Protein Techniques 1 APPENDIX TO CHAPTER 5

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1 Protein Techniques 1 APPENDIX T CHAPTER 5 Dialysis and Ultrafiltration If a solution of protein is separated from a bathing solution by a semipermeable membrane, small molecules and ions can pass through the semipermeable membrane to equilibrate between the protein solution and the bathing solution, called the dialysis bath or dialysate (Figure 5A.1). This method is useful for removing small molecules from macromolecular solutions or for altering the composition of the protein-containing solution. Ultrafiltration is an improvement on the dialysis principle. Filters with pore sizes over the range of biomolecular dimensions are used to filter solutions to select for molecules in a particular size range. Because the pore sizes in these filters are microscopic, high pressures are often required to force the solution through the filter. This technique is useful for concentrating dilute solutions of macromolecules. The concentrated protein can then be diluted into the solution of choice. Ion Exchange Chromatography Can Be Used to Separate Molecules on the Basis of Charge Charged molecules can be separated using ion exchange chromatography, a process in which the charged molecules of interest (ions) are exchanged for another ion (usually a salt ion) on a charged solid support. In a typical procedure, solutes in a liquid phase, usually water, are passed through a column filled with a porous solid phase composed of synthetic resin particles containing charged groups. Resins containing positively charged groups attract negatively charged solutes and are referred to as anion exchange resins. Resins with negatively charged groups are cation exchangers. Figure 5A.2 shows several typical anion and cation exchange resins. Weakly acidic or basic groups on ion exchange resins exhibit charges that are dependent on the ph of the bathing solution. Changing the ph will alter the ionic interaction between the resin groups Semipermeable bag containing protein solution Dialysate Stir bar Magnetic stirrer for mixing FIGURE 5A.1 A dialysis experiment. The solution of macromolecules to be dialyzed is placed in a semipermeable membrane bag, and the bag is immersed in a bathing solution. A magnetic stirrer gently mixes the solution to facilitate equilibrium of diffusible solutes between the dialysate and the solution contained in the bag. 1 Although this appendix is titled Protein Techniques, these methods are also applicable to other macromolecules such as nucleic acids.

2 128 Chapter 5 Proteins: Their Primary Structure and Biological Functions (a) Cation Exchange Media Structure Strongly acidic, polystyrene resin (Dowex-50) S Weakly acidic, carboxymethyl (CM) cellulose C Weakly acidic, chelating, polystyrene resin (Chelex-100) N C C (b) Anion Exchange Media Structure CH 3 Strongly basic, polystyrene resin (Dowex-1) + N CH 3 CH 3 Weakly basic, diethylaminoethyl (DEAE) cellulose CH 3 + N H CH 3 FIGURE 5A.2 Cation (a) and anion (b) exchange resins commonly used for biochemical separations. and the bound ions. In all cases, the bare charges on the resin particles must be counterbalanced by oppositely charged ions in solution (counterions); salt ions (e.g., Na or Cl ) usually serve this purpose. The separation of a mixture of several amino acids on a column of cation exchange resin is illustrated in Figure 5A.3. Increasing the salt concentration in the solution passing through the column leads to competition between the cationic amino acid bound to the column and the cations in the salt for binding to the column. Bound cationic amino acids that interact weakly with the charged groups on the resin wash out first, and those interacting strongly are washed out only at high salt concentrations. Size Exclusion Chromatography Size exclusion chromatography is also known as gel filtration chromatography or molecular sieve chromatography. In this method, fine, porous beads are packed into a chromatography column. The beads are composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P ). The pore sizes of these beads approximate the dimensions of macromolecules. The total bed volume (Figure 5A.4) of the packed chromatography column, V t, is equal to the volume outside the porous beads (V o ) plus the volume inside the beads (V i ) plus the volume actually occupied by the bead material (V g ): V t V o V i V g. (V g is typically less than 1% of V t and can be conveniently ignored in most applications.) As a solution of molecules is passed through the column, the molecules passively distribute between V o and V i, depending on their ability to enter the pores (that is,

3 Chapter 5 Appendix 129 Sample containing several amino acids Elution column containing cation exchange resin beads The elution process separates amino acids into discrete bands Eluant emerging from the column is collected Some fractions do not contain amino acids Amino acid concentration Elution time ACTIVE FIGURE 5A.3 The separation of amino acids on a cation exchange column. Test yourself on the concepts in this figure at their size). If a molecule is too large to enter at all, it is totally excluded from V i and emerges first from the column at an elution volume, V e, equal to V o (Figure 5A.4). If a particular molecule can enter the pores in the gel, its distribution is given by the distribution coefficient, K D : K D (V e V o )/V i where V e is the molecule s characteristic elution volume (Figure 5A.4). The chromatography run is complete when a volume of solvent equal to V t has passed through the column. Electrophoresis Electrophoretic techniques are based on the movement of ions in an electrical field. An ion of charge q experiences a force F given by F Eq/d, where E is the voltage (or electrical potential ) and d is the distance between the electrodes. In a vacuum,

4 130 Chapter 5 Proteins: Their Primary Structure and Biological Functions (a) Small molecule Large molecule Porous gel beads Elution column (b) Protein concentration Elution profile of a large macromolecule (excluded from pores) (V e V o ) A smaller macromolecule V o Volume (ml) V e V t FIGURE 5A.4 (a) A gel filtration chromatography column. Larger molecules are excluded from the gel beads and emerge from the column sooner than smaller molecules, whose migration is retarded because they can enter the beads. (b) An elution profile. F would cause the molecule to accelerate. In solution, the molecule experiences frictional drag, F f, due to the solvent: F f 6 r where r is the radius of the charged molecule, is the viscosity of the solution, and is the velocity at which the charged molecule is moving. So, the velocity of the charged molecule is proportional to its charge q and the voltage E, but inversely proportional to the viscosity of the medium and d, the distance between the electrodes. Generally, electrophoresis is carried out not in free solution but in a porous support matrix such as polyacrylamide or agarose, which retards the movement of molecules according to their dimensions relative to the size of the pores in the matrix. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS is sodium dodecylsulfate (sodium lauryl sulfate) (Figure 5A.5). The hydrophobic tail of dodecylsulfate interacts strongly with polypeptide chains. The number of SDS molecules bound by a polypeptide is proportional to the length (number of amino acid residues) of the polypeptide. Each dodecylsulfate contributes two negative charges. Collectively, these charges overwhelm any intrinsic charge that the protein might have. SDS is also a detergent that disrupts protein folding (pro- Na + S Na + CH 3 FIGURE 5A.5 The structure of sodium dodecylsulfate (SDS).

5 Chapter 5 Appendix 131 tein 3 structure). SDS-PAGE is usually run in the presence of sulfhydryl-reducing agents such as -mercaptoethanol so that any disulfide links between polypeptide chains are broken. The electrophoretic mobility of proteins upon SDS-PAGE is inversely proportional to the logarithm of the protein s molecular weight (Figure 5A.6). SDS-PAGE is often used to determine the molecular weight of a protein. Isoelectric Focusing Isoelectric focusing is an electrophoretic technique for separating proteins according to their isoelectric points (pis). A solution of ampholytes (amphoteric electrolytes) is first electrophoresed through a gel, usually contained in a small tube. The migration of these substances in an electric field establishes a ph gradient in the tube. Then a protein mixture is applied to the gel, and electrophoresis is resumed. As the protein molecules move down the gel, they experience the ph gradient and migrate to a position corresponding to their respective pis. At its pi, a protein has no net charge and thus moves no farther. Log molecular weight Relative electrophoretic mobility FIGURE 5A.6 A plot of the relative electrophoretic mobility of proteins in SDS-PAGE versus the log of the molecular weights of the individual polypeptides. Two-Dimensional Gel Electrophoresis This separation technique uses isoelectric focusing in one dimension and SDS- PAGE in the second dimension to resolve protein mixtures. The proteins in a mixture are first separated according to pi by isoelectric focusing in a polyacrylamide gel in a tube. The gel is then removed and laid along the top of an SDS-PAGE slab, and the proteins are electrophoresed into the SDS polyacrylamide gel, where they are separated according to size (Figure 5A.7). The gel slab can then be stained to reveal the locations of the individual proteins. Using this powerful technique, researchers have the potential to visualize and construct catalogs of virtually all the Isoelectric focusing gel 10 ph ph 4 ph 10 High MW 4 Direction of electrophoresis Low MW SDS-polyacrylamide slab Protein spot FIGURE 5A.7 A two-dimensional electrophoresis separation. A mixture of macromolecules is first separated according to charge by isoelectric focusing in a tube gel. The gel containing separated molecules is then placed on top of an SDS-PAGE slab, and the molecules are electrophoresed into the SDS-PAGE gel, where they are separated according to size.

6 132 Chapter 5 Proteins: Their Primary Structure and Biological Functions proteins present in particular cell types. The ExPASy server ( provides access to a two-dimensional polyacrylamide gel electrophoresis database named SWISS-2DPAGE. This database contains information on proteins, identified as spots on two-dimensional electrophoresis gels, from many different cell and tissue types. A protein interacts with a metabolite. The metabolite is thus a ligand that binds specifically to this protein Protein + Metabolite The metabolite can be immobilized by covalently coupling it to an insoluble matrix such as an agarose polymer. Cell extracts containing many individual proteins may be passed through the matrix. Specific protein binds to ligand. All other unbound material is washed out of the matrix. Adding an excess of free metabolite that will compete for the bound protein dissociates the protein from the chromatographic matrix. The protein passes out of the column complexed with free metabolite. Purifications of proteins as much as 1000-fold or more are routinely achieved in a single affinity chromatographic step like this. FIGURE 5A.8 Diagram illustrating affinity chromatography. Hydrophobic Interaction Chromatography Hydrophobic interaction chromatography (HIC) exploits the hydrophobic nature of proteins in purifying them. Proteins are passed over a chromatographic column packed with a support matrix to which hydrophobic groups are covalently linked. Phenyl Sepharose, an agarose support matrix to which phenyl groups are affixed, is a prime example of such material. In the presence of high salt concentrations, proteins bind to the phenyl groups by virtue of hydrophobic interactions. Proteins in a mixture can be differentially eluted from the phenyl groups by lowering the salt concentration or by adding solvents such as polyethylene glycol to the elution fluid. High-Performance Liquid Chromatography The principles exploited in high-performance (or high-pressure) liquid chromatography (HPLC) are the same as those used in the common chromatographic methods such as ion exchange chromatography or size exclusion chromatography. Very-highresolution separations can be achieved quickly and with high sensitivity in HPLC using automated instrumentation. Reverse-phase HPLC is a widely used chromatographic procedure for the separation of nonpolar solutes. In reverse-phase HPLC, a solution of nonpolar solutes is chromatographed on a column having a nonpolar liquid immobilized on an inert matrix; this nonpolar liquid serves as the stationary phase. A more polar liquid that serves as the mobile phase is passed over the matrix, and solute molecules are eluted in proportion to their solubility in this more polar liquid. Affinity Chromatography Affinity purification strategies for proteins exploit the biological function of the target protein. In most instances, proteins carry out their biological activity through binding or complex formation with specific small biomolecules, or ligands, as in the case of an enzyme binding its substrate. If this small molecule can be immobilized through covalent attachment to an insoluble matrix, such as a chromatographic medium like cellulose or polyacrylamide, then the protein of interest, in displaying affinity for its ligand, becomes bound and immobilized itself. It can then be removed from contaminating proteins in the mixture by simple means such as filtration and washing the matrix. Finally, the protein is dissociated or eluted from the matrix by the addition of high concentrations of the free ligand in solution. Figure 5A.8 depicts the protocol for such an affinity chromatography scheme. Because this method of purification relies on the biological specificity of the protein of interest, it is a very efficient procedure and proteins can be purified several thousand-fold in a single step. Ultracentrifugation Centrifugation methods separate macromolecules on the basis of their characteristic densities. Particles tend to fall through a solution if the density of the solution is less than the density of the particle. The velocity of the particle through the medium is proportional to the difference in density between the particle and the solution. The tendency of any particle to move through a solution under centrifugal force is given by the sedimentation coefficient, S: S ( p m )V/ƒ

7 Chapter 5 Appendix 133 where p is the density of the particle or macromolecule, m is the density of the medium or solution, V is the volume of the particle, and f is the frictional coefficient, given by ƒ F f /v where v is the velocity of the particle and F f is the frictional drag. Nonspherical molecules have larger frictional coefficients and thus smaller sedimentation coefficients. The smaller the particle and the more its shape deviates from spherical, the more slowly that particle sediments in a centrifuge. Centrifugation can be used either as a preparative technique for separating and purifying macromolecules and cellular components or as an analytical technique to characterize the hydrodynamic properties of macromolecules such as proteins and nucleic acids.