H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 37
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Finally, we consider several techniquesfor characterizing a protein’s mass, sequence, and threedimensional structure.Several allosteric mechanisms act as switches, turningprotein activity on and off in a reversible fashion.Centrifugation Can Separate Particles andMolecules That Differ in Mass or DensityThe binding of allosteric ligand molecules may lead tothe conversion of a protein from one conformational/The first step in a typical protein purification scheme iscentrifugation. The principle behind centrifugation is that■■3.6 • Purifying, Detecting, and Characterizing Proteinstwo particles in suspension (cells, organelles, or molecules) with different masses or densities will settle to thebottom of a tube at different rates.
Remember, mass is theweight of a sample (measured in grams), whereas densityis the ratio of its weight to volume (grams/liter). Proteinsvary greatly in mass but not in density. Unless a proteinhas an attached lipid or carbohydrate, its density will notvary by more than 15 percent from 1.37 g/cm3, the average protein density. Heavier or more dense molecules settle, or sediment, more quickly than lighter or less densemolecules.A centrifuge speeds sedimentation by subjecting particlesin suspension to centrifugal forces as great as 1,000,000times the force of gravity g, which can sediment particles assmall as 10 kDa.
Modern ultracentrifuges achieve theseforces by reaching speeds of 150,000 revolutions per minute(rpm) or greater. However, small particles with masses of5 kDa or less will not sediment uniformly even at such highrotor speeds.Centrifugation is used for two basic purposes: (1) as apreparative technique to separate one type of material fromothers and (2) as an analytical technique to measure physical properties (e.g., molecular weight, density, shape, andequilibrium binding constants) of macromolecules. The sedimentation constant, s, of a protein is a measure of its sedimentation rate. The sedimentation constant is commonlyexpressed in svedbergs (S): 1 S 1013 seconds.Differential Centrifugation The most common initial step inprotein purification is the separation of soluble proteins frominsoluble cellular material by differential centrifugation.
Astarting mixture, commonly a cell homogenate, is pouredinto a tube and spun at a rotor speed and for a period of timethat forces cell organelles such as nuclei to collect as a pelletat the bottom; the soluble proteins remain in the supernatant(Figure 3-31a). The supernatant fraction then is poured offand can be subjected to other purification methods to separate the many different proteins that it contains.Rate-Zonal Centrifugation On the basis of differences intheir masses, proteins can be separated by centrifugationthrough a solution of increasing density called a density gradient. A concentrated sucrose solution is commonly used toform density gradients. When a protein mixture is layered ontop of a sucrose gradient in a tube and subjected to centrifugation, each protein in the mixture migrates down the tubeat a rate controlled by the factors that affect the sedimentation constant.
All the proteins start from a thin zone at thetop of the tube and separate into bands, or zones (actuallydisks), of proteins of different masses. In this separation technique, called rate-zonal centrifugation, samples are centrifuged just long enough to separate the molecules ofinterest into discrete zones (Figure 3-31b). If a sample is centrifuged for too short a time, the different protein moleculeswill not separate sufficiently.
If a sample is centrifuged much87longer than necessary, all the proteins will end up in a pelletat the bottom of the tube.Although the sedimentation rate is strongly influenced byparticle mass, rate-zonal centrifugation is seldom effectivein determining precise molecular weights because variationsin shape also affect sedimentation rate.
The exact effects ofshape are hard to assess, especially for proteins and singlestranded nucleic acid molecules that can assume many complex shapes. Nevertheless, rate-zonal centrifugation hasproved to be the most practical method for separating manydifferent types of polymers and particles. A second densitygradient technique, called equilibrium density-gradient centrifugation, is used mainly to separate DNA or organelles(see Figure 5-37).Electrophoresis Separates Molecules on the Basisof Their Charge : Mass RatioElectrophoresis is a technique for separating molecules in amixture under the influence of an applied electric field.
Dissolved molecules in an electric field move, or migrate, at aspeed determined by their charge:mass ratio. For example,if two molecules have the same mass and shape, the one withthe greater net charge will move faster toward an electrode.SDS-Polyacrylamide Gel Electrophoresis Because manyproteins or nucleic acids that differ in size and shape havenearly identical charge:mass ratios, electrophoresis ofthese macromolecules in solution results in little or noseparation of molecules of different lengths.
However,successful separation of proteins and nucleic acids can beaccomplished by electrophoresis in various gels (semisolidsuspensions in water) rather than in a liquid solution.Electrophoretic separation of proteins is most commonlyperformed in polyacrylamide gels. When a mixture ofproteins is applied to a gel and an electric current is applied, smaller proteins migrate faster through the gel thando larger proteins.Gels are cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously cross-linking the chainsinto a semisolid matrix.
The pore size of a gel can be variedby adjusting the concentrations of polyacrylamide and thecross-linking reagent. The rate at which a protein movesthrough a gel is influenced by the gel’s pore size and thestrength of the electric field. By suitable adjustment ofthese parameters, proteins of widely varying sizes can beseparated.In the most powerful technique for resolving proteinmixtures, proteins are exposed to the ionic detergent SDS(sodium dodecylsulfate) before and during gel electrophoresis (Figure 3-32).
SDS denatures proteins, causing multimeric proteins to dissociate into their subunits, andall polypeptide chains are forced into extended conformations with similar charge:mass ratios. SDS treatment thus88CHAPTER 3 • Protein Structure and Function(a) Differential centrifugation1 Sample is poured into tube(b) Rate-zonal centrifugation1 Sample is layered on top of gradientLarger particleMore dense particleSmaller particleLess dense particle2CentrifugeParticles settleaccording tomassSucrosegradient2CentrifugalforceCentrifugeParticles settleaccording tomassCentrifugal force3Stop centrifugeDecant liquidinto container3Stop centrifugeCollect fractionsand do assayDecreasing mass of particles▲ EXPERIMENTAL FIGURE 3-31 Centrifugation techniquesseparate particles that differ in mass or density. (a) Indifferential centrifugation, a cell homogenate or other mixture isspun long enough to sediment the denser particles (e.g., cellorganelles, cells), which collect as a pellet at the bottom of thetube (step 2 ).
The less dense particles (e.g., soluble proteins,nucleic acids) remain in the liquid supernatant, which can betransferred to another tube (step 3 ). (b) In rate-zonalcentrifugation, a mixture is spun just long enough to separatemolecules that differ in mass but may be similar in shape anddensity (e.g., globular proteins, RNA molecules) into discretezones within a density gradient commonly formed by aconcentrated sucrose solution (step 2 ). Fractions are removedfrom the bottom of the tube and assayed (step 5 ).eliminates the effect of differences in shape, and so chainlength, which corresponds to mass, is the sole determinant ofthe migration rate of proteins in SDS-polyacrylamide electrophoresis.
Even chains that differ in molecular weight byless than 10 percent can be separated by this technique.Moreover, the molecular weight of a protein can be estimated by comparing the distance that it migrates through agel with the distances that proteins of known molecularweight migrate.Two-Dimensional Gel Electrophoresis Electrophoresis ofall cellular proteins through an SDS gel can separate proteinshaving relatively large differences in mass but cannot resolveproteins having similar masses (e.g., a 41-kDa protein froma 42-kDa protein). To separate proteins of similar masses,another physical characteristic must be exploited.
Most commonly, this characteristic is electric charge, which is determined by the number of acidic and basic residues in aprotein. Two unrelated proteins having similar masses are3.6 • Purifying, Detecting, and Characterizing Proteins1Denature sample withsodium dodecylsulfatePlace mixture of proteins on gel,apply electric field_Cross-linkedpolyacrylamidegelPartiallyseparatedproteinsDirection of migration+3Stain to visualizeseparated bandsDecreasingsizeunlikely to have identical net charges because their sequences, and thus the number of acidic and basic residues,are different.In two-dimensional electrophoresis, proteins are separated sequentially, first by their charges and then by theirmasses (Figure 3-33a).
In the first step, a cell extract isfully denatured by high concentrations (8 M) of urea andthen layered on a gel strip that contains an continuous pHgradient. The gradient is formed by ampholytes, a mixtureof polyanionic and polycationic molecules, that are castinto the gel, with the most acidic ampholyte at one endand the most basic ampholyte at the opposite end. Acharged protein will migrate through the gradient until itreaches its isoelectric point (pI), the pH at which the netcharge of the protein is zero.
This technique, called iso- EXPERIMENTAL FIGURE 3-32 SDSpolyacrylamide gel electrophoresis separatesproteins solely on the basis of their masses. Initialtreatment with SDS, a negatively charged detergent,dissociates multimeric proteins and denatures all thepolypeptide chains (step 1 ).
During electrophoresis,the SDS-protein complexes migrate through thepolyacrylamide gel (step 2 ). Small proteins are ableto move through the pores more easily, and faster,than larger proteins. Thus the proteins separate intobands according to their sizes as they migrate throughthe gel. The separated protein bands are visualized bystaining with a dye (step 3 ).electric focusing (IEF), can resolve proteins that differ byonly one charge unit. Proteins that have been separated onan IEF gel can then be separated in a second dimension onthe basis of their molecular weights.
To accomplish thisseparation, the IEF gel strip is placed lengthwise on a polyacrylamide slab gel, this time saturated with SDS. When anelectric field is imposed, the proteins will migrate from theIEF gel into the SDS slab gel and then separate accordingto their masses.The sequential resolution of proteins by charge and masscan achieve excellent separation of cellular proteins (Figure3-33b). For example, two-dimensional gels have been veryuseful in comparing the proteomes in undifferentiated anddifferentiated cells or in normal and cancer cells because asmany as 1000 proteins can be resolved simultaneously.MEDIA CONNECTIONSTechnique Animation: SDS Gel ElectrophoresisSDS-coatedproteins28990CHAPTER 3 • Protein Structure and Function1Isoelectricfocusing (IEF)pH 10.0Apply first gelto top of secondpH 4.02pH 10.0)Separatein firstdimensionby chargeIsoelectric focusing ( 1 )(b)pH 4.0366SDS electrophoresis (ProteinmixtureMolecular weight 103(a)433016Separatein seconddimensionby size3SDSelectrophoresis▲ EXPERIMENTAL FIGURE 3-33 Two-dimensional gelelectrophoresis can separate proteins of similar mass.
(a) Inthis technique, proteins are first separated on the basis of theircharges by isoelectric focusing (step 1 ). The resulting gel strip isapplied to an SDS-polyacrylamide gel and the proteins areseparated into bands by mass (step 3 ). (b) In this two-Liquid Chromatography Resolves Proteinsby Mass, Charge, or Binding AffinityA third common technique for separating mixtures of proteins, as well as other molecules, is based on the principlethat molecules dissolved in a solution will interact (bind anddissociate) with a solid surface.
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