In the Laboratory
Purification of Bovine Carbonic Anhydrase by Affinity Chromatography An Undergraduate Biochemistry Laboratory Experiment C. Larry Bering* and Jennifer J. Kuhns Department of Chemistry, Clarion University, 840 Wood Street, Clarion, PA 16214 Roger Rowlett Department of Chemistry, Colgate University, 13 Oak Drive, Hamilton, NY 13346
Affinity chromatography, originally conceived and developed by Cuatrecasas et al. (1) is a very important biochemical purification technique. The principles of affinity chromatography are discussed in every major textbook and laboratory manual (e.g., 2–5). However, there are few experiments that utilize the power of affinity chromatography and can fit within the time and financial constraints of the undergraduate biochemistry laboratory. We describe an inexpensive but highly specific procedure for isolating bovine carbonic anhydrase (CA) from erythrocytes in a single 3-hour laboratory period. The enzyme shows very high activity as determined by a simple assay and exhibits a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Affinity chromatography is similar to ion exchange chromatography in that a substance binds tightly to a resin and can be eluted after all other proteins or contaminants have been removed. However, in affinity chromatography the basis of binding is not ionic interactions, which would include any species of charge opposite to that of the resin. Rather, the binding is based on biological specificity. For instance, if one wanted to isolate a particular hormone receptor from a mixture of membrane proteins, one could covalently attach the hormone (the ligand) for that receptor to a chromatographic support such as polydextran or agarose, and add the mixture to the column. All unwanted components would pass through the column leaving the desired receptor bound to the immobilized hormone. The receptor could then be eluted by changes in pH or ionic strength, or by addition of a competitor to the ligand. Other examples of affinity chromatography include isolation of antibodies (using bound antigens), and enzymes (using bound substrate or substrate analogs.) Affinity chromatography has the characteristic of very high specificity. While this is distinctly advantageous for the rapid purification of a substance from a complex mixture, it generally means that for every protein or enzyme one wishes to purify it is necessary to construct a specific affinity medium. However, there are some affinity media (group specific) that will bind a family of proteins—for instance, NAD+-containing enzymes (6 ). Group-specific resins (7 ) are less selective and more convenient to use—many prepared media are commercially available—but may yield an undesirable mixture of proteins. The most useful affinity chromatography media are those that bind one and only one protein. This means that the application of affinity chromatography can be time consuming and perhaps expensive: the chromatography medium
must be custom tailored to a given application. It was our purpose to develop an affinity chromatography teaching laboratory that combined high selectivity, low cost, and ease of use. The purification of bovine carbonic anhydrase from erythrocytes is an excellent choice for a number of reasons. First, erythrocytes are rich in the enzyme; CA is the second most abundant protein after hemoglobin. Second, the enzyme is extremely robust and can tolerate handling at room temperature, obviating the need to carry out operations in a cold room. Third, the enzyme has high activity and can be detected at low concentrations using a simple assay. Finally, the enzyme binds tightly and selectively to aromatic sulfonamides that can be used to construct highly efficient affinity chromatography media. Carbonic anhydrase is a zinc metalloenzyme ubiquitous in nature. It catalyzes the reaction of eq 1 with high efficiency: the turnover number k cat is approximately 1 × 106 s᎑1 and the second-order rate constant kcat /Km (Km is the Michaelis constant) is approximately 1 × 108 M᎑1 s᎑1, making it one of the most efficient enzymes known (8). HCO3᎑ + H+
CO2 + H2O
(1)
2+
In the animal carbonic anhydrases, the Zn ion in the active site has three histidine residue ligands and a fourth coordination site occupied by a catalytically required water molecule (9). This water molecule can be displaced by a variety of inhibitors, including monovalent anions (Cl᎑, NO3᎑, SCN᎑, N3᎑, etc.) and sulfonamides, 1: R
1
The best inhibitors of erythrocyte carbonic anhydrase have R as an aromatic moiety. Toluenesulfonamide, 2, has a Ki (the dissociation constant of the enzyme–inhibitor complex) of approximately 0.1 µ M for erythrocyte carbonic anhydrase (10), and one of the most potent inhibitors is benzolamide, 3, which has an IC50 (concentration of inhibitor that causes a 50% reduction in rate) of approximately 0.4 nM (11) under optimum conditions.
2
3
*Corresponding author.
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The “business end” of the sulfonamide inhibitors is the sulfonamide group itself, which has been shown by 15N and 113 Cd NMR studies to be bound to the enzyme as the anion RSO 2NH᎑, even though at neutral pH the predominant species in solution is the neutral species RSO2NH2 (12). The large aromatic portion of the molecule is important for tight binding to the active site, but can generally be chemically altered without causing drastic changes in the IC50 of the inhibitor. For example, 4-aminomethylbenzenesulfonamide, 4, is a derivative of 2 that has essentially identical inhibitory capacity, but is easily covalently attached to a carboxylate-bearing resin (eq 2) using the water-soluble coupling agent 1-ethyl3-(3-dimethylaminopropyl) carbodiimide (EDAC) (13):
(2)
When a solution containing erythrocyte carbonic anhydrase is applied to the 4-aminomethylbenzenesulfonamide column, carbonic anhydrase sticks quite tightly, and all other proteins are washed away. The bound carbonic anhydrase can subsequently be eluted by adding a high concentration of an inexpensive competitive inhibitor, such as a salt that contains a monovalent anion. Historically, sodium azide or potassium iodide has been used for this purpose (11), but the former is highly toxic and a hazardous waste disposal problem, and the latter is easily oxidized to iodine in the presence of air and must be replaced frequently. We have chosen to use potassium thiocyanate, which is equally effective and does not pose the problems of sodium azide or potassium iodide. The anions that bind tightly to the active site of carbonic anhydrase can be removed by exhaustive dialysis if necessary. However, the presence of these anions does not appreciably affect the activity of the assay provided the enzyme is sufficiently diluted in the assay mixture. The binding capacity of affinity chromatography media is much larger than one might suspect. Typical ligand loading capacities for affinity chromatography supports like carboxymethyl agarose can approach 20–30 meq per milliliter of wet gel. This corresponds to 600–900 mg of 30-kDa protein per milliliter of chromatography medium! Real-life loading factors are somewhat lower, but a typical 5-mL affinity column can be expected to bind up to 2 g (!) or more of a typical protein. The laboratory experiment we describe here will allow students to carry out the isolation and assay the enzyme in a single laboratory period. In a subsequent laboratory they can examine the homogeneity of the purified enzyme by SDSPAGE. The power of affinity chromatography is clearly illustrated as the students begin with bovine blood and have a pure enzyme in about three hours. The experiment can be expanded if the students prepare the affinity gel by coupling the sulfonamide to the support. However, it is more practical and cost-effective for the instructor to prepare the affinity
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gel ahead of time and describe the coupling procedure during one of the breaks in the lab period. Experimental Procedure
Materials Bovine blood was obtained from a local slaughterhouse and stored at 4 °C in the presence of 1% heparin. CM-BioGel A was purchased from BioRad. EDAC and heparin were purchased from Sigma. The affinity ligand 4-aminomethylbenzenesulfonamide was purchased from Aldrich. Electrophoresis was carried out using precast 10% polyacrylamide gels purchased from Jules Biotech, Inc., and run on a Hoefer Mighty Small unit. Absorbance of fractions at 280 nm was obtained using a Shimadzu UV160 spectrophotometer. Preparation of Affinity Gel The coupling reaction, essentially as described by Khalifah et al. (13), was performed by the instructor approximately one week before the laboratory. The sulfonamide ligand 4-aminomethylbenzenesulfonamide (1.5 g) was dissolved in 100 mL of 50% acetone and added to 125 mL of CM-BioGel A that had been washed in 50% acetone. The pH was adjusted to 4.8 with HCl. EDAC (2.5 g in 5 mL of 50% acetone) was added dropwise with gentle stirring. The pH was monitored during the addition and adjusted to 4.8 as necessary during the first two hours of the reaction. (The pH change is rapid at first and stabilizes after about 2 hours.) The mixture was then stirred overnight to allow coupling to complete. The coupled gel was washed thoroughly with 50% acetone to remove excess ligand, then several times with distilled water. After the final wash the gel was washed and resuspended in an equal volume 25 mM Tris, pH 8.3/0.25 M Na2SO4. For prolonged storage, the gel should be placed at 4 °C and NaN3 should be added to a final concentration of 0.02%. The gel should be washed and resuspended in azidefree buffer before use. Properly stored and regenerated affinity gel is reusable indefinitely. Hemolysis and Purification of Carbonic Anhydrase Just before the laboratory period, whole blood was spun in a centrifuge at 5000 × g for 10 min at 4 °C to pellet the erythrocytes. The packed cells were then resuspended in cold saline (0.15 M NaCl) and centrifuged again. This washing procedure was repeated twice. The final pellet was presented to the students, who began the experiment at this point. Packed cells from approximately 25–50 mL of whole blood were used for each student or group. The remainder of the purification can be carried out at the bench at room temperature, if desired. Students resuspended the pellet in approximately 100 mL of distilled water. The suspension was placed in a beaker and 1 mL (approximately 1%) of toluene was added. This was allowed to stir at room temperature for 1 h. At the end of the hour, the hemolysate was spun in a centrifuge at 5000 × g for 10 min to remove cellular debris and the supernatant was collected. The pH of the supernatant was adjusted to a value between 7 and 9 using solid Tris. The supernatant was then placed in a 250-mL Erlenmeyer flask and 5 mL of the affinity gel was added. The flask was stoppered and shaken on a wrist shaker for one hour. (Alternatively, the flask can
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be swirled intermittently by hand.) After shaking, the resin was collected by vacuum filtration using a sintered-glass filter. The unbound hemoglobin appears in the red filtrate, and the gel was washed on the filter several times with Tris/Na 2SO4 buffer, until virtually all of the red color was removed from the affinity gel. The washed gel was resuspended in an equal volume of buffer, poured into a 15-cm chromatography column, packed by gravity flow, and washed by passing 2–3 column volumes of buffer through the column. During the packing and washing, samples of the column effluent were collected every few milliliters and the absorbance at 280 nm was measured. Most of the unbound protein was removed in one or two column volumes of buffer and the absorbance decreased to a low value. At this point, the students switched to the elution buffer (25 mM Tris, pH 8.3; 0.4 M KSCN) and immediately began collecting 1-mL fractions. The 280 nm absorbance of the fractions was determined and fractions exhibiting a high absorbance were pooled for subsequent CA assays. It was found that a very large peak of protein was eluted in fractions 3–6 after the addition of the KSCN-containing buffer. The column was then washed with the eluting buffer until the absorbance returned to near baseline. The affinity gel can be reused after re-equilibration of the gel in the starting buffer.
Enzyme Assay The activity assay is derived from the procedure of Rickli et al. (14). During the coupling and chromatography steps, the instructor prepares saturated carbon dioxide in water for the CA enzyme assay. To do this, CO2 is bubbled into a 1-L Erlenmeyer flask filled with distilled water and chilled in an ice-water bath to 0 °C. The CO 2 should be bubbled into the water using a sparger (or an aquarium bubbler) for about 45 min to 1 h before use to assure saturation. This process should yield a solution containing CO2 at 60–70 mM. To test for the presence of CA, students added 2 mL of 0.025 M Tris, pH 8.3 (containing sufficient bromothymol blue to give a distinct and visible blue color) to two 13 × 100 mm test tubes chilled in an ice bath to 0 °C. To one tube, 10–50 µL of the enzyme from the column was added, and an equivalent amount of buffer was added to the second tube to serve as a control. Using a 5-mL syringe and a long needle or cannula, 2 mL of CO2 solution was added very quickly and smoothly to the bottom of each tube. Simultaneously with the addition of the CO2 solution, a stopwatch was started. The time required for the solution to change from blue to yellow was recorded. (The production of hydrogen ion during the CO2 hydration reaction of eq 1 lowers the pH of the solution until the color transition point of the bromothymol blue is reached. The time required for the color change is inversely related to the quantity of CA present in the sample.) The tubes must remain immersed in the ice bath for the duration of the assay for results to be reproducible. The best way to view the color change is by looking into the tubes from above. If the color change is too subtle to detect, additional bromothymol blue may have to be added to the buffer solution. The same procedure was carried out using the blank. Typically, the uncatalyzed reaction (the blank) takes approximately 2 min for the color change to occur, whereas the enzyme catalyzedreaction is complete in 5–15 s, depending upon the amount of enzyme added. Detecting the color change is somewhat
subjective, but results for each individual are fairly reproducible and sufficient for the purpose of locating the active fractions. The “activity units” of the enzyme can be calculated according to eq 3,
Activity units = t1 – t1 × 1000 c u
(3)
where tc represents the time in seconds for the catalyzed reaction and tu the time for the uncatalyzed reaction.
Characterization by SDS-PAGE To determine the purity and the approximate molecular weight of the CA, students performed SDS-PAGE (15) in a subsequent laboratory. Precast SDS gels (10% acrylamide) purchased from Jules Biotech, Inc., were made available to the students. Five hundred microliters of the protein was added to 500 µ L of a double-strength protein buffer containing 0.125 M Tris-HCl, pH 6.8–4% SDS–20% glycerol–10% β-mercaptoethanol–1% bromophenol blue. The protein solution can be prepared in advance and stored in 500-µ L aliquots in microfuge tubes in the freezer. The enzyme in the protein solution was placed in a boiling water bath for 1–2 min. Samples of the treated enzyme (5 or 10 µL) were then placed in sample wells of the precast gel. A set of molecular weight standards in the range of 14.4–97.4 kDa was added in one or two lanes. The electrophoresis was carried out and the gel was stained with Coomassie blue. A standard curve of log molecular weight vs distance was prepared using the molecular weight markers in the standard lane. From this, the molecular weight of the enzyme can be determined. A single band, corresponding to a molecular weight of 31 kDa, was found in the active fractions from the affinity column that matches the expected molecular weight of bovine CA. Indeed, one of the commercial molecular weight markers in the standard lane is carbonic anhydrase, and the isolated enzyme matched that band (Fig. 1). Conclusions This experiment was conducted during the last few weeks of the biochemistry laboratory course. During this time, students were rotated through three experiments that did not require extensive supervision by the instructor. Students had previously been exposed to centrifugation, electrophoresis, and ion exchange chromatography. These techniques were taught to the class as a whole, and students followed a “cookbook” procedure that took most of the lab periods. An important method of learning is by application of a technique to a real problem. This experiment allowed the students plenty of independence and served as a “capstone” experiment. Two new techniques were introduced in this laboratory—the isolation of an enzyme from a biological source, and affinity chromatography—that built on basic technical skills learned in previous laboratories. For this experiment, students worked in pairs and there were usually only three groups working at a time while the rest of the class was involved with other experiments. Thus students were required to be relatively self-sufficient and work out problems as a team. There are points during the experiment at which students are not very busy: the hemolysis and the binding of CA to
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the affinity gel. This time can be advantageously used to discuss affinity chromatography, techniques for isolation of biomolecules starting with whole cells or tissues, and the chemistry of the coupling reaction. The electrophoresis cannot be completed during the three-hour lab, but student groups were asked to return on a subsequent day to run their gels. This presented no problems and was well received by the students, since they could work in the lab by themselves with only the instructor present. At the end of the semester, students rated their selfconfidence in several techniques covered in the course. They were asked how they would feel if they were in a job or research situation and were asked to carry out certain techniques. On a sliding scale, they indicated whether they felt “very confident”, “capable of performing the task with some initial supervision”, or “uncomfortable performing the task”. For all techniques in the course, student responses were from the middle to the very confident level. Techniques that were repeated in the semester and in which students were given more independence consistently scored in the very confident range. This experiment can be performed readily at a reasonable cost, in a reasonable time frame, and with minimal hazards.
Figure 1. 10% SDS-polyacrylamide gel of eluant from affinity column (lane 2).
Literature Cited 1. Cuatrecasas, P.; Wilchek, M.; Anfinsen, C. D. Proc. Natl. Acad. Sci. USA 1968, 61, 636–643. 2. Cooper, T. G. The Tools of Biochemistry; Wiley-Interscience: New York, 1977. 3. Boyer, R. F. Modern Experimental Biochemistry, 2nd ed.; Benjamin/ Cummings: Redwood City, CA, 1993. 4. Voet, D.; Voet, J. G. Biochemistry, 2nd ed; Wiley: Somerset, NJ, 1995. 5. Stryer, L. Biochemistry, 3rd ed; Freeman: New York, 1988. 6. Scopes, R. In Protein Purification: Principles and Practice; Springer: New York, 1982. 7. Freifelder, D. In Physical Biochemistry; Freeman: New York, 1982. 8. Hewlett, R. S.; Silverman, D. N. J. Am. Chem. Soc. 1982, 104, 6737–6741.
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9. Silverman, D. N.; Lindskog, S. Acc. Chem. Res. 1988, 21, 30–36. 10. King, R. W.; Burgen, A. S. V. Proc. R. Soc. London B, 1976, 193, 107–125. 11. Sanyal, G.; Swenson, E. R.; Pessay, N. I.; Maren, T. H. Mol. Pharmacol. 1982, 22, 211–220. 12. Blackburn, G. M.; Mann, B. E.; Taylor, B. F.; Worrall, A. F. Eur. J. Biochem. 1985, 153, 53–58. 13. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson, S. M. Biochemistry 1977, 16, 2241–2247. 14. Rickli, E. E.; Ghazanfar, S. A. S.; Gibbons, B. H.; Edsall, J. T. J. Biol. Chem. 1964, 239, 1065–1078. 15. Laemmli, U. K. Nature 1970, 227, 680–685.
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