Experiment 3. Protein Size

Transcription

1 Biological Sciences 11 Spring 2000 Experiment 3. Protein Size Ultracentrifugation - Sedimentation Coefficient Size Exclusion Chromatography - Stokes Radius Voet & Voet, Biochemistry, Chap 5, pp Voet & Voet, Chap 5, pp Alberts et al. MBOC, pp start start after 20 hours after 20 min Introduction. When one isolates a protein, there are several characteristics to determine, including the specific activity, molecular weight, the size, shape, and subunit composition. In BioSci 11, we are using β-galactosidase as our model protein and we are characterizing it by several techniques. In Expts 1 and 2, we have measured its concentration and activity. In this experiment we will measure its sedimentation coefficient by centrifugation and measure its Stokes radius by size exclusion chromatography. The sedimentation coefficient and radius provide the information needed to determine molecular weight. The subunit composition will be addressed by crosslinking and polyacrylamide gel electrophoresis in Expt 4.

2 Biochemists have a keen interest in the elucidation of molecular structure and its correlation with activity. Structural information can be derived from electron microscopy, and the detailed coordinates of amino acid residues can be obtained by x-ray diffraction of single crystals of proteins. Both of these techniques provide information on solid-state specimens. Inside the cell, proteins in solution interact with one another and react with substrates. Centrifugation and chromatography offer the capability to study proteins in buffered solutions, closer to their native environment. Moreover, proteins can be isolated in sufficient quantities for functional assays such as enzyme activity. Today s samples: Fractions from centrifugation on a sucrose gradient. 1. A mixture of marker proteins and an unknown protein was loaded onto a 14-mL stabilizing sucrose gradient, 5% to 25%, in 20 mm imidazole, 100 mm NaCl, ph 7.0, and the tubes were spun in a swinging bucket rotor for 20 hours at 38,000 rpm at 6 C. Fractions, 750 µl, were collected from the gradient by puncturing the bottom of the tube, as shown in Voet and Voet. 2. Reference proteins. Reference subunit mw (kd) expected on PAGE Sedimentation coefficient, S 20,w (Svedbergs) µg loaded onto each gradient tube Thyroglobulin (TG) Catalase (CAT) Lactate dehydrogenase (LDH) Bovine serum albumin (BSA)

3 3. Analyze the fractions. A. Enzyme activity. Prepare a set of 14 cuvettes containing 900 µl Z-DTT buffer + 90 µl ONPG (4 mg/ml stock). Add 10 µl of each fraction. Wait until yellow color develops in 1 or 2 tubes. Then add 250 µl 1 M Na 2 CO 3 to raise ph and slow the reaction. Read A 410 in one cuvette after the other to construct a profile of the cuvettes (fractions) containing enzyme. B. Identify proteins in each fraction by PAGE. Make a set of Eppendorf tubes To a 20-µL aliquot of each fraction, add 2 µl of 10X sample buffer. [Sample buffer contains a reducing agent, and glycerol to make the sample more dense than buffer.] Heat samples for 5 min at 95 C. Load 15 µl of each sample into the wells of the gel. TFs will demonstrate loading. Lane 1 markers -- use as is, without heating them, but mix well. M A R K E R F R A C 1 F R A C C. Reference proteins. TFs will run a gel of the marker proteins. On the back side of one electrophoresis apparatus, assemble a 4-20% polyacrylamide gel of the same type as in part B, and load 2 different volumes of 4 reference proteins and 1 unknown protein. Use 1 well for the pre-stained marker proteins. D. Electrophoresis for approx. 60 min at 100 volts. Stop when blue tracking dye reaches bottom. E. Detection. Wearing gloves, disassemble plastic plates and place polyacrylamide gel in rectangular dish with lid. Rinse 4 times with 100 ml water, to remove SDS detergent. First rinse: Pour off water after 30 sec. 2,3,4: Place the dish on the rocker for 5 min for each rinse cycle.

4 Option 1. Stain with E-zinc. Add 20 ml E-zinc reversible stain solution and place on the rocker for 10 min., then pour off this solution into a recycle bottle. Add 20 ml developer solution. Wait 2 min or longer, until the polyacrylamide gel is opaque, and the bands are transparent. Pour off this solution into a recycle bottle. Rinse with 100 ml water for 1 min (longer is OK). Make an image of this gel on the scanner with the cover open, so that transparent bands look black. Save the file in a folder with your TF s name, then print a copy. Look at your image and identify the bands present in each lane. If some bands are faint, darker staining can be achieved with GelCode Blue. But first you must remove the zinc stain by adding 20 ml E-zinc eraser to your gel in its tray. Place on rocker and wait 10 min, until the gel is clear, and rinse 3 times with water, 30 sec each time. Option 2. Add GelCode blue dye; 1 squirt, just enough to cover the gel. Place on rocker for 15 min. Rinse with water, 100 ml. 3 times for 5 min each. With spatula, transfer gel from water to Kodak photographic apparatus light box. Double-click on 1D icon. Turn on light. Take photo. Save in BS11 file folder with your TF s name. Consult instructions near the camera to analyze data later; check with TF. Option 3. Stain with SYPRO Ruby. Take photo on a Kodak apparatus equipped with fluorescent light table. Prestained molecular weight markers in lane #1. 10 bands -- the 4th one is pink and others are blue. Look ahead: This distinctive pattern will be visible on the Zinc stained gel. After GelCode blue staining, all bands will be blue, so note the position of the pink band and mark it on your scanned image. BenchMark Ladder., Lot No Refer to the color image on our web site. Band MW (kd) Band MW (kd) pink

5 Analysis of fractions Fraction Reference proteins, indicate [highest conc] in [ ] Enzyme activity (arbitrary units such as A 420 after x min) For the centrifugation part of your report (1) Show an image of your gel, with the proteins identified in each lane. (2) Make a calibration plot of s for each marker protein vs fraction number. Indicate the fraction containing the highest concentration of β-galactosidase. (3) Make a plot of enzyme activity vs fraction number. Use the same x-axis length as in the calibration plot. (4) Show your calculation of s for β-galactosidase. In the results paragraph, describe results, referring to your data in tables and graphs. In the discussion paragraph, note the correlation between fractions having high enzyme activity and those having a band on the gel that corresponds to a β-galactosidase subunit.

6 4. We need more data to determine the size of our unknown protein, so we measure the Stokes radius (r p ) by size exclusion chromatography and use r p to calculate the frictional coefficient, ƒ. Size exclusion chromatography to be done while PAGE is running. To do... Principles of liquid chromatography -- overview of experiment and demonstration of equipment by teaching fellows. Obtain a printed profile of protein concentration vs fraction number. Identify proteins in each fraction and find β-galactosidase-containing fractions by enzyme activity assay. Use information on known marker proteins to make a calibration plot, similar to Voet and Voet, Fig. 5-13, p Calibration proteins. Ve: Where is peak of highest concentration in ml. 2. Void volume. Blue dextran elution profile 3. Total volume. Change in conductivity (alternatively -- elution volume of unique solvent such as acetone). 3. Unknown; β-galactosidase. Identify peak; calculate elution volume in ml. Make plot of data: protein concentration (ordinate) vs fraction number or elution volume in ml (abscissa). Analyze contents of the tubes in your collected fractions for possible enzyme activity. Add (10 µl; verify with TF) of each fraction to cuvettes containing 900 µl buffer and 90µL ONPG. Mix. After (5) min, (or when color develops) add 500 µl Na 2 CO 3. Measure A 420 for all tubes in set. Make graphs for your report. Protein concentration in each fraction. Enzyme activity in each fraction. Identity of proteins in each fraction, based on chromatogram. Make list. Elution volume (highest concentration) of marker proteins; make calibration plot of Stokes radius vs elution volume. Calculate Stokes radius of β-galactosidase. Calculate partition coefficient K av of β-galactosidase.

7 5. Enzyme activity assay. To a set of cuvettes containing 900 µl Z-DTT buffer and 90 µl ONPG, add 10 µl from each fraction. After yellow color develops, add 250 µl Na 2 CO 3. Read A 420. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Calibration Standards for our columns Protein Molecular Weight (kd) Stokes Radius (Angstroms) Subunit mol wgt Thyroglobulin Catalase Lactate dehydrogenase Ovalbumin Chymotrypsinogen A Blue Dextran 2000 Extra large 1 In each run, we select calibration proteins that give well-separated peaks on the column that we use (Superdex or Superose). Please check with your TF for today s proteins. The void volume Vo is measured as the elution volume Ve for Blue Dextran, a polymer too large to enter the pores of the beads. The total volume Vt accessible to solvent and small molecules is determined by introducing the proteins in a high salt buffer and measuring the change in conductivity. The elution buffer is lower in salt concentration than the protein buffer. An alternative way to estimate the total volume is by including acetone in the mixture of protein solutions applied to the column. Any soluble substance with UV/visible absorption or conductivity characteristics different from the elution buffer can be used. This information will be provided by TFs from the run made on your lab day. Blue dextran Change in conductivity

8 For the size exclusion chromatorgraphy portion of your report 6. Make 3 plots, all with elution volume as the x-axis (same length). a) Identify the calibration proteins, and plot Stokes radius vs elution volume. Remember that we collected the first 5 ml of elution buffer in bulk rather than in a tube because we knew from previous runs not to expect proteins in the early effluent. Convert fraction number (number of your tube) to elution volume to make the print out correspond to your enzyme assay. b) Inclue the plot A 280, which is proportional to protein concentration, vs fraction number (or elution volume) that is given to you from the FPLC-1 or FPLC-2. Identify the type of column packing. c) Activity vs fraction number. 7. Determine the elution volume of the unknown protein, β-galactosidase. Calculate its Stokes radius, r p and use r p to calculate the frictional coefficient, ƒ. 8. Calculate the molecular weight of β-galactosidase, as determined from your data, s and ƒ. Refer to the table of definitions below (also given in Voet and Voet). Note: Some texts use cgs units -- r p is in cm. For reference. Information is from Voet and Voet. s N ƒ s N 6 π η r p M = = (1 - Ÿ ρ) (1 - Ÿ ρ) Eq. 5.15, p. 98 (rearranged) s = Svedberg coefficient, in units of sec M = molecular weight Ÿ = partial specific volume in cm 3 g approx 0.73 cm 3 g -1 for most proteins. Examples are given in Table 5.5. ρ = density of water = 1 g/ml Each protein molecule displaces an equivalent volume of solution. N = Avogadro s number x ƒ = frictional coefficient ƒ = 6 π η r p Eq η = viscosity of water = 0.01 poise r p = Stokes radius of the protein to be determined by size exclusion chromatography

9 9. We have used elution volume for a specific column to compare β-galactosidase to reference proteins. Use of the partition coefficient Kav allows this data to be compared with results on other columns of different dimensions. Calculate Kav for β-galactosidase. 10. In your results paragraph, describe your data by referring to it in tables or figures. Indicate your value for molecular weight and describe its calculation in words. In the discussion paragraph, describe any improvements to the experimental procedure that you might make, were you to repeat the experiment. For example, could the sedimentation coefficient be determined with greater accuracy if more fractions of smaller volume had been examined. You may think of other variations and postulate their effect upon the experimental outcome. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Background Information on size exclusion chromatography. Proteins are polypeptides that fold into unique structures suited to perform specific tasks. The special biological properties that each protein possesses are conferred by the three-dimensional structure and by the distribution of hydrophobic, nonpolar, positive and negative charged regions on the surface and in active site crevices. Biochemists make use of these characteristics to separate a given protein from a mixture of cellular components. Let s consider a hypothetical isolation of a soluble protein from the cell cytosol. The first step is to rupture the cell plasma membrane by methods such as osmotic shock, mechanical abrasion with glass beads, or sonication. Next, the crude lysate is centrifuged at low speed to pellet nuclei, mitochondria, and membrane fragments. The supernatant from this first centrifugation step contains a mixture of many soluble proteins. What to do next? Researchers often fractionate the soluble proteins into groups by performing a high speed zonal centrifugation. The protein mixture is applied to the top of a stabilizing sucrose gradient and centrifuged for several hours until each protein resides in a different sucrose density zone. In each zone of buffered sucrose, one can expect to find proteins of different size, different surface charge distributions, and different ligand binding characteristics. At this point, the separation power of chromatography comes into play. There are three major types of liquid chromatography -- size exclusion, ion exchange, and affinity chromatography. Each of these methods offers selected advantages. Size exclusion chromatography is often used first to analyze the proteins in a mixture, and then proteins in each size group are secondarily segregated on the basis of charge by ion exchange (anion or cation exchange) chromatography. Alternatively, proteins that bind to specific ligands can be isolated by binding to polymer beads containing covalently bound ligands. This is termed affinity chromatography.

10 Size exclusion chromatography (also called gel filtration chromatography or gel permeation chromatography) is a method of separating molecular mixtures on the basis of their distribution between a mobile and a stationary phase. The stationary phase is made of spherical particles, such as cross-linked dextrans ("Sephadex") or dextran-agarose copolymers ("Superdex"), of a particular size and porosity that are packed in a column. The mobile phase is the buffer which is passed through the column. The primary separation process is the diffusional partitioning of solute molecules between the mobile solvent phase and the interior solvent spaces of the gel particles. Thus, molecules are fractionated on the basis of their size and shape. Size refers to Stokes radius. Small molecules can diffuse into pores in the gel particles and their movement through the column is retarded; large molecules, on the other hand, are excluded from the gel particles because their size is larger than the pore size of the particles and they move rapidly through the space between the gel particles. In general, the elution position of a molecule is proportional to its apparent size, with large molecules eluting first and small molecules at greater elution volumes. The total volume (Vt) of a gel filtration column is given approximately by : V t = V 0 + V i where Vo, the void volume, is the volume of solvent outside the gel particles, and Vi, the internal volume, is the volume inside the gel beads. A molecule that is completely excluded from the gel beads elutes at a volume equal to V 0. Small molecules (such as sugars and buffer salts) that freely diffuse into the pores of gel beads elute as volume V t has passed through the column. Molecules with intermediate sizes elute at elution volumes, V e, between V 0 and V t.

11 globular ( ~ spherical) proteins small medium large large protein ellipsoid Figure 3-1. Schematic diagram of a column containing inert beads with pores of various sizes that admit or exclude solute molecules on the basis of size for separation by "size exclusion chromatography". Large molecules like blue dextran are excluded from the beads, large proteins can enter large pores, small proteins can enter large or small pores, and low molecular weight solutes such as DTT or salt can diffuse freely throughout the beads and the bulk solvent. By using proteins of known sizes with similar globular shape, a column can be calibrated by plotting the elution volume of each protein, Ve, versus the logarithm of the molecular weight, Mr, as shown in Figure 4-2 (similar to Figure 5-13 in Voet and Voet). One can then determine the apparent Mr of a protein of interest from its Ve. Typically, the behavior of a protein in gel filtration is characterized by its partition coefficient, K av, which is defined as : Ve - Vo K av = Vt - Vo

12 The void volume, Vo, is determined by measuring the elution position of Blue Dextran, a polymer of average molecular weight 2 x 106. The teaching staff has run dextran under the same conditions as used in this experiment. In principle Vt may be experimentally found by identifying the fraction where some small molecular component of the sample solution is no longer present. Typical small molecules used to mark Vt include amino acids (such as Tyr, Trp, or Phe, UV chromophores) or dithiothreitol, a reducing agent that yields a colored product when reacted with Ellman s reagent, or a solvent such as acetone. In our FPLC apparatus, Vt is simply obtained as the change in conductivity of the sample buffer. The sample is prepared in a buffer with two-fold higher salt concentration than the elution buffer. Teaching fellows will provide these data from the chromatography run on each column performed earlier in the day. A plot of log K av versus log Mr gives a linear relationship in the range of values of K av between 0.1 and 0.9. An example of such a plot is shown below. At the beginning of the lab period we (the teaching fellows) will run a standard mixture to obtain data for a calibration curve (plotted by students) for each column on its respective FPLC apparatus. Our experiment is more straightforward than the plot shown in Figure 4-2. Figure 3-2. A typical calibration plot for a column (not our BioSci 11 column). The calibration plot must be made for each column, each buffer system, and each flow rate. There is a similar graph in Voet and Voet, page 84, Figure 5-13.

13 In our experiment, a mixture of solutes will be separated by size exclusion on Superdex 200 HR 10/30, a highly cross-linked agarose polymer covalently bonded to dextran and formed into 13 µm beads. The porosity of the beads allows the fractionation of proteins having Mr between 10,000 and 600,000. Technical note: Agarose is a carbohydrate polymer of [D-galactose anhydrogalactose] repeating units. In BioSci 10 we used agarose gels (pore size approx Å) to separate the stiff polymer DNA. The gels were prepared by heating agarose solutions and allowing them to cool, a process that facilitates formation of orderly hydrogen bonds between neighboring sugar residues. For the size exclusion beads, agarose is strengthened by cross-linking it with chemicals such as divinyl sulfone so that it can withstand moderate flow rates. Additional mechanical strength is conferred by covalently bonding the cross-linked agarose to dextran. Dextran is also a polysaccharide, with D-glucose as the primary repeating unit. Pore size is smaller in agarose-dextran, from Å. agarose dextran Superdex beads :: crosslinked agarose with covalently bonded dextran Figure 3-3. The size exclusion column is packed with Superdex beads. The extent of crosslinking of agarose governs the size of proteins that may be admitted into the pores of the beads. For example, highly crosslinked agarose has small pores and it is used for separation of proteins in the 10 kd to 50 kd range. Chymotrypsinogen (25 kd) and ovalbumin (42 kd) are well resolved on such beads, but thyroglobulin (670 kd) and blue dextran (2,000 kd) are poorly resolved. Conversely, on beads with low extent of crosslinking and large pores, blue dextran and thyroglobulin are nicely separated, but the lower molecular weight proteins come off the column at nearly the same elution volume.