Voet D., Voet Ju.G. Biochemistry

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thereby “focused” into a narrow band about its isoelectric

point that may be as thin as 0.01 pH unit. Hence, this

process is called isoelectric focusing (IEF).

A pH gradient produced by mixing two different buffers

together in continuously varying ratios is unstable in an

electric field because the buffer ions migrate to the elec-

trode of opposite polarity. Rather, the pH gradient in IEF

is formed by a mixture of low molecular mass (600–900 D)

oligomers bearing aliphatic amino and carboxylic acid

groups (Fig. 6-26) that have a range of isoelectric points.

Under the influence of an electric field in solution, these

ampholytes (amphoteric electrolytes) will each migrate to

their isoelectric points. Consequently, the most acidic am-

pholytes gather at the anode and the progressively more

basic ones position themselves ever closer to the cathode.

The pH gradient, which is maintained by the ⬃1000 V elec-

tric field, arises from the buffering action of these am-

pholytes. The convection of the pH gradient is prevented

by carrying out IEF in a lightly cross-linked polyacryl-

amide gel in the form of a rod or slab. IEF gels often con-

tain ⬃6M urea, a powerful protein denaturing agent that,

unlike SDS, is uncharged and hence cannot directly affect

the charge of a protein.

An alternative form of IEF utilizes gels containing im-

mobilized pH gradients. These are made from acrylamide

derivatives that are covalently linked to ampholytes.

Through the use of a gradient maker (e.g., Fig. 6-7), a gel

containing an immobilized pH gradient is polymerized

from a continuously varying mixture of acrylamide deriva-

tives with different pK’s so that the gel’s pH varies

smoothly from one end to the other.

The fact that IEF separates proteins into sharp bands

makes it a useful analytical and preparative tool. In fact,

many protein preparations once thought to be homogen-

eous have been resolved into several components by IEF.

IEF can be combined with electrophoresis in an extremely

powerful two-dimensional separation technique named

Section 6-4. Electrophoresis 151

Molecular mass (kD)

0.2

Relative mobility

0.4 0.6 0.8 1.0

–

Figure 6-24 SDS–PAGE. The SDS–polyacrylamide disc

electrophoretogram shows separation of proteins from the

supernatant (left) and membrane fractions (right) of various

strains of the bacterium Salmonella typhimurium. Samples of

200 ␮g of protein each were run in parallel lanes on a 35-cm-long,

0.8-mm-thick slab gel containing 10% polyacrylamide. The lane

marked MW contains molecular weight standards. [Courtesy of

Giovanna F. Ames, University of California at Berkeley.]

Figure 6-25 Logarithmic relationship between the molecular

mass of a protein and its relative electrophoretic mobility in

SDS–PAGE. This relationship is plotted for 37 polypeptides

ranging from 11 to 70 kD. [After Weber, K. and Osborn, M., J.

Biol. Chem. 244, 4406 (1969).]

Figure 6-26 General formula of the ampholytes used in

isoelectric focusing.

(CH

)

(CH

)

N N

(CH

)

COOH

⫽ 2 or 3

R ⫽ H or

JWCL281_c06_129-162.qxd 2/22/10 2:25 PM Page 151

two-dimensional (2D) gel electrophoresis; Fig. 6-27). Up

to 5000 proteins can be observed on a single two-dimen-

sional electrophoretogram. Hence two-dimensional gel

electrophoresis is a valuable tool for proteomics (the

study of the proteome, which, in analogy with the term

“genome,” is defined as the aggregate of all the proteins

expressed by a cell or organism, but with emphasis on

their quantitation, localization, modifications, interactions,

and activities, as well as their identification). Individual

protein bands in a stained gel can be cut out from the gel

(with a scalpel or by a robot guided by a digitized image of

the gel acquired by an optical scanner or a digital camera),

destained, and the protein eluted from the gel fragment

for identification and/or characterization, often by mass

spectrometry (Section 7-1J).Variant proteins can be found

by comparing the positions and intensities of the bands in

2D gels of similar preparations. This can be done with the

aid of a computer after acquiring digitized images of the

stained gels. Numerous reference 2D gels are publicly

available for this purpose in the Web-accessible databases

listed at http://www.expasy.org/swiss-2dpage?de. These

databases contain images of 2D gels of a variety of organ-

isms and tissues and identify many of their component

proteins.

E. Capillary Electrophoresis

Although gel electrophoresis in its various forms is a com-

mon and highly effective method for separating charged

molecules, it typically requires an hour or more for a run

and is difficult to quantitate and automate.These disadvan-

tages are largely overcome through the use of capillary

electrophoresis (CE), a technique in which electrophoresis

is carried out in very thin (10 to 100 ␮m inner diameter)

capillary tubes made of silica, glass, or plastic. Such narrow

capillaries rapidly dissipate heat and hence permit the use

of high electric fields (typically 100 to 300 V ⴢ cm

–1

, about

10 times that of most other electrophoretic techniques),

which reduces separation times to a few minutes. These

rapid separations, in turn, minimize band broadening

caused by diffusion, thereby yielding extremely sharp

separations. Capillaries can be filled with buffer (as in

moving boundary electrophoresis, but here the capillary’s

narrow bore all but eliminates convective mixing),

SDS–polyacrylamide gel (separation according to molecu-

lar mass; Section 6-4C), or ampholytes (isoelectric focus-

ing; Section 6-4D). These CE techniques have extremely

high resolution and can be automated in much the same

way as is HPLC, that is, with automatic sample loading and

on-line sample detection. Since CE can only separate small

amounts of material, it is largely limited to use as an analyt-

ical technique.

5 ULTRACENTRIFUGATION

If a container of sand and water is shaken and then allowed

to stand quietly, the sand will rapidly sediment to the bot-

tom of the container due to the influence of Earth’s gravity

(an acceleration g equal to 9.81 m

–2

). Yet macromole-

cules in solution, which experience the same gravitational

field, do not exhibit any perceptible sedimentation because

their random thermal (Brownian) motion keeps them uni-

formly distributed throughout the solution. Only when they

are subjected to enormous accelerations will the sedimenta-

tion behavior of macromolecules begin to resemble that of

sand grains.

The ultracentrifuge was developed around 1923 by the

Swedish biochemist The Svedberg. Using this instrument,

Svedberg first demonstrated that proteins are macromole-

cules with homogeneous compositions and that many pro-

teins are composed of subunits.Within a few decades, ultra-

centrifugation became an indispensable tool for the

fractionation of proteins, nucleic acids, and subcellular par-

ticles. Modern ultracentrifuges can attain rotational speeds

as high as 150,000 rpm (revolutions per minute) so as to

generate centrifugal fields in excess of 1 million ⫻ g.In this

section we outline the theory and practice of ultracentrifu-

gation.

A. Sedimentation

The rate at which a particle sediments in the ultracen-

trifuge is related to its mass. The force, F

sedimentation

, acting to

sediment a particle of mass m that is located a distance r

from a point about which it is revolving with angular veloc-

ity ␻ (in radians ⴢ s

–1

) is the centrifugal force (m␻

r) on the

152 Chapter 6. Techniques of Protein and Nucleic Acid Purification

Figure 6-27 Two-dimensional (2D) gel electrophoresis. This

autoradiogram shows the separation of E. coli proteins by 2D gel

electrophoresis (isoelectric focusing horizontally and

SDS–PAGE vertically). A 10-␮g sample of proteins from E. coli

that had been labeled with

C-amino acids was subjected to

isoelectric focusing in a 2.5 ⫻ 130–mm tube of a urea-containing

polyacrylamide gel.The gel was then extruded from its tube,

placed in contact with one edge of an SDS–polyacrylamide slab

gel, and subjected to electrophoresis. Over 1000 spots were

counted on the original autoradiogram, which resulted from an

825-h exposure. [Courtesy of Patrick O’Farrell, University of

California at San Francisco.]

JWCL281_c06_129-162.qxd 2/22/10 2:25 PM Page 152

particle less the buoyant force (V

␳␻

r) exerted by the so-

lution:

[6.10]

Here V

is the particle volume and ␳ is the density of the

solution. However, the motion of the particle through the

solution, as we have seen in our study of electrophoresis, is

opposed by the frictional force:

[6.7]

where v ⫽ dr/dt is the rate of migration of the sedimenting

particle and f is its frictional coefficient. The particle’s fric-

tional coefficient can be determined from measurements

of its rate of diffusion.

Under the influence of gravitational (centrifugal) force,

the particle accelerates until the forces on it exactly bal-

ance:

[6.11]

The mass of 1 mol of particles, M, is

[6.12]

where N is Avogadro’s number (6.022 ⫻ 10

). Thus, a par-

ticle’s volume, V

, may be expressed in terms of its molar

mass:

[6.13]

where , the particle’s partial specific volume, is the vol-

ume change when 1 g (dry weight) of particles is dissolved

⫽ Vm ⫽

M ⫽ mN

m␻

r ⫺ V

␳␻

r ⫽ vf

friction

⫽ vf

sedimentation

⫽ m␻

r ⫺ V

␳␻

in an infinite volume of the solute. For most proteins dis-

solved in pure water at 20°C, is near 0.73 cm

ⴢ g

–1

(Table

6-4). Indeed, for proteins of known amino acid composi-

tion, is closely approximated by the sum of the partial

specific volumes of its component amino acid residues,

thereby indicating that the atoms in proteins are closely

packed (Section 8-3Bc).

a. A Particle May Be Characterized by Its

Sedimentation Rate

Substituting Eqs. [6.12] and [6.13] into Eq. [6.11] yields

[6.14]

Now define the sedimentation coefficient, s, as

[6.15]

The sedimentation coefficient, a quantity that is analo-

gous to the electrophoretic mobility (Eq. [6.9]) in that it

is a velocity per unit force, is usually expressed in units of

–13

s, which are known as svedbergs (S). For the sake of

uniformity, the sedimentation coefficient is customarily

corrected to the value that would be obtained at 20°C in

a solvent with the density and viscosity of pure water.

This is symbolized s

20,w

. Table 6-4 and Fig. 6-28 indicate

the values of s

20,w

in svedbergs for a variety of biological

materials.

Equation [6.15] indicates that a particle’s mass, m ⫽

M/N, can be determined from the measurement of its sedi-

mentation coefficient, s, and the solution density, ␳, if its

s ⫽

␻

⫽

␻

d ln r

b⫽

M(1 ⫺ V

␳)

vf ⫽

M(1 ⫺ V

␳)␻

Section 6-5. Ultracentrifugation 153

Table 6-4 Physical Constants of Some Proteins

Partial Specific Sedimentation

Molecular

Volume,

Coefficient, Frictional

Protein Mass (kD) (cm

ⴢ g

–1

) s

20,w

(S) Ratio, f/f

Lipase (milk) 6.7 0.714 1.14 1.190

Ribonuclease A (bovine pancreas) 12.6 0.707 2.00 1.066

Cytochrome c (bovine heart) 13.4 0.728 1.71 1.190

Myoglobin (horse heart) 16.9 0.741 2.04 1.105

␣-Chymotrypsin (bovine pancreas) 21.6 0.736 2.40 1.130

Crotoxin (rattlesnake) 29.9 0.704 3.14 1.221

Concanavalin B (jack bean) 42.5 0.730 3.50 1.247

Diphtheria toxin 70.4 0.736 4.60 1.296

Cytochrome oxidase (P. aeruginosa) 89.8 0.730 5.80 1.240

Lactate dehydrogenase H (chicken) 150 0.740 7.31 1.330

Catalase (horse liver) 222 0.715 11.20 1.246

Fibrinogen (human) 340 0.725 7.63 2.336

Hemocyanin (squid) 612 0.724 19.50 1.358

Glutamate dehydrogenase (bovine liver) 1015 0.750 26.60 1.250

Turnip yellow mosaic virus protein 3013 0.740 48.80 1.470

20,w

Source: Smith, M.H., in Sober, H.A. (Ed.), Handbook of Biochemistry and Molecular Biology (2nd ed.), p. C-10, CRC Press (1970).

JWCL281_c06_129-162.qxd 2/22/10 2:25 PM Page 153

frictional coefficient, f, and its partial specific volume, ,

are known. Indeed, before about 1970, most macromolecu-

lar mass determinations were made using the analytical ul-

tracentrifuge, a device in which the sedimentation rates of

molecules under centrifugation can be optically measured

(the masses of macromolecules are too high to be accurately

determined by such classic physical techniques as melting

point depression or osmotic pressure measurements).

Although the advent of much simpler molecular mass deter-

mination methods, such as gel filtration chromatography

(Section 6-3Ba) and SDS–PAGE (Section 6-4C), had caused

analytical ultracentrifugation to largely fade from use, re-

cently developed instrumentation has led to a resurgence

in the use of analytical ultracentrifugational measure-

ments. They are particularly useful in characterizing sys-

tems of associating macromolecules.

154 Chapter 6. Techniques of Protein and Nucleic Acid Purification

Figure 6-28 Sedimentation coefficients in svedbergs (S) for some biological materials.

[After a diagram supplied by Beckman Coulter, Inc.]

100

200

400

600

800

1000

2000

4000

6000

8000

10,000

20,000

40,000

60,000

80,000

100,000

Collagen

Albumin

Luteinizing hormone

Immunoglobulin G

Aldolase

Catalase

-Macroglobulin

Rous sarcoma

Feline leukemia

Bacteriophage T2

Soluble proteins

Yeast tRNA

Nucleic acids

E. coli tRNA

Calf liver DNA

Vesicular stomatitis virus RNA

Bacteriophage T5 DNA

Bacteriophage T2 & T4 DNAs

Ribosomal subunits

Broad bean mottleRibosomes

Polysomes

Polio

Tobacco mosaic

Equine encephalitis

Viruses

Microsomes

Subcellular

particles

Plasma

membranes

Mitochondria

Svedbergs

Cytochrome c

JWCL281_c06_129-162.qxd 2/22/10 2:25 PM Page 154

b. The Frictional Ratio Is Indicative of Molecular

Solvation and Shape

For an unsolvated spherical particle of radius r

the fric-

tional coefficient is determined according to the Stokes

equation:

[6.16]

where ␩ is the viscosity of the solution. Solvation increases

the frictional coefficient of a particle by increasing its effec-

tive or hydrodynamic volume. Furthermore, f is minimal

when the particle is a sphere.This is because a nonspherical

particle has a larger surface area than a sphere of equal vol-

ume and therefore must, on the average, present a greater

surface area toward the direction of movement than a

sphere.

The frictional coefficient, f, of a particle of known mass

and partial specific volume can be ultracentrifugationally

determined using Eq. [6.15]. The effective or Stokes radius

of a particle in solution can be calculated by solving Eq.

[6.16] for r

, given the experimentally determined values of

f and ␩. Conversely, the minimal frictional coefficient of a

particle, f

, can be calculated from the mass and the partial

specific volume of the particle by assuming it to be spheri-

cal and unsolvated:

[6.17]

If the frictional ratio, f/f

, of a particle is much greater than

unity, it must be concluded that the particle is highly sol-

vated and/or significantly elongated. The frictional ratios

of a selection of proteins are presented in Table 6-4. The

globular proteins, which are known from structural studies

to be relatively compact and spheroidal (Section 8-3B),

have frictional ratios ranging up to ⬃1.5. Fibrous mole-

cules such as DNA and the blood clotting protein fibrino-

gen (Section 35-1Aa) have larger frictional ratios. On de-

naturation, the frictional coefficients of globular proteins

increase by as much as twofold because denatured pro-

teins assume flexible and fluctuating random coil confor-

mations in which all parts of the molecule are in contact

with solvent (Section 8-1D).

B. Preparative Ultracentrifugation

Preparative ultracentrifuges, which as their name implies

are designed for sample preparation, differ from analytical

ultracentrifuges in that they lack sample observation facil-

ities. Preparative rotors contain cylindrical sample tubes

whose axes may be parallel, at an angle,or perpendicular to

the rotor’s axis of rotation, depending on the particular ap-

plication (Fig. 6-29).

In the derivation of Eq. [6.15], it was assumed that sedi-

mentation occurred through a homogeneous medium. Sed-

imentation may be carried out in a solution of an inert sub-

stance, however, such as sucrose or CsCl, in which the

concentration, and therefore the density, of the solution in-

creases from the top to the bottom of the centrifuge tube.

⫽ 6␲␩ a

3MV

4␲N

1>3

⫽

␲r

)

f ⫽ 6␲␩r

The use of such density gradients greatly enhances the re-

solving power of the ultracentrifuge. Two applications of

density gradients are widely employed: (1) zonal ultracen-

trifugation and (2) equilibrium density gradient ultracen-

trifugation.

a. Zonal Ultracentrifugation Separates Particles

According to Their Sedimentation Coefficients

In zonal ultracentrifugation, a macromolecular solu-

tion is carefully layered on top of a density gradient pre-

pared by use of a device resembling that diagrammed in

Fig. 6-7. The purpose of the density gradient is to allow

smooth passage of the various macromolecular zones by

damping out convective mixing of the solution. Sucrose,

which forms a syrupy and biochemically benign solution,

is commonly used to form a density gradient for zonal ul-

tracentrifugation. The density gradient is normally rather

shallow because the maximum density of the solution

must be less than that of the least dense macromolecule

of interest. Nevertheless, consideration of Eq. [6.15] indi-

cates that the sedimentation rate of a macromolecule is a

more sensitive function of molecular mass than density.

Consequently, zonal ultracentrifugation separates simi-

larly shaped macromolecules largely on the basis of their

molecular masses.

During centrifugation, each species of macromolecule

moves through the gradient at a rate largely determined by

its sedimentation coefficient and therefore travels as a

zone that can be separated from other such zones as is dia-

grammed in Fig. 6-30. After centrifugation, fractionation is

commonly effected by puncturing the bottom of the cellu-

loid centrifuge tube with a needle, allowing its contents to

drip out, and collecting the individual zones for subsequent

analysis.

Section 6-5. Ultracentrifugation 155

Figure 6-29 A selection of preparative ultracentrifuge rotors.

The sample tubes of the swinging bucket rotors (rear) are hinged

so that they swing from the vertical to the horizontal position as

the rotor starts spinning, whereas the sample tubes of the other

rotors have a fixed angle relative to the rotation axis. [Courtesy

of Beckman Coulter, Inc.]

JWCL281_c06_129-162.qxd 2/22/10 2:25 PM Page 155

b. Equilibrium Density Gradient Ultracentrifugation

Separates Particles According to Their Densities

In equilibrium density gradient ultracentrifugation [al-

ternatively, isopycnic ultracentrifugation (Greek: isos,

equal ⫹ pyknos, dense)], the sample is dissolved in a rela-

tively concentrated solution of a dense, fast-diffusing (and

therefore low molecular mass) substance, such as CsCl or

, and is spun at high speed until the solution

achieves equilibrium. The high centrifugal field causes the

low molecular mass solute to form a steep density gradient

(Fig. 6-31) in which the sample components band at posi-

tions where their densities are equal to that of the solution;

that is, where in Eq. [6.15] is zero (Fig. 6-32).

These bands are collected as separate fractions when the

sample tube is drained as described above.The salt concen-

tration in the fractions and hence the solution density is

easily determined with an Abbé refractometer, an optical

instrument that measures the refractive index of a solution.

The equilibrium density gradient technique is often the

method of choice for separating mixtures whose compo-

nents have a range of densities. These substances include

nucleic acids, viruses, and certain subcellular organelles

such as ribosomes. However, isopycnic ultracentrifugation

is rather ineffective for the fractionation of protein mix-

tures because most proteins have similar densities (high

salt concentrations also salt out or possibly denature pro-

teins). Density gradient ultracentrifugation in an analytical

ultracentrifuge was used to show that DNA is semiconser-

vatively replicated (Section 5-3B).

6 NUCLEIC ACID FRACTIONATION

In the preceding parts of this chapter we considered the

most commonly used procedures for isolating and, to some

extent, characterizing proteins. Most of these methods, of-

ten with some modification, are also regularly used to frac-

tionate nucleic acids according to size, composition, and se-

quence.There are also many techniques that are applicable

only to nucleic acids. In this section we outline some of the

most useful of the procedures employed in the separation

of nucleic acids.

A. Solution Methods

Nucleic acids in cells are invariably associated with pro-

teins. Once cells have been broken open (Section 6-1B),

(1 ⫺ V

␳)

156 Chapter 6. Techniques of Protein and Nucleic Acid Purification

Stabilizing

sucrose

gradient

Sample

centrifugation

fractionation

Fast-sedimenting

component

Slow-sedimenting

component

1.78

1.74

1.70

1.66

1.62

6.0 6.2 6.4 6.6 6.8 7.0 7.2

Radius (cm)

Density (g

•

mL)

Figure 6-30 Zonal ultracentrifugation. The sample is layered

onto a sucrose gradient (left). During centrifugation (middle),

each particle sediments at a rate that depends largely on its mass.

Figure 6-31 Equilibrium density distribution of a CsCl

solution in an ultracentrifuge spinning at 39,460 rpm. The initial

density of the solution was 1.7 g ⴢ mL

–1

. [After Ifft, J.B.,Voet,

D.H., and Vinograd, J., J. Phys. Chem. 65, 1138 (1961).]

After the end of the run, the centrifugation tube is punctured

and the separated particles (zones) are collected (right).

JWCL281_c06_129-162.qxd 2/22/10 2:25 PM Page 156

their nucleic acids must be deproteinized. This can be ac-

complished by shaking (very gently if high molecular mass

DNA is being isolated; Section 5-3D) the aqueous solution

containing the protein–nucleic acid complex with a 25:24:1

mixture of phenol, chloroform, and isoamyl alcohol. The

protein is thereby denatured and extracted into the water-

immiscible organic phase, which is separated from the nu-

cleic acid–containing aqueous phase by centrifugation

(when a large amount of protein is present, it forms a white

precipitate between the organic and aqueous phases). Al-

ternatively (or in addition), protein can be dissociated from

the nucleic acids by such denaturing agents as detergents,

guanidinium chloride, or high salt concentrations, and/or it

can be enzymatically degraded by proteases. In all cases,

the nucleic acids, a mixture of RNA and DNA, can then be

isolated by precipitation with ethanol.The RNA can be re-

covered from such a precipitate by treating it with pancre-

atic DNase to eliminate the DNA. Conversely, the DNA

can be freed of RNA by treatment with RNase. Alterna-

tively, RNA and DNA may be separated by ultracentrifu-

gation (Section 6-6D).

In all these and subsequent manipulations, the nucleic

acids must be protected from degradation by nucleases

that occur both in the experimental materials and on hu-

man hands. Nucleases may be inhibited by the presence of

chelating agents such as ethylenediaminetetraacetic acid

(EDTA),

Ethylenediaminetetraacetic acid (EDTA)

COOHCH

HOOC

which sequester the divalent metal ions that nucleases re-

quire for activity. In cases where no nuclease activity can be

tolerated, all glassware must be autoclaved to heat dena-

ture the nucleases and the experimenter should wear plas-

tic gloves. Nevertheless, nucleic acids are generally easier

to handle than proteins because their lack, in most cases, of

a complex tertiary structure makes them relatively tolerant

of extreme conditions.

B. Chromatography

Many of the chromatographic techniques that are used to

separate proteins (Section 6-3) are also applicable to nu-

cleic acids. Oligonucleotides can be readily separated

HPLC, particularly that using reverse-phase chromatogra-

phy. Larger nucleic acids are often separated by procedures

that include ion exchange chromatography and gel filtra-

tion chromatography.

a. Hydroxyapatite Can Be Used to Isolate and

Fractionate DNA

Hydroxyapatite (a form of calcium phosphate; Section

6-3Db) is particularly useful in the chromatographic purifi-

cation and fractionation of DNA. Double-stranded DNA

binds to hydroxyapatite more tightly than do most other

molecules. Consequently, DNA can be rapidly isolated by

passing a cell lysate through a hydroxyapatite column,

washing the column with a phosphate buffer of concentra-

tion low enough to release only the RNA and proteins, and

then eluting the DNA with a more concentrated phosphate

solution. In addition, single-stranded DNA elutes from hy-

droxyapatite at a lower phosphate concentration than does

double-stranded DNA.

Section 6-6. Nucleic Acid Fractionation 157

Centrifugation2.Uniform mixture

of sample and

gradient-forming

substance

1. Gradient is

formed and

samples band at

their isopycnic

positions

Figure 6-32 Isopycnic ultracentrifugation. The centrifugation

starts with a uniform mixture of a macromolecular sample

dissolved in a solution of a dense, fast-diffusing solute such as

CsCl (left).At equilibrium in a centrifugal field, the solute forms

a density gradient in which the macromolecules migrate to their

positions of buoyant density (right).

JWCL281_c06_129-162.qxd 2/22/10 2:25 PM Page 157

b. Messenger RNAs Can Be Isolated

by Affinity Chromatography

Affinity chromatography (Section 6-3C) is useful in iso-

lating specific nucleic acids. For example, most eukaryotic

messenger RNAs (mRNAs) have a poly(A) sequence at

their 3¿ ends (Section 5-4Ac). They can be isolated on an

agarose or cellulose matrix to which poly(U) has been co-

valently attached. The poly(A) sequences specifically bind

to the complementary poly(U) in high salt and at low tem-

peratures and can later be released by altering these condi-

tions. Moreover, if the (partial) sequence of an mRNA is

known (e.g., as inferred from the corresponding protein’s

amino acid sequence), the complementary DNA strand may

be synthesized (via methods discussed in Section 7-6Aa)

and used to isolate that particular mRNA.

C. Electrophoresis

Nucleic acids of a given type may be separated by poly-

acrylamide gel electrophoresis (Sections 6-4B and 6-4C)

because their electrophoretic mobilities in such gels vary

inversely with their molecular masses. However, DNAs of

more than a few thousand base pairs are too large to pene-

trate even a weakly cross-linked polyacrylamide gel. This

difficulty is partially overcome through the use of agarose

gels. By using gels with an appropriately low agarose con-

tent, relatively large DNAs in various size ranges may be

fractionated. In this manner, plasmids, for example, may be

separated from the larger chromosomal DNA of bacteria.

a. Duplex DNA Is Detected by Selectively Staining It

with Intercalation Agents

The various DNA bands in a gel must be detected if

they are to be isolated. Double-stranded DNA is readily

stained by planar aromatic cations such as ethidium ion

and acridine orange.

These dyes bind to duplex DNA by intercalation (slip-

ping in between the stacked base pairs), where they exhibit

a fluorescence under UV light that is far more intense than

that of the free dye. As little as 50 ng of DNA may be de-

tected in a gel by staining it with ethidium bromide (Fig.6-33).

N(CH

)

(CH

)

Ethidium

Acridine orange

Single-stranded DNA and RNA also stimulate the fluores-

cence of ethidium ion but to a lesser extent than does du-

plex DNA. Disadvantages of using these dyes are that they

are mutagens and therefore must be handled and disposed

of with caution and that exposure to UV light damages

DNA (Section 30-5Aa). SYBR Safe, an equally sensitive

nonmutagenic dye that fluoresces when it is bound to dou-

ble-stranded DNA and excited by blue light, avoids these

difficulties.

b. Very Large DNAs Are Separated by Pulsed-Field

Gel Electrophoresis

The sizes of the DNAs that can be separated by conven-

tional gel electrophoresis are limited to ⬃100,000 bp even

when gels containing as little as 0.1% agarose (which

makes an extremely fragile gel) are used. However, the de-

velopment of pulsed-field gel electrophoresis (PFGE) by

Charles Cantor and Cassandra Smith extended this limit to

DNAs with up to 10 million bp (6.6 million kD). The elec-

trophoresis apparatus used in PFGE has two or more pairs

of electrodes arrayed around the periphery of an agarose

slab gel. The different electrode pairs are sequentially

pulsed for times varying from 0.1 to 1000 s depending on

the sizes of the DNAs being separated. Gel electrophoresis

of DNA requires that these elongated molecules worm

their way through the gel’s labyrinthine channels more or

less in the direction from the cathode to the anode. If the

direction of the electric field abruptly changes, these DNAs

must reorient their long axes along the new direction of the

field before they can continue their passage through the

gel. The time required to reorient very long gel-embedded

158 Chapter 6. Techniques of Protein and Nucleic Acid Purification

Figure 6-33 Agarose gel electrophoretogram of double helical

DNA. After electrophoresis, the gel was soaked in a solution of

ethidium bromide, washed, and photographed under UV light.

The fluorescence of the ethidium cation is strongly enhanced

by binding to DNA, so each fluorescent band marks a

different sized DNA fragment. [Klaus Guldbrandsen/Photo

Researchers, Inc.]

JWCL281_c06_129-162.qxd 6/3/10 8:24 AM Page 158

DNA molecules evidently increases with their size. Conse-

quently, a judicious choice of electrode distribution and

pulse lengths causes shorter DNAs to migrate through the

gel faster than longer DNAs, thereby effecting their sepa-

ration (Fig. 6-34).

D. Ultracentrifugation

Equilibrium density gradient ultracentrifugation (Section

6-5Bb) in CsCl constitutes one of the most commonly used

DNA separation procedures. The buoyant density, ␳, of

double-stranded Cs

⫹

DNA depends on its base composi-

tion:

[6.18]

where is the mole fraction of G ⫹ C. Hence a CsCl

density gradient fractionates DNA according to its base com-

position. For example,eukaryotic DNAs often contain minor

fractions that band separately from the major species. Some

of these satellite bands consist of mitochondrial and chloro-

plast DNAs. Another important class of satellite DNA is

composed of repetitive sequences that are short segments of

DNA tandemly repeated hundreds, thousands, and in some

cases millions of times in a genome (Section 34-2B). Like-

wise, plasmids may be separated from bacterial chromosomal

DNA by equilibrium density gradient ultracentrifugation.

Single-stranded DNA is ⬃0.015 g ⴢ cm

–3

denser than the

corresponding double-stranded DNA so that the two may

be separated by equilibrium density gradient ultracentrifu-

gation. RNA is too dense to band in CsCl but does so in

solutions. RNA–DNA hybrids will band in CsCl

but at a higher density than the corresponding duplex

DNA.

RNA may be fractionated by zonal ultracentrifugation

through a sucrose gradient (Section 6-5Ba). RNAs are sep-

arated by this technique largely on the basis of their size. In

fact, ribosomal RNA, which constitutes the major portion

G⫹C

␳⫽1.660 ⫹ 0.098X

G⫹C

of cellular RNA, is classified according to its sedimentation

rate; for example, the RNA of the E. coli small ribosomal

subunit is known as 16S RNA (Section 32-3A).

Chapter Summary 159

Figure 6-34 Pulsed-field gel electrophoresis (PFGE) of a set of

yeast chromosomes. The yeast chromosomes, which were run as

identical samples in the 13 inner lanes, have sizes 260, 290, 370,

460, 580/600, 700, 780, 820, and 850 kb. The two outer lanes, which

provide molecular mass standards, contained twenty successively

larger multimers of a ⬃43.5-kb bacteriophage ␭ DNA (top to

bottom) to an observed limit of ⬃850 kb. [Electrophoretogram by

Margit Burmeister, University of Michigan, in Wilson, K. and

Walker, J., Principles and Techniques of Biochemistry and Molecular

Biology (6th ed.), p. 477, Cambridge University Press (2005).]

1 Protein Isolation Macromolecules in cells are solubil-

ized by disrupting the cells by various chemical or mechanical

means such as detergents or blenders. Partial purification by

differential centrifugation is used after cell lysis to remove cell

debris or to isolate a desired subcellular component.When out

of the protective environment of the cell, proteins and other

macromolecules must be treated so as to prevent their de-

struction by such influences as extremes of pH and tempera-

ture, enzymatic and chemical degradation, and rough mechan-

ical handling.The state of purity of a substance being isolated

must be monitored throughout the purification procedure by a

specific assay.

2 Solubilities of Proteins Proteins are conveniently pu-

rified on a large scale by a fractional precipitation process

called salting out, in which protein solubilities are varied by

changing the salt concentration or pH.

3 Chromatographic Separations Ion exchange chro-

matography employs support materials such as cellulose or

cross-linked dextran gels. Separations are based on differential

electrostatic interactions between charged groups on the ion

exchange materials and those on the substances being sepa-

rated. Molecules may be located through their UV absorbance,

fluorescence, radioactivity, or enzymatic activity. In gel filtra-

tion chromatography, molecules are separated according to

their size and shape through the use of cross-linked dextran,

polyacrylamide, or agarose beads that have pores of molecular

dimensions. A calibrated gel filtration column can be used to

estimate the molecular masses of macromolecules. Affinity

chromatography separates biomolecules according to their

unique biochemical abilities to bind other molecules specific-

ally. High-performance liquid chromatography (HPLC) uti-

lizes any of the foregoing separation techniques but uses high-

resolution chromatographic materials, high solvent pressures,

and automatic solvent mixing and monitoring systems so as to

obtain much greater degrees of separation than are achieved

with the more conventional chromatographic procedures.

CHAPTER SUMMARY

JWCL281_c06_129-162.qxd 2/22/10 2:26 PM Page 159

General

Ahmed, H., Principles and Reactions of Protein Extraction, Purifi-

cation, and Characterization, CRC Press (2005).

Bonner, P.L.R., Protein Purification, Taylor & Francis (2007).

Boyer, R.F., Biochemistry Laboratory: Modern Theory and Tech-

niques, Benjamin-Cummings (2006).

Burgess, R.R. and Deutscher, M.P. (Eds.), Guide to Protein Purifi-

cation (2nd ed.), Methods Enzymol. 463, (2009).

Harding, S.E. and Chowdhry, B.Z. (Eds.)., Protein Ligand Interac-

tions: Structure and Spectroscopy. A Practical Approach,

Oxford University Press (2001). [Contains descriptions of a

variety of physical techniques for studying proteins and their

interactions with other molecules.]

Meyers, R.A., Proteins. From Analytics to Structural Genomics,

Vol. 2, Chapters 20–24, Wiley–VCH (2007).

Ninfa,A.J., Ballou, D.P., and Benore, M., Fundamental Laboratory

Approaches for Biochemistry and Biotechnology (2nd ed.),

Wiley (2010).

Pingoud,A., Urbanke, C., Hoggett, J., and Jeltsch,A., Biochemical

Methods. A Concise Guide for Students and Researchers,

Wiley–VCH (2002).

Roe, S. (Ed.), Protein Purification Techniques. A Practical Ap-

proach (2nd ed.); and Protein Purification Applications. A

Practical Approach (2nd ed.), Oxford University Press (2001).

Tinoco, I., Sauer, K., Wang, J.C., and Puglisi, J.C., Physical Chem-

istry. Principles and Applications in Biological Sciences (4th ed.),

Chapter 6, Prentice-Hall (2002).

Simpson, R.J.,Adams, P.D., and Golemis, E.A. (Eds.), Basic Meth-

ods in Protein Purification and Analysis. A Laboratory Man-

ual, Cold Spring Harbor Laboratory Press (2009).

Structural Genomics Consortium, et al., Protein production and

purification, Nature Methods 5, 135–146 (2008). [Discusses

methods for most efficiently producing and purifying recombi-

nant proteins.]

Walker, J.M. (Ed.), The Protein Protocols Handbook (2nd ed.),

Humana Press (2002).

Wilson, K. and Walker, J.M. (Eds.), Principles and Techniques of

Biochemistry and Molecular Biology (6th ed.), Chapters 10

and 11, Cambridge University Press (2005).

Solubility and Crystallization

Arakawa, T. and Timasheff, S.N., Theory of protein solubility,

Methods Enzymol. 114, 49–77 (1985).

Ducruix, A. and Giegé, R. (Eds.), Crystallization of Nucleic Acids

and Proteins. A Practical Approach, (2nd ed.), Oxford Univer-

sity Press (1999).

McPherson, A., Crystallization of Biological Macromolecules,

Cold Spring Harbor Laboratory Press (1999).

Chromatography

Dean, P.D.G., Johnson, W.S., and Middle, F.A. (Eds.), Affinity

Chromatography. A Practical Approach, IRL Press (1985).

Fischer, L., Gel filtration chromatography (2nd ed.), in Work, T.S.

and Burdon, R.H. (Eds.), Laboratory Techniques in Biochem-

istry and Molecular Biology, Vol. 1, Part II, North-Holland

Biomedical Press (1980).

Meyer,V.R., Practical High-Performance Liquid Chromatography

(2nd ed.),Wiley (1994).

Oliver, R.W.A. (Ed.), HPLC of Macromolecules. A Practical

Approach (2nd ed.), IRL Press (1998).

Rossomando, E.F., HPLC in Enzymatic Analysis (2nd ed.), Wiley

(1998).

Weston, A. and Brown, P.R., HPLC and CE. Principles and Prac-

tice,Academic Press (1997).

Electrophoresis

Altria, K.D., Capillary Electrophoresis Guidebook, Humana Press

(1996).

Baker, D.R., Capillary Electrophoresis, Wiley (1995).

Burmeister, M. and Ulanovsky, L., Pulsed-Field Gel Electrophore-

sis, Humana Press (1992).

Gersten, D.M., Gel Electrophoresis: Proteins, Wiley (1996).

Griffin, T.J. and Aebersold, R., Advances in proteome analysis

by mass spectrometry, J. Biol. Chem. 276, 45497–45500

(2001).

Hames, B.D. (Ed.), Gel Electrophoresis of Proteins. A Practical

Approach (3rd ed.), IRL Press (1998).

Jones, P., Gel Electrophoresis: Essential Techniques,Wiley (1999).

REFERENCES

Adsorption chromatography, thin layer chromatography (TLC),

reverse-phase chromatography (RPC), hydrophobic interac-

tion chromatography (HIC), and metal chelate affinity chrom-

atography also have valuable biochemical applications.

4 Electrophoresis In electrophoresis, charged molecules

are separated according to their rates of migration in an electric

field on a solid support such as paper, cellulose acetate, cross-

linked polyacrylamide, or agarose. Gel electrophoresis employs

a cross-linked polyacrylamide or agarose gel support, so that

molecules are separated according to size by gel filtration as

well as according to charge.The separated molecules may be vis-

ualized by means of stains, autoradiography, or immunoblot-

ting.The anionic detergent sodium dodecyl sulfate (SDS) dena-

tures proteins and uniformly coats them so as to give most

proteins a similar charge density and shape. SDS–PAGE may be

used to estimate macromolecular masses. In isoelectric focusing

(IEF), macromolecules are immersed in a stable pH gradient

and subjected to an electric field that causes them to migrate to

their isoelectric positions. In capillary electrophoresis, the use of

thin capillary tubes and high electric fields permits rapid and

highly resolved separations of small amounts of material.

5 Ultracentrifugation In ultracentrifugation, molecules

are separated by subjecting them to gravitational fields large

enough to counteract diffusional forces. Molecules may be

separated and their molecular masses estimated from their

rates of sedimentation through a solvent or a preformed gra-

dient of an inert low molecular mass material such as sucrose.

Alternately, molecules may be separated according to their

buoyant densities in a solution with a density gradient of a

dense, fast-diffusing substance such as CsCl. The deviation of

a molecule’s frictional ratio from unity is indicative of its de-

grees of solvation and elongation.

6 Nucleic Acid Fractionation Nucleic acids can be frac-

tionated by many of the techniques that are used to separate

proteins. Hydroxyapatite chromatography separates single-

stranded DNA from double-stranded DNA. Polyacrylamide or

agarose gel electrophoresis separates DNA largely on the basis

of size. Very large DNAs can be separated by pulsed-field gel

electrophoresis (PFGE) on agarose gels. DNAs may be fraction-

ated according to their base composition by CsCl density gradi-

ent ultracentrifugation. Different species of RNA can be sepa-

rated by zonal ultracentrifugation through a sucrose gradient.

160 Chapter 6. Techniques of Protein and Nucleic Acid Purification

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