the tape in the path of the light. [ DO NOT WRITE DIRECTLY ON THE CUVETTES !] 3. Precut many small square pieces of parafilm. 4. Into each cuvette add 990 μl of the reaction mix.

Lab 3-20 5. Make sure the spectrophotometer is zeroed at 480 nm, which is the absorbance maximum of tetraguaiacol using one of the cuvettes that contains only reaction mixture. 6. If there is a need, your instructor may ask that you dilute some samples by 1/10 th and use these diluted samples in the peroxidase assay. The samples that will likely need to be diluted are as follows: CH, DS, Super, F1, F2 and possibly F3. Ask your instructor. If you do dilute your samples, you will need to correct for this dilution by multiplying your measured absorbance values by 10. However, if you do not need to dilute the sample then DO NOT multiply by 10. To make the dilutions do the following: Into clean microfuge tubes, add 100 microliter of a sample (e.g. CH) to 900 microliter of water and mix. This new diluted sample can then be treated the same as normal in the peroxidase assay (see below). 7. Once zeroed, do the following very fast : a. Add 10 μl of the sample to the first cuvette and very quickly place a small piece of parafilm on top of the opening and mix by inverting very fast 3 times. b. Place the tube in the spectrophotometer and write down the first number you see after a short stabilization period this is the time zero measurement. NOW start the stop watch. c. After 10 seconds take another reading. Continue to take readings every 10 seconds until 60 seconds have elapsed. d. [Please note that some samples (for example the CH, DS and others) may have such high peroxidase levels that the reaction proceeds too fast for you to take readings before the spectrophotometer maxes out. in this case, you will have to dilute that sample. A good way to do this is make a 1/100 dilution of the sample using water: Add 0.99ml water to a clean microcentrifuge tube and then add 0.01ml of the sample to the water and mix. Now repeat the peroxidase assay as before. Of course, if you did this dilution, you will have to multiply the absorbances you get from the diluted sample by 100 to arrive at the correct absorbance .] Time (sec) 0 10 20 30 40 50 60 Time (min) 0 0.17 0.33 0.5 0.67 0.83 1 Crude Homogenate (CH) Supernatant Desalted (DS) F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12

Lab 3-21 F13 F14 F15 F16 F17 F18 F19 F20

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Lab 3-22 8. Next prepare a scatter plot of all the samples that seem to show positive slopes (that show there is a reaction taking place). Do not plot samples that show no reaction (the ones that have a negative slope or that do not have a consistent upward trend in absorbance over time). The graph should have time (in minutes) on the X-axis and absorbance on the Y-axis. Place the data for all samples on the same graph ( one graph with multiple plots ).

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Lab 3-23 9. Next, determine the slope of each plot from the graph you prepared on the previous page. Again, ONLY do this for samples that had a positive slope. This slope represents the initial reaction rate for each sample that you analyzed. Place this slope in the second column in the table below. 10. Next , determine the activity of peroxidase in units (U) for each sample you plotted by using the formula: 𝑈𝑈𝑝𝑝𝑝𝑝𝑝𝑝𝑈𝑈 ( 𝑈𝑈 ) = 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑓𝑓 𝑝𝑝𝑟𝑟𝑝𝑝 𝑈𝑈𝑓𝑓𝑓𝑓𝑝𝑝𝑠𝑠 ( 𝑝𝑝𝑎𝑎𝑈𝑈 ∙ 𝑚𝑚𝑝𝑝𝑝𝑝 −1 ) [0.0266 ( 𝜇𝜇𝑚𝑚𝑓𝑓𝑓𝑓𝑠𝑠 𝐿𝐿 ) −1 ∙ 𝑝𝑝𝑚𝑚 −1 ] ∙ 𝑝𝑝𝑚𝑚 × 10 −3 𝐿𝐿 = 𝜇𝜇𝑚𝑚𝑓𝑓𝑓𝑓𝑠𝑠 ∙ 𝑚𝑚𝑝𝑝𝑝𝑝 −1 = 𝑈𝑈 The value 0.0266 ( 𝜇𝜇𝜇𝜇𝜇𝜇𝜇𝜇𝜇𝜇 𝐿𝐿 ) −1 ∙ 𝑝𝑝𝑚𝑚 −1 is the molar extinction coefficient of tetraguaiacol at 480 nm and it allows us to convert changes in absorbance of tetraguaiacol to changes in amount of tetraguaiacol at that wavelength. Write the result in the table above. 11. Next, determine the relative activity (U/ml) of each sample by dividing by the volume of sample used: And write the results in the table above. Be sure to write down all your calculations on an extra sheet of paper. 12. Next, determine the specific activity of the samples by dividing the relative activity by the concentration of protein in the sample that you determined in lab 2B (see table on page 12). 13. Finally, determine the fold purification of each sample, which is calculated as the specific activity of a sample divided by the specific activity of the crude homogenate (so by definition, the fold purification of the CH will be 1). Write all the results in the table on the next page. Specific Activity = Relative Activity (U / ml) Total Corrected Protein Concentration ( g / ml) = U g protein µ µ

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Lab 3-24 Sample Slope of the line = Initial Reaction Rate (as Δ Absorbance/Δ min) Activity (U) Relative Activity (U/ml) Specific Activity (U/μg protein) Fold Purification Crude Homogenate 1 Supernatant Desalted F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20

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Lab 3-25 Questions 1. Which sample had the highest peroxidase purity? How do you know (explain)? 2. Does your answer in # 1 above correspond with what you expected from your ion exchange chromatography purification? Yes or no and explain. 3. What was the purpose of the guaiacol and H 2 O 2 solutions in the determination of peroxidase specific activity? 4. Why do you think we set the spectrophotometer to 480 nm? What is this wavelength indicative of? 5. 0.0266 μM -1 cm -1 is the molar extinction coefficient of tetraguaiacol. Why was this extinction coefficient used?

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Lab 3-26 6. In your own words, explain why Increasing concentrations of KCl were used during the IEC procedure? What was the purpose of the KCl? What did it accomplish? 7. Write a brief one page description of your conclusions from lab 2. Your conclusions should be based on the actual results we got and what they are telling us about our success in purifying peroxidase from other proteins and macromolecules found in horseradish. Be sure to briefly describe each result you got and what that result is telling you. Type your response, print it and attach it to this document when you turn it in to your instructor.

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Lab3-Protein Purification

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