component would be too small to be useful. But if the B lymphocytes that pro(cid:173)
`duce the various components of this antiserum are made into hyb ridomas, it
`becomes possible to screen individual hybridoma clones from the large mixture
`10 select one that produces the desired type of monoclonal antibody and co
`propagate the selected hybridoma indefinitely so as to produce that antibody in
`unlimited quantities. In principle, therefore, a monoclonal antibody can be
`made against any protein in a biological sample.
`Once an antibody has been made, it can be used as a specific probe-both
`to track down and loca lize its protein antigen and to purify that protein in order
`to study its structure and function. Because only a small fraction of the estimated
`I 0,000 -20,000 proteins in a typical mammalian cell have thus far been isola ted,
`many monoclonal antibodies made against impure protein mixtures in frac(cid:173)
`tionated cell extracts identify new proteins. With the use of monoclonal an ti(cid:173)
`bodies and the rapid protein identification methods we shall describe hortly, it
`is no longer difficult to identify and characterize novel proteins and genes. The
`major problem is instead co determine their function , using a sec of powerful
`cools chat we discuss in the lase sections of chis chapter.
`
`Summary
`Tissues can be dissociated into their component cells, from which individual cell
`types can be purified and used for biochemical analysis or for the establishment of
`cell cultures. Many animal and plant cells survive and proliferate in a culture dish
`if they are provided with a suitable medium containing nutrients and specific protein
`growth factors. Althollgh most animal cells die after a finite number of divisions,
`immortal cells that arise spontaneously in culture-or are generated by adding
`genes throllgh genetic manipulation-can be maintained indefinitely as cell lines.
`Clones ca11 be derived from a single ancestor cell, making it possible to isolate uni(cid:173)
`form populations of mutant cells with defects in a single protein. Two cells can be
`fused to prodL1ce heterocaryons with two nuclei, enabling interactions between the
`components of the original two cells to be examined. Heterocaryons eventually form
`hybrid cells with a single fused nucleus. Because such cells lose chromosomes, they
`ca11 provide a convenient method for assigning genes to specific chromosomes. One
`type of hybrid cell, called a hybridoma, is widely employed to produce unlimited
`qua.ntities of 1111iform monoclonal antibodies, which are widely used to detect and
`purify cellular proteins.
`
`FRACTIONATION OF CELLS
`Although biochemical analysis requires disruption of the anatomy of the cell,
`gentle fractionation techniques have been devised to separate the various cell
`components while preserving their individual functions. Just as a tissue can be
`separated into its living constituent cell types, so the cell can be separated into
`its functioning organelles and macromolecules. In this section we con ider the
`methods that allow organelles a nd proteins to be purified and analyzed bio(cid:173)
`chemically.
`
`Organelles and Macromolecules Can Be Separated
`by Ultracentrifugation
`Cells can be broken up in various ways: they can be subjected to osmotic shock
`or ultrasonic vi bra tion, forced through a small orifice, or ground up in a blender.
`These procedures break many of the membranes of the cell (including the plas(cid:173)
`ma membrane and membran es of the endoplasmic reticulum) into fragments
`that immediately reseal to form small clo ed vesicles. If carefully applied, how(cid:173)
`ever, the disruption procedures leave organelles such as nuclei, mitochondria,
`the Golgi apparatus, lysosomes, and peroxisomes largely intact. The suspension
`of cells is thereby reduced to a thick slurry (called a homogenate or extract) that
`contains a variety of membrane-enclosed organelles, each with a distincti ve
`
`Figure 8-7 The preparative ultracentrifuge. The sample is contained in
`tubes that are inserted into a ring of cylindrical holes in a metal rotor. Rapid
`rotation of the rotor generates enormous centrifugal forces, which cause
`particles in the sample to sediment. The vacuum reduces friction, preventing
`heating of the rotor and allowing the refrigeration system to maintain the
`sample at 4°C.
`
`size, charge, and density. Provided that the homogenization medium has been
`carefully chosen (by trial and error for each organelle), the various compo(cid:173)
`nents-including the vesicles derived from the endoplasmic reticulum, called
`microsomes-retain most of the ir original biochemical properties.
`The different components of the homogenate must then be separated. Such
`cell fractiona tions became possible only after the commercial development in
`the early 1940s of an instrument known as the preparative ultracentrifuge, in
`which extracts of broke n cells are rotated at high speeds (Figure 8-7). This treat(cid:173)
`ment separates cell components by size and density: in general. the largest u nits
`experience the largest centri fugal force and move the most rapidly. At relatively
`low speed, large components such as nuclei sediment to form a pellet at the bot(cid:173)
`tom of the centrifuge cube; at slightly higher speed, a pellet of mitochondria is
`deposited; and at even higher speeds and with longer pe riods of cen trifugation,
`first the small closed vesicles a nd then the ribosomes can be collected (Figure
`8-8). All of these fractions are impure, but many of the contaminants can be
`removed by resuspending the pellet and repeating the centrifugation procedure
`several times.
`Centrifugation is the first step in most fractionations, but it separates only
`components that differ greatly in size. A finer degree of separation can be
`achieved by layering the homogenate in a thin band on lop of a dilute salt solu(cid:173)
`tion that fills a centrifuge tube. When centrifuged, the various components in
`the mixture move as a series of distinct bands through the salt solution, each at
`a different rate, in a process called uelocity sedimentation (Figure 8-9A). For the
`procedure to work effectively, the bands must be protected from convective mix(cid:173)
`ing, which would normally occur whenever a denser solut ion (for example, one
`containing organelles) fin ds itself on top of a lighter one (the salt solution). This
`is achieved by filling the centrifuge tube with a shallow gradient of sucrose pre(cid:173)
`pared by a special mixing device. The resulting density gradient-with the dense
`end at the bottom of the tube-keeps each region of the salt solution denser
`than any solution above it, and it thereby prevents convective m ixing from dis(cid:173)
`torting the sepa ration.
`When sedi mented through such dilute sucrose gradients, different cell com(cid:173)
`ponents separate into distinct bands that can be collected individually. The rel(cid:173)
`ative rate at which each componen t sed iments depends primarily on its size and
`shape-being normally described in terms of its sedimentation coefficient, or s
`value. Present-day ultracentrifuges rotate at speeds ofup to 80,000 rpm and pro(cid:173)
`duce fo rces as high as 500,000 times gravity. With these enormous forces, even
`small macromolecules, such as tRNA molecules and simple enzymes, can be
`driven to sediment at an appreciable rate and so can be separated from one
`another by size. Measurements of sedimentation coefficients are routinely used
`to help in determining the size and subunit composition of the organized
`assemblies of macromolecules found in cells.
`The ultracentrifuge is also used to separate cellular componen ts on the basis
`of their buoyant density, independently of their size and shape. In this case the
`
`Figure 8-8 Cell fractionation by centrifugation. Repeated
`centr ifugatio n at progressively higher speeds will fractionate homogenates
`of cells into their components. In general, the smaller the subcellular
`component. the greater is the centrifugal force required to sediment it.
`Typical values fo r the various centrifugation steps referred to in the
`figure are:
`
`low speed
`medium speed
`high speed
`very high speed
`
`I 000 times gravity for IO minutes
`20,000 times gravity for 20 minutes
`80,000 times gravity for I hour
`I 50.000 times gravity for 3 hours
`
`478
`
`Chapter 8 : MANIPULATING PROTEINS. D NA. AND RNA
`
`FRACTIONATION OF Ch.LS
`
`armored chamber
`
`sedimenting material
`
`t
`
`refrigeration
`
`vacuum
`
`motor
`
`ill ·.-;.~ ·~:
`
`... ·•
`..• _ .
`·--~·-··
`I
`LOW-SPEED CENTRIFUGATION
`•
`
`cell
`homogenate
`
`pellet contains
`who, ce Is
`nuclei
`cytoskeletons
`
`SUPERNATANT SUBJECTED TO
`MEDIUM-SPEED CENTRIFUGATION
`
`l ..... : . :
`
`pellet contains
`r t
`:hond,
`, ... ..,~vml. ..
`perox,somes
`
`SUPERNATANT SU BJECTED TO
`HIGH-SPEED CENTR IFUGATION
`
`l
`
`,.
`: . . · ..
`-:.: _: :_::
`.... .. .
`.. ·.::··,:
`~t:--
`
`l
`
`SUPERNATANT SUBJECTED TO VERY
`HIGH-SPEED CENTRIFUGATION
`
`pellet contains
`
`pellet contains
`ribosomes
`viruses
`'-\\y( - - - ~ic~omolecules
`
`479
`
`00029
`
`EX1010, pt.2
`
`

`

`(A)
`
`, ,.,,, ...... , .. ,
`
`sample
`
`(B)
`
`stabilizing
`sucrose
`gradient
`
`I
`CENTRIFUGATION
`
`l
`
`. ,,,~,, . .,,,,~
`
`slow-sedimenting
`component
`
`fast-sedimenting
`component
`
`I
`FRACTIONATION
`l
`
`sample
`
`steep
`sucrose
`g radient
`(e.g., 20-70%)
`
`low-buoyant(cid:173)
`density
`component
`
`- high-buoyant(cid:173)
`density
`component
`
`-~,,.~,,,,,,,
`
`Figure 8-9 Comparison of velocity
`sedimentation and e quilibrium
`sedimentation. In ve locity
`sedimentation (A) subcellu lar components
`sedime nt at different speeds according to
`their size and shape when layered over a
`dilute solution containing sucrose. To
`stabilize the sedimenting bands against
`convective mixing caused by small
`diffe rences in temperature or solute
`concentration, the tube contains a
`continuous shallow gradient of sucrose
`that increases in concentration toward the
`bottom of the tube (typically from 5% to
`20% sucrose). Following centrifugation. the
`differe nt components can be collected
`individually, most simply by puncturing the
`plastic ce ntrifuge tube and collecting
`drops from the bottom, as illustrated
`here. In equilibrium sedimentation (B)
`subcellular components move up or down
`when centrifuged in a gradient until they
`reach a position where their density
`matches their surround ings. Although a
`sucrose grad ient is shown here, denser
`gradients, which are especially useful fo r
`protein and nucleic acid separation, can b
`formed from cesium chloride. The final
`bands, at equilibrium, can be collected
`as in (A).
`
`sample is usually sedimented th ro ugh a steep density gradient that contains a
`very high concentration of sucrose or cesium chloride. Each cellula r compon ent
`begins to m ove down the gradient as in Figure 8-9A, but it even tually reaches a
`positio n whe re the density of the solution is equal to its own density. At this
`poin t the component floats and can move no farther. A series of disti nct bands
`is thereby prod uced in the centrifuge tube, with the bands closest to the bottom
`of the tube containing the compo ne nts of highest buoyant density (Figure
`8-9 B). This method, called equilibrium sedimentation, is so sensitive that it is
`capable of separating macromolecules tha t h ave incorporated heavy isotopes,
`such as 13C or 15N, from the same macromolecules that contain the lighter, com (cid:173)
`mon isotop es (12C or 14N). ln fact, th e cesium-chloride method was developed
`in 1957 to separate the labeled from the unlabeled DNA produced after exposure
`of a growin g population of bacteria to nucleotide precursors containing 15N; this
`classic experimen t provided direct evidence for the semiconservative replica(cid:173)
`tio n of DNA (see Figure 5-5).
`
`The Molecular Details of Complex Cellular Processes
`Can Be Deciphered in Cell-Free Systems
`Studies of organelles and other large subcellular componen ts isolated in the
`ultrace ntrifuge have contributed enormously to our understanding of the func(cid:173)
`tions of different cellular compone nts. Experiments on mitochondria and
`chloroplasts purified by centrifugation , for example, demonstrated the cen tral
`function of these organelles in convert ing en ergy into forms that the cell can
`use. Similarly, resealed vesicles fo rmed from fragments of ro ugh and smooth
`endoplasmic reticulum (microsomes) have been separated from each other and
`analyzed as functional models of these compa rtments of the intact cell .
`
`480
`
`Chapter 8 : MANIPULATIN G PROTEINS, D N A. A N D RN A
`
`An extension of this approach makes it possible to study many other biolog(cid:173)
`ical processes free from all of the complex side reactions that occur in a living
`cell, by using purified cell-free systems. In this case, cell homogenates are frac(cid:173)
`tionated with the aim of purifying each of the individual macromolecules that
`are needed to catalyze a biological process of interest. For example, the mecha(cid:173)
`nisms of protein synthesis were deciphered in experiments that began with a
`cell h omogena te that could translate RNA molecules to produce proteins. Frac(cid:173)
`tionation of this homogenate, step by step, produced in turn the ribosomes,
`tRNAs, and various enzymes that together constitute the protein-synthetic
`machinery. Once individual pure components were available, each could be
`added or withheld separately to define its exact role in the overall process. A
`major goal today is the reconstitution of every biological process in a purified
`cell-free system, so as to be able to define all of its components and their mech(cid:173)
`anism of action. Some lan dmarks in the development of this critical approach
`for understanding the cell are listed in Table 8-4 .
`Much of what we know about the molecular biology of the cell has been dis(cid:173)
`covered by studying cell-free systems. As a few of many examples, they have
`been used to decipher th e molecular details of D A replication and D A tran(cid:173)
`scription, RNA splicing, protein translation, muscle contraction, and particle
`transport along microtubules. Cell-free systems have even been used to study
`such com plex and highly organized processes as the cell-division cycle, the sep(cid:173)
`aration of chromosomes on the mitotic spindle, and the vesicular-transport
`steps involved in the movement of proteins from the endoplasmic reticulum
`through th e Golgi apparatus to the plasma membrane.
`Cell h omogenates also provide, in principle, the starting material for the
`complete separation of all of the individual macromolecular components from
`the cell. We now consider how this separation is achieved, focusing on proteins.
`
`Proteins Can Be Separated by Chromatography
`Proteins are most often fractionated by column chromatography, in which a
`mixture of proteins in solution is passed through a column containing a porous
`solid matrix. The different proteins are relarded to different extents by lheir
`
`TABLE 8-4 Some Major Events in the Development of Cell-Free Systems
`
`1897
`
`1926
`
`1935
`
`1938
`
`1939
`1949
`
`1951
`1954
`1954
`
`1957
`
`1975
`1976
`1983
`1984
`
`Buchner shows that ceU-free extracts of yeast can ferment sugars to form carbon dioxide and ethanol, laying
`the foundations of enzymology.
`Svedberg develops the first analytical ultracentrifuge and uses it to estimate the mass of hemoglobin
`as 68,000 daltons.
`Pickets and Beams introduce several new features of centrifuge design that lead to its use as a preparative
`instrument.
`Behrens employs differential centrifugation to separate nuclei and cytoplasm from liver cells, a technique
`fu rther developed for the fractionation of cell organelles by Claude, Brachet, Hogeboom, and others in the
`1940s and early 1950s.
`Hill shows that isolated chloroplasts, when illuminated, can perform the reactions of photosynthesis.
`Szent-Gyorgyi shows that isolated myofibrils from skeletal muscle cells contract upon the addition of ATP. In
`1955 a similar cell-free system was developed for ciliary beating by Hofmann-Berling.
`Brakke uses density-gradient centrifugation in sucrose solutions to purify a plant virus.
`de Duve isolates lysosomes and, later, peroxisomes by centrifugation.
`Zamecnik and colleagues develop the first cell-free system to perform protein synthesis. A decade of intense
`research activity, during which the genetic code is elucidated, follows.
`Meselson, Stahl, and Vinograd develop equilibrium density-gradient centrifugation in cesium chloride
`solutions for separating nucleic acids.
`Dobberstein and Blobel demonstrate protein translocation across membranes in a cell-free system.
`Neher and Sakrnann develop patch-clamp recording to measure the activity of single ion channels.
`Lohka and Masui makes concentrated extracts from frog eggs that performs the entire cell cycle in vitro.
`Rothman and colleagues reconstitute Golgi vesicle trafficking in vitro with a cell-free system.
`
`FRACTIONATION OF CELLS
`
`481
`
`•
`
`00030
`
`

`

`poorer separation by conventional chromatography. HPLC has therefore become
`the method of choice for separating many proteins and small molecules.
`
`Affinity Chromatography Explo its Specific
`Binding Sites on Proteins
`
`lf one starts with a complex mixture of proteins, these types of column chro(cid:173)
`matography do not produce very highly pu rified fractions: a single passage
`through the column generally increases the proportion of a given protein in the
`mixture no more than twentyfold. Because most individual proteins represent
`less than l /1000 of the total cellular protein, it is usually necessary to use several
`different types of column in succession to attain sufficient purity (Figure 8-12).
`A more efficient procedure, known as affinity chromatography, takes advan(cid:173)
`tage of the biologically important binding interactions that occur on protein
`surfaces. If a substrate molecule is covalently coupled to an inert matrix such as
`a polysaccharide bead, for example, the enzyme that operates on that substrate
`will often be specifically retained by the matrix and can then be eluted (washed
`out) in nearly pure form. Likewise, short DNA oligonucleotides of a specifically
`designed sequence can be immobilized in this way and used to purify D A(cid:173)
`binding proteins that normally recognize th is sequence of nucleotides in chro(cid:173)
`mosomes (see Figure 7-30). Alternatively, specific antibodies can be coupled to
`a matrix to purify protein molecules recognized by the antibodies. Because of
`the great specificity of all such affinity columns, 1000- to 10,000-fold purifica(cid:173)
`tions can sometimes be achieved in a s ingle pass.
`Any gene can be modified, using the recombinant DNA methods discussed
`in the next section, to produce its protein with a molecular tag attached to it,
`making subsequent purification of the protein by affinity chromatography sim(cid:173)
`ple and rapid (see Figure 8-48, below). For example, the amino acid histidine
`
`solvent flow
`
`. .-.-•-.-
`
`+ + +
`• + _
`positively
`e +
`• -
`-e
`e+
`+
`h
`d
`-
`c arge
`+e- bead
`+++
`+
`- . +
`+
`+ + + +
`e+ bound
`+
`.
`-
`•
`+
`+
`• + + + e + 4?
`negatively
`+ + + e --- charged
`+ •
`e+ •
`+ e+ molecule
`+
`e+ • \
`free
`+ _
`• + . +• + + + • e:_ positively
`e+
`charged
`molecule
`
`•
`
`•
`
`•
`•
`•
`• : ..•...
`•
`• •••
`• . . --
`•••
`• • •
`• •• •
`•
`• • •
`• • •
`
`porous beads
`
`retarded
`small molecu le
`
`unretarded
`large molecule
`
`solvent continuously
`applied to the top of
`sample column from a large
`applied
`reservoir of solvent
`
`Figure 8- 10 The separat ion of
`m o lecule s by co lumn
`chromatography. The sample, a mixture
`of different molecules, is applied to the
`top of a cylindr ical glass or plastic column
`filled with a permeable solid matrix, such
`as cellulose, immersed in solvent.A large
`amount of solvent is then pumped slowly
`through the column and collected in
`separate tubes as it emerges from the
`bottom. Because various components of
`the sample travel at different rates
`through the column, they are fractionated
`into different tubes.
`
`6
`
`solid
`
`plug
`
`test
`tu be
`
`"""' u 0
`
`time
`
`6
`
`'
`'
`0 i i
`
`fractionated molecules
`eluted and collected
`
`interaction with the matrix, and they can be collected separately as they flow
`out of the bottom of the column (Figure 8- 10). Depending on the choice of
`matrix, proteins can be separated according to their charge (ion-exchange
`chromatography), their hydrophobicity (hydrophobic chromatography), their
`size (gel-filtration chromatography), or their ability to bind to particular small
`molecules or to other macromolecules (affinity chromatography).
`Many types of matrices are commercially available (Figure 8-11 ). Ion(cid:173)
`exchange columns are packed with small beads that carry either a positive or
`negative charge, so that proteins arc fractionated according to the arrangement
`of charges on their surface. Hydrophobic columns are packed with beads from
`which hydrophobic side chains protrude, so tha t proteins with exposed
`hydrophobic regions are retarded. Gel-filtration columns, which separate pro(cid:173)
`teins according lo their size, are packed with tiny porous beads: molecules that
`are small enough to enter the pores linger inside successive beads as they pass
`down the column, wh ile larger molecules remain in the solution flowing
`between the beads and therefore move more rapidly, emerging from the column
`first. Besides providi ng a means of separating molecules, gel-filtration chro(cid:173)
`matography is a convenient way to determine their size.
`The resolution of conventional column chromatography is limited by
`inhomogeneities in the matrices (such as cellulose), which cause an uneven
`flow of solvent through the column. Newer chromatography resins (usually sil(cid:173)
`ica-based) have bee n developed in the form of tiny spheres (3 to 10 µm in
`diameter) that can be packed with a special apparatus to form a uniform col(cid:173)
`umn bed. A high degree of resolution is attainable on such high -performance
`liquid chromatography (HPLC) columns. Because they contain such tightly
`packed particles, HPLC columns have negligible flow rates unless high pressures
`are applied. For this reason these columns a re typically packed in steel cylinders
`and require an elaborate system of pumps and valves to force the solvent
`through them at sufficient pressure to produce the desired rapid flow rates of
`about one column volume per minute. In conventional column chromatogra phy,
`flow rates must be kept slow (often about one column volume per hour) to give
`the solutes being fractionated time to equilibrate with the interior of the large
`matrix particles. In HPLC the solutes equilibrate very rapidly with the interior of
`the tiny spheres, so solutes with different affinities for the matrix are effi ciently
`separated from one another even at fast flow rates. This allows most fract ion(cid:173)
`ations to be carried out in minutes, whereas hours are required to obtain a
`
`solvent flow
`
`solvent flow
`
`(\
`
`\ .,
`
`-
`
`\ f)
`C
`bead with
`f)
`/
`covalently
`-.. ~ a ttached
`W
`'.,,.
`s ubstrate
`ft& G) '
`, , I
`,
`/
`I
`e--~ other proteins
`
`bound
`enzyme
`molecule
`
`•
`
`,
`
`I
`
`\
`
`, ~ pass through
`
`(A)
`
`ION EXCHANGE CHROMATOGRAPHY
`
`(B) GEL-FILTRATION CHROMATOGRAPHY
`
`(C) AFFINITY CHROMATOGRAPHY
`
`Figure 8-11 Three types of matrices used for chromatography. In ion-exchange chromatography
`(A) the insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge.
`Matrices used for separating proteins include diethylaminoethylcellulose (DEAE-cellulose), which is positively
`charged, and carboxymethylcellulose (CM-cellulose) and phosphocellulose, which are negatively charged.
`Analogous matrices based on agarose or other polymers are also frequently used. The strength of the
`association between the dissolved molecules and the ion-exchange matrix depends on both the ionic
`strength and the pH of the solution that is passing down the column, which may therefore be varied
`systematically (as in Figure 8-12) to achieve an effective separation. In gel-filtration chromatography (8 ) the
`matrix is inert but porous. Molecules that are small enough to penetrate into the matrix are thereby delayed
`and travel more slowly through the column. Beads of cross-linked polysaccharide (dextran, agarose, or
`acrylamide) are available commercially in a wide range of pore sizes, making them suitable for the
`fractionation of molecules of various molecular weights, from less than 500 to more than S x I 06. Affinity
`chromatography (C) uses an insoluble matrix that is covalently linked to a specific ligand, such as an antibody
`molecule or an enzyme substrate, that will bind a specific protein. Enzyme molecules that bind to immobilized
`substrates on such columns can be eluted with a concentrated solution of the free form of the substrate
`molecule, while molecules that bind to immobilized antibodies can be eluted by dissociating the
`antibody-antigen comple x with concentrated salt solutions or solutions of high or low pH. High degrees of
`purification are often achieved in a single pass through an affinity column.
`
`482
`
`Chapt er 8 : MANIPULATING PROTEINS, DNA. AND RNA
`
`FRACTIONATION OF CELLS
`
`483
`
`00031
`
`

`

`binds to certain metal ions, including nickel and copper. If genetic engineering
`techniques are used to attach a short string of histidine residues to either end of
`a protein, the slightly modified protein can be retained selectively on an affinity
`column containing immobilized nickel ions. Metal affinity chromatography can
`thereby be used to purify that modified protein from a complex molecular mix(cid:173)
`ture. In other cases, an entire protein is used as the molecular tag. When the
`small enzyme glutathione S-transferase (GST) is attached to a target protein, the
`resulting fusion protein can be purified using an affinity column containing
`glutathione, a substrate molecule that binds specifically and tightly to GST (see
`Figure 8-50, below).
`As a further refinement of this last technique, an amino acid sequence that
`forms a cleavage site for a highly specific protease can be engineered between
`the protein of choice and the histidine or GST tag. The cleavage sites for the pro(cid:173)
`teases that are used, such as factor X that functions during blood clotting, are
`very rarely found by chance in proteins. Thus, the tag can later be specifically
`removed by cleavage at the cleavage site without destroying tl1e purified protein.
`
`(A) ION-EXCHANGE CHROMATOGRAPHY
`
`protein
`
`E
`::,
`0
`
`E ., .,
`-~ .;
`~
`
`.J:_activity
`
`fraction number -
`
`L__J
`pool these fractions and apply them
`to the next column below
`
`(B) GEL-FILTRATION CHROMATOGRAPHY
`
`E
`::,
`0
`E ., .,
`. ., .,
`~
`
`>
`
`fraction number -
`
`L__J
`pool these fractions and apply them
`to the next column below
`
`(C) AFFINITY CHROMATOGRAPHY
`
`E
`::,
`0
`
`E ., .,
`-~ .;
`~
`
`activity
`
`protein
`
`eluting
`solution
`applied
`
`to column l
`
`fraction number -
`
`pool these fractions, which now contain the
`highly purified protein
`
`Figure 8-12 Protein purification by
`chromatography. Typical results
`obtained when three different
`chromatographic steps are used in
`succession to purify a protein. In this
`example a homogenate of cells was first
`fractionated by allowing it to percolate
`through an ion-exchange resin packed into
`a column (A). The column was washed.
`and the bound proteins were then eluted
`by passing a solution containing a gradually
`increasing concentration of salt onto the
`top of the column. Proteins with the
`lowest affinity for the ion-exchange resin
`passed directly through the column and
`were collected in the earliest fractions
`eluted from the bottom of the column.
`The remaining proteins were eluted in
`sequence according to their affinity for the
`resin-those proteins binding most tightly
`to the resin requiring the highest
`concentration of salt to remove them. The
`protein of interest was eluted in several
`fractions and was detected by its
`enzymatic activity. The fractions with
`activity were pooled and then applied to
`second. gel-filtration column (B). The
`elution position of the still-impure protein
`was again determined by its enzymatic
`activity and the active fractions were
`pooled and purified to homogeneity on an
`affinity column (C) that contained an
`immobilized substrate of the enzyme.
`(D) Affinity purification of cyclin-binding
`proteins from S. cerevisiae, as analyzed by
`SDS polyacrylamide-gel electrophoresis
`(see Figure 8-14). Lane I is a total cell
`extract; lane 2 shows the proteins eluted
`from an affinity column containing cyclin
`B2; lane 3 shows one major protein eluted
`from a cyclin B3 affinity column. Proteins
`in lanes 2 and 3 were eluted with salt and
`the gels was stained with Coomassie blue
`(D. from D. Kellogg et al.,). Cell Biol.
`130:675-685, 1995. © The Rockefeller
`University Press.)
`
`2
`
`3
`
`(D)
`
`220-
`
`96- -
`--
`
`•
`
`T he Size and Subunit Composition of a Protein Can Be
`D etermined by SOS Polyacrylamide-Gel Electrophoresis
`
`Proteins usually possess a net positive or negative charge, depending on tl1e
`mixture of charged amino acids they contain. When an electric field is applied to
`a solution containing a protein molecule, the protein migrates at a rate that
`depends on its net charge and on its size a nd shape. This technique, known as
`electrophoresis, was originally used to separate mixtures of proteins either in
`free aqueous solution or in solutions held in a solid porous matrix such as
`starch.
`In ilie mid-1960s a modified version of thi s method-which is known as SDS
`polyacrylamide-gel electrophoresis (SDS-PAGE)-was developed that has rev(cid:173)
`olutionized routine protein analysis. It uses a highly cross-linked gel of poly(cid:173)
`acrylamide as the inert matrix through which the proteins migrate. The gel is
`prepared by polymerization from monomers; the pore size of the gel can be
`adjusted so that it is small enough to retard the migration of the protein
`molecules of interest. The proteins themselves are not in a simple aqueous solu(cid:173)
`tion but in one that includes a powerful negatively charged detergent, sodium
`dodecyl sulfate, or SDS (Figure 8-13). Because this detergent binds to hydropho(cid:173)
`bic regions of tl1e protein molecules, causing them to unfold into extended
`polypeptide chains, tl1e individual protein molecules are released from their
`associations with oilier proteins or lipid molecules and rendered freely soluble
`in the detergent solution. In addition, a reducing agent such as P-mercapto(cid:173)
`ethanol (see Figure 8-13) is usually added to break any S-S linkages in ilie pro(cid:173)
`teins, so that all of ilie constituent polypeptides in multisubunit molecules can
`be analyzed separately.
`What happens when a mixture of SDS-solubilized proteins is run through a
`slab of polyacrylamide gel? Each protein molecule binds large numbers of tl1e
`negatively charged detergent molecules, which mask the protein's intrinsic
`charge and cause it to migrate toward the positive electrode when a voltage is
`applied. Proteins of the same size tend to move through the gel with similar
`speeds because (1) their native structure is completely unfolded by the SOS, so
`that ilieir shapes are the same, and (2) they bind the same amount of SOS and
`therefore have the same amount of negative charge. Larger proteins, with more
`charge, will be subjected to larger electrical forces and also to a larger drag. In
`free solution tl1e two effects would cancel out, but in the mesh of the polyacryl(cid:173)
`amide gel, which acts as a molecular sieve, large proteins are reta rded much
`more than small ones. As a result, a complex mixture of proteins is fractionated
`into a series of discrete protein bands arranged in order of molecular weight
`(Figure 8-14). The major proteins are readily detected by staining the proteins in
`the gel with a dye such as Coomassie blue, and even minor proteins are seen in
`gels treated witl1 a silver or gold stain (with which as little as 10 ng of protein can
`be detected in a band).
`SDS polyacrylamide-gel electrophoresis is a more powerful procedure than
`any previous method of protein analysis principally because it can be used to
`separate all types of proteins, including those that are insoluble in water. Mem(cid:173)
`brane proteins, protein components of the cytoskeleton, and proteins that are
`part of large macromolecular aggregates can all be resolved. Because the
`meiliod separates polypeptides by size, it also provides