Dialysis and UltrafiltrationIf a solution of protein is separated from a bathing solution by a semipermeablemembrane, small molecules and ions can pass through the semipermeable mem-brane to equilibrate between the protein solution and the bathing solution, calledthe dialysis bath or dialysate (Figure 5A.1). This method is useful for removing smallmolecules from macromolecular solutions or for altering the composition of theprotein-containing solution.

Ultrafiltration is an improvement on the dialysis principle. Filters with pore sizesover the range of biomolecular dimensions are used to filter solutions to select formolecules in a particular size range. Because the pore sizes in these filters are mi-croscopic, high pressures are often required to force the solution through the filter.This technique is useful for concentrating dilute solutions of macromolecules. Theconcentrated protein can then be diluted into the solution of choice.

Ion Exchange Chromatography Can Be Used to Separate Molecules on the Basis of ChargeCharged molecules can be separated using ion exchange chromatography, a process inwhich the charged molecules of interest (ions) are exchanged for another ion (usuallya 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 composedof synthetic resin particles containing charged groups. Resins containing positivelycharged groups attract negatively charged solutes and are referred to as anion ex-change resins. Resins with negatively charged groups are cation exchangers. Figure 5A.2shows several typical anion and cation exchange resins. Weakly acidic or basic groupson ion exchange resins exhibit charges that are dependent on the pH of the bathingsolution. Changing the pH will alter the ionic interaction between the resin groups

APPENDIX TO CHAPTER 5

Protein Techniques1

1Although this appendix is titled Protein Techniques, these methods are also applicable to other macro-molecules such as nucleic acids.

Dialysate

Stir bar

Semipermeable bagcontaining protein solution

Magnetic stirrerfor mixing

FIGURE 5A.1 A dialysis experiment. The solution of macromolecules to be dialyzed is placed in a semiperme-able membrane bag, and the bag is immersed in a bathing solution. A magnetic stirrer gently mixes the solu-tion to facilitate equilibrium of diffusible solutes between the dialysate and the solution contained in the bag.

128 Chapter 5 Proteins: Their Primary Structure and Biological Functions

and the bound ions. In all cases, the bare charges on the resin particles must be coun-terbalanced by oppositely charged ions in solution (counterions); salt ions (e.g., Na! orCl") usually serve this purpose. The separation of a mixture of several amino acids ona column of cation exchange resin is illustrated in Figure 5A.3. Increasing the saltconcentration in the solution passing through the column leads to competition be-tween the cationic amino acid bound to the column and the cations in the salt forbinding to the column. Bound cationic amino acids that interact weakly with thecharged groups on the resin wash out first, and those interacting strongly are washedout only at high salt concentrations.

Size Exclusion ChromatographySize exclusion chromatography is also known as gel filtration chromatography or molecularsieve chromatography. In this method, fine, porous beads are packed into a chromatog-raphy column. The beads are composed of dextran polymers (Sephadex), agarose(Sepharose), or polyacrylamide (Sephacryl or BioGel P ). The pore sizes of these beads ap-proximate the dimensions of macromolecules. The total bed volume (Figure 5A.4) ofthe packed chromatography column, Vt, is equal to the volume outside the porousbeads (Vo) plus the volume inside the beads (Vi) plus the volume actually occupied bythe bead material (Vg): Vt # Vo ! Vi ! Vg. (Vg is typically less than 1% of Vt and can beconveniently ignored in most applications.)

As a solution of molecules is passed through the column, the molecules passivelydistribute between Vo and Vi, depending on their ability to enter the pores (that is,

Structure

Strongly acidic, polystyrene resin (Dowex-50) S O–

O

O

O CH2 C

O–

O

Weakly acidic, carboxymethyl (CM) cellulose

CH2 N

CH2C

CH2CWeakly acidic, chelating, polystyrene resin(Chelex-100)

Structure

Strongly basic, polystyrene resin (Dowex-1) CH3CH2 N

CH3

CH3

Weakly basic, diethylaminoethyl (DEAE) cellulose

HOCH2CH2 N

CH2CH3

CH2CH3

+

O

O

O–

O–

+

(a) Cation Exchange Media

(b) Anion Exchange Media

FIGURE 5A.2 Cation (a) and anion (b) exchange resins commonly used for biochemical separations.

Chapter 5 Appendix 129

their size). If a molecule is too large to enter at all, it is totally excluded from Vi andemerges first from the column at an elution volume, Ve, equal to Vo (Figure 5A.4).If a particular molecule can enter the pores in the gel, its distribution is given by thedistribution coefficient, KD:

KD # (Ve " Vo)/Vi

where Ve is the molecule’s characteristic elution volume (Figure 5A.4). The chro-matography run is complete when a volume of solvent equal to Vt has passedthrough the column.

ElectrophoresisElectrophoretic 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,

The elution processseparates amino acidsinto discrete bands

Eluant emergingfrom the columnis collected

Am

ino

acid

conc

entr

atio

n

Elution time

Some fractionsdo not contain

amino acids

Samplecontainingseveral amino acids

Elution columncontainingcation exchangeresin beads

ACTIVE FIGURE 5A.3 The separation of amino acids on a cation exchange column. Test yourself on the con-cepts in this figure at www.cengage.com/login

130 Chapter 5 Proteins: Their Primary Structure and Biological Functions

F would cause the molecule to accelerate. In solution, the molecule experiences fric-tional drag, Ff, due to the solvent:

Ff # 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 chargedmolecule is proportional to its charge q and the voltage E, but inversely proportionalto 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 sup-port matrix such as polyacrylamide or agarose, which retards the movement of mol-ecules 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 hydro-phobic tail of dodecylsulfate interacts strongly with polypeptide chains. The num-ber of SDS molecules bound by a polypeptide is proportional to the length (num-ber of amino acid residues) of the polypeptide. Each dodecylsulfate contributes twonegative charges. Collectively, these charges overwhelm any intrinsic charge thatthe protein might have. SDS is also a detergent that disrupts protein folding (pro-

Prot

ein

conc

entr

atio

n

VtVolume (mL)

A smallermacromolecule

VeVo

(b)

Elution profile of a large macromolecule(excluded from pores) (Ve ! Vo)

(a)

Smallmolecule

Largemolecule

Porousgel beads

Elutioncolumn

FIGURE 5A.4 (a) A gel filtration chromatography column. Larger molecules are excluded from the gel beadsand emerge from the column sooner than smaller molecules, whose migration is retarded because they canenter the beads. (b) An elution profile.

Na+ –O S O

O–

O

CH2

CH2CH2

CH2CH2

CH2CH2

CH2CH2

CH2CH2

CH3

Na+

FIGURE 5A.5 The structure of sodium dodecylsulfate (SDS).

Chapter 5 Appendix 131

tein 3° structure). SDS-PAGE is usually run in the presence of sulfhydryl-reducingagents such as $-mercaptoethanol so that any disulfide links between polypeptidechains are broken. The electrophoretic mobility of proteins upon SDS-PAGE is in-versely proportional to the logarithm of the protein’s molecular weight (Figure5A.6). SDS-PAGE is often used to determine the molecular weight of a protein.

Isoelectric FocusingIsoelectric focusing is an electrophoretic technique for separating proteins ac-cording to their isoelectric points (pIs). A solution of ampholytes (amphoteric elec-trolytes) is first electrophoresed through a gel, usually contained in a small tube.The migration of these substances in an electric field establishes a pH gradientin the tube. Then a protein mixture is applied to the gel, and electrophoresis isresumed. As the protein molecules move down the gel, they experience the pHgradient and migrate to a position corresponding to their respective pIs. At itspI, a protein has no net charge and thus moves no farther.

Two-Dimensional Gel ElectrophoresisThis separation technique uses isoelectric focusing in one dimension and SDS-PAGE in the second dimension to resolve protein mixtures. The proteins in a mix-ture are first separated according to pI by isoelectric focusing in a polyacrylamidegel 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 theyare separated according to size (Figure 5A.7). The gel slab can then be stained toreveal the locations of the individual proteins. Using this powerful technique, re-searchers have the potential to visualize and construct catalogs of virtually all the

Log

mol

ecul

arw

eigh

t

Relative electrophoreticmobility

FIGURE 5A.6 A plot of the relative electrophoretic mo-bility of proteins in SDS-PAGE versus the log of the mol-ecular weights of the individual polypeptides.

10

Isoelectricfocusing gel

Direction of electrophoresis

4

pH

HighMW

LowMW

Protein spotSDS-poly-acrylamideslab

pH 4 pH 10

FIGURE 5A.7 A two-dimensional electrophoresis separa-tion. A mixture of macromolecules is first separated ac-cording to charge by isoelectric focusing in a tube gel.The gel containing separated molecules is then placedon top of an SDS-PAGE slab, and the molecules are elec-trophoresed into the SDS-PAGE gel, where they are sepa-rated according to size.

132 Chapter 5 Proteins: Their Primary Structure and Biological Functions

proteins present in particular cell types. The ExPASy server (http://us.expasy.org)provides access to a two-dimensional polyacrylamide gel electrophoresis databasenamed SWISS-2DPAGE. This database contains information on proteins, identi-fied as spots on two-dimensional electrophoresis gels, from many different celland tissue types.

Hydrophobic Interaction ChromatographyHydrophobic interaction chromatography (HIC) exploits the hydrophobic nature of pro-teins in purifying them. Proteins are passed over a chromatographic column packedwith a support matrix to which hydrophobic groups are covalently linked. PhenylSepharose, an agarose support matrix to which phenyl groups are affixed, is a primeexample of such material. In the presence of high salt concentrations, proteins bindto the phenyl groups by virtue of hydrophobic interactions. Proteins in a mixturecan be differentially eluted from the phenyl groups by lowering the salt concentra-tion or by adding solvents such as polyethylene glycol to the elution fluid.

High-Performance Liquid ChromatographyThe principles exploited in high-performance (or high-pressure) liquid chromatography(HPLC) are the same as those used in the common chromatographic methods suchas ion exchange chromatography or size exclusion chromatography. Very-high-resolution separations can be achieved quickly and with high sensitivity in HPLC usingautomated instrumentation. Reverse-phase HPLC is a widely used chromatographic pro-cedure for the separation of nonpolar solutes. In reverse-phase HPLC, a solution ofnonpolar solutes is chromatographed on a column having a nonpolar liquid immobi-lized on an inert matrix; this nonpolar liquid serves as the stationary phase. A more po-lar liquid that serves as the mobile phase is passed over the matrix, and solute moleculesare eluted in proportion to their solubility in this more polar liquid.

Affinity ChromatographyAffinity purification strategies for proteins exploit the biological function of the tar-get protein. In most instances, proteins carry out their biological activity throughbinding or complex formation with specific small biomolecules, or ligands, as inthe case of an enzyme binding its substrate. If this small molecule can be immo-bilized through covalent attachment to an insoluble matrix, such as a chromato-graphic medium like cellulose or polyacrylamide, then the protein of interest, indisplaying affinity for its ligand, becomes bound and immobilized itself. It canthen be removed from contaminating proteins in the mixture by simple meanssuch as filtration and washing the matrix. Finally, the protein is dissociated oreluted from the matrix by the addition of high concentrations of the free ligandin solution. Figure 5A.8 depicts the protocol for such an affinity chromatographyscheme. Because this method of purification relies on the biological specificity ofthe protein of interest, it is a very efficient procedure and proteins can be puri-fied several thousand-fold in a single step.

UltracentrifugationCentrifugation methods separate macromolecules on the basis of their characteris-tic densities. Particles tend to “fall” through a solution if the density of the solutionis less than the density of the particle. The velocity of the particle through themedium is proportional to the difference in density between the particle and thesolution. The tendency of any particle to move through a solution under centrifu-gal force is given by the sedimentation coefficient, S:

S # (%p " %m)V/ƒ

A protein interacts with a metabolite. Themetabolite is thus a ligand that binds specificallyto this protein

Protein Metabolite

The metabolite can be immobilized by covalentlycoupling it to an insoluble matrix such as anagarose polymer. Cell extracts containing manyindividual proteins may be passed through the matrix.

Specific protein binds to ligand. All otherunbound material is washed out of the matrix.

+

Adding an excess of free metabolite that willcompete for the bound protein dissociates theprotein from the chromatographic matrix. Theprotein passes out of the column complexed withfree metabolite.

Purifications of proteins asmuch as 1000-fold or more areroutinely achieved in a singleaffinity chromatographic steplike this.

FIGURE 5A.8 Diagram illustrating affinitychromatography.

Chapter 5 Appendix 133

where %p is the density of the particle or macromolecule, %m is the density of themedium or solution, V is the volume of the particle, and f is the frictional coeffi-cient, given by

ƒ # Ff/v

where v is the velocity of the particle and Ff is the frictional drag. Nonspherical mol-ecules have larger frictional coefficients and thus smaller sedimentation coeffi-cients. The smaller the particle and the more its shape deviates from spherical, themore slowly that particle sediments in a centrifuge.

Centrifugation can be used either as a preparative technique for separating andpurifying macromolecules and cellular components or as an analytical technique tocharacterize the hydrodynamic properties of macromolecules such as proteins andnucleic acids.