《生物化学》课程教学资源（文献资料）Harper’s Illustrated Biochemistry，26th edition，Robert K. Murray Daryl K. Granner Peter A. Mayes Victor W. Rodwell

Proteins:Determination of4Primary StructureVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDColumnChromatographyBIOMEDICALIMPORTANCEProteins perform multiple critically important roles. AnColumn chromatography of proteins employs as thestationary phase a column containing small sphericalinternal protein network, the cytoskeleton (Chapterbeads of modified cellulose, acrylamide, or silica whose49),maintains cellular shape and physical integritysurface typically has been coated with chemical func-Actin and myosin filaments formthe contractile ma-tional groups.These stationary phase matrices interactchinery of muscle (Chapter 49).Hemoglobin transwith proteins based on their charge, hydrophobicity,ports oxygen (Chapter 6), while circulating antibodiessearch outforeign invaders (Chapter 50).Enzymes cat-and ligand-binding properties. A protein mixture is ap-plied to the column and the liquid mobile phase is per-alyze reactions that generate energy,synthesize and de-colated through it. Small portions of the mobile phasegrade biomolecules, replicate and transcribe genes,process mRNAs, etc (Chapter 7). Receptors enable cellsor eluant are collected as they emerge (Figure 41).to sense and respond to hormones and other environ-mental cues (Chapters 42 and 43). An important goalPartitionChromatographyof molecularmedicineis theidentification of proteinswhose presence, absence, or deficiency is associatedColumn chromatographic separations depend on thewith specific physiologic states or diseases.The primaryrelative affinity of different proteins for a given station-sequence of a protein provides both a molecular fingerary phase and for the mobile phase.Association be-print for its identification and information that can between each protein and the matrix is weak and tran-used to identify and clone the gene or genes that en-sient. Proteins that interact more strongly with thecodeit.stationary phase are retained longer.The length of timethataproteinisassociatedwiththestationaryphase isafunction of the composition of both the stationary andmobile phases.Optimal separation of the protein of in-PROTEINS&PEPTIDESMUSTBEterest from other proteins thus can be achieved by care-PURIFIEDPRIORTOANALYSISful manipulation of the composition of thetwo phases.Highly purified protein is essential for determination ofits amino acid sequence.Cells contain thousands of difSize Exclusion Chromatographyferent proteins, each in widely varying amounts. TheSize exclusion-or gel filtration-isolation ofa specific protein in quantities sufficient for-chromatography sep-analysis thus presents a formidable challenge that mayarates proteins based on their Stokes radius, the diamrequire multiple successive purification techniqueseter of the sphere they occupy as they tumble in solu-Classic approaches exploitdifferences in relative solu-tion.TheStokesradiusisafunctionof molecularmassbility of individual proteins as a function of pH (isoandshape.Atumblingelongatedproteinoccupiesaelectric precipitation),polarity(precipitation withlarger volumethana spherical proteinof the samemass.ethanol oracetone),or salt concentration (salting outSize exclusion chromatography employs porous beadswith ammonium sulfate).Chromatographic separations(Figure 4-2). The pores are analogous to indentationspartitionmolecules betweentwo phases,onemobilein a river bank. As objects move downstream, those thatand the other stationary.For separation of amino acidsenter an indentation are retarded until they drift backinto the main current.Similarly,proteins with Stokesor sugars, the stationary phase, or matrix, may be asheet of filter paper (paper chromatography) or a thinradii too large to enter the pores (excluded proteins) re-layer of cellulose, silica, or alumina (thin-layer chro-main in the flowing mobile phase and emerge beforematography;TLC).proteins that can enter the pores (included proteins).21

Proteins: Determination of Primary Structure 4 21 Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD BIOMEDICAL IMPORTANCE Proteins perform multiple critically important roles. An internal protein network, the cytoskeleton (Chapter 49), maintains cellular shape and physical integrity. Actin and myosin filaments form the contractile ma￾chinery of muscle (Chapter 49). Hemoglobin trans￾ports oxygen (Chapter 6), while circulating antibodies search out foreign invaders (Chapter 50). Enzymes cat￾alyze reactions that generate energy, synthesize and de￾grade biomolecules, replicate and transcribe genes, process mRNAs, etc (Chapter 7). Receptors enable cells to sense and respond to hormones and other environ￾mental cues (Chapters 42 and 43). An important goal of molecular medicine is the identification of proteins whose presence, absence, or deficiency is associated with specific physiologic states or diseases. The primary sequence of a protein provides both a molecular finger￾print for its identification and information that can be used to identify and clone the gene or genes that en￾code it. PROTEINS & PEPTIDES MUST BE PURIFIED PRIOR TO ANALYSIS Highly purified protein is essential for determination of its amino acid sequence. Cells contain thousands of dif￾ferent proteins, each in widely varying amounts. The isolation of a specific protein in quantities sufficient for analysis thus presents a formidable challenge that may require multiple successive purification techniques. Classic approaches exploit differences in relative solu￾bility of individual proteins as a function of pH (iso￾electric precipitation), polarity (precipitation with ethanol or acetone), or salt concentration (salting out with ammonium sulfate). Chromatographic separations partition molecules between two phases, one mobile and the other stationary. For separation of amino acids or sugars, the stationary phase, or matrix, may be a sheet of filter paper (paper chromatography) or a thin layer of cellulose, silica, or alumina (thin-layer chro￾matography; TLC). Column Chromatography Column chromatography of proteins employs as the stationary phase a column containing small spherical beads of modified cellulose, acrylamide, or silica whose surface typically has been coated with chemical func￾tional groups. These stationary phase matrices interact with proteins based on their charge, hydrophobicity, and ligand-binding properties. A protein mixture is ap￾plied to the column and the liquid mobile phase is per￾colated through it. Small portions of the mobile phase or eluant are collected as they emerge (Figure 4–1). Partition Chromatography Column chromatographic separations depend on the relative affinity of different proteins for a given station￾ary phase and for the mobile phase. Association be￾tween each protein and the matrix is weak and tran￾sient. Proteins that interact more strongly with the stationary phase are retained longer. The length of time that a protein is associated with the stationary phase is a function of the composition of both the stationary and mobile phases. Optimal separation of the protein of in￾terest from other proteins thus can be achieved by care￾ful manipulation of the composition of the two phases. Size Exclusion Chromatography Size exclusion—or gel filtration—chromatography sep￾arates proteins based on their Stokes radius, the diam￾eter of the sphere they occupy as they tumble in solu￾tion. The Stokes radius is a function of molecular mass and shape. A tumbling elongated protein occupies a larger volume than a spherical protein of the same mass. Size exclusion chromatography employs porous beads (Figure 4–2). The pores are analogous to indentations in a river bank. As objects move downstream, those that enter an indentation are retarded until they drift back into the main current. Similarly, proteins with Stokes radii too large to enter the pores (excluded proteins) re￾main in the flowing mobile phase and emerge before proteins that can enter the pores (included proteins). ch04.qxd 2/13/2003 2:02 PM Page 21

22/CHAPTER4cFigure4-1.Componentsofasimpleliquidchromatographyapparatus.R:Reser-voirofmobilephaseliquid,deliveredeitherbygravityorusingapump.C:Glassoplasticcolumncontainingstationaryphase.F:Fractioncollectorforcollectingportions, called fractions, of theeluant liquid in separatetest tubes.Proteins thus emerge from a gel filtration column in de-lonExchangeChromatographyscending order of their Stokes radii.In ion exchange chromatography,proteins interact withthe stationary phase by charge-charge interactions. Pro-AbsorptionChromatographyteins with a net positive charge at a given pH adhere toFor absorption chromatography,the protein mixture isbeads with negatively charged functional groups such ascarboxylates or sulfates (cation exchangers). Similarly,applied to a column under conditions where the pro-proteins with a net negative charge adhere to beads withtein of interest associates with the stationary phase sopositively charged functional groups, typically tertiary ortightly that its partition coefficient is essentially unityquaternary amines (anion exchangers).Proteins, whichNonadheringmoleculesarefirstelutedanddiscardedProteins are then sequentially released bydisrupting thearepolyanions,compete againstmonovalentionsforbinding to the support-thus the term"ion exchange."forces that stabilize the protein-stationary phase comFor example,proteins bind to diethylaminoethylplex, most often by using a gradient of increasing salt(DEAE)cellulose by replacing the counter-ions (generconcentration.The composition of the mobile phase isallyCIorCH,COO)thatneutralizetheprotonatedaltered gradually so that molecules are selectively re-amine. Bound proteins are selectively displaced by grad-leased in descending order of their affinity for the sta-ually raising the concentration of monovalent ions intionary phase

22 / CHAPTER 4 R C F Figure 4–1. Components of a simple liquid chromatography apparatus. R: Reser￾voir of mobile phase liquid, delivered either by gravity or using a pump. C: Glass or plastic column containing stationary phase. F: Fraction collector for collecting por￾tions, called fractions, of the eluant liquid in separate test tubes. Proteins thus emerge from a gel filtration column in de￾scending order of their Stokes radii. Absorption Chromatography For absorption chromatography, the protein mixture is applied to a column under conditions where the pro￾tein of interest associates with the stationary phase so tightly that its partition coefficient is essentially unity. Nonadhering molecules are first eluted and discarded. Proteins are then sequentially released by disrupting the forces that stabilize the protein-stationary phase com￾plex, most often by using a gradient of increasing salt concentration. The composition of the mobile phase is altered gradually so that molecules are selectively re￾leased in descending order of their affinity for the sta￾tionary phase. Ion Exchange Chromatography In ion exchange chromatography, proteins interact with the stationary phase by charge-charge interactions. Pro￾teins with a net positive charge at a given pH adhere to beads with negatively charged functional groups such as carboxylates or sulfates (cation exchangers). Similarly, proteins with a net negative charge adhere to beads with positively charged functional groups, typically tertiary or quaternary amines (anion exchangers). Proteins, which are polyanions, compete against monovalent ions for binding to the support—thus the term “ion exchange.” For example, proteins bind to diethylaminoethyl (DEAE) cellulose by replacing the counter-ions (gener￾ally Cl− or CH3COO− ) that neutralize the protonated amine. Bound proteins are selectively displaced by grad￾ually raising the concentration of monovalent ions in ch04.qxd 2/13/2003 2:02 PM Page 22

PROTEINS:DETERMINATIONOFPRIMARYSTRUCTURE123ABcFiqure4-2.Size-exclusion chromatography.A:Amixtureof largemolecules(diamonds)andsmall molecules (circles)areappliedtothetopofagelfiltrationcolumn.B:Upon entering the column,the small molecules enterpores in the sta-tionary phase matrix from which the large molecules are excluded. C: As the mo-bile phase flows down the column, the large, excluded molecules flow with itwhilethe small molecules,which are temporarily shelteredfrom the flow when in-sidethepores,lagfartherandfartherbehind.themobilephase.Proteins elutein inverseorderof thefied by affinity chromatography using immobilized substrength of their interactions with the stationary phase.strates,products, coenzymes, or inhibitors.In theorySince the net charge on a protein is determined byonly proteins that interact with the immobilized ligandthepH (see Chapter3),sequential elution of proteinsadhere. Bound proteins are then eluted either by compe-tition with soluble ligand or, less selectively, by disrupt-may be achieved by changing the pH of the mobilephase. Alternatively, a protein can be subjected to con-ing protein-ligand interactions using urea, guanidinesecutive rounds of ion exchange chromatography, eachhydrochloride, mildly acidic pH, or high salt concentra-at a different pH, such that proteins that co-elute at onetions.Stationary phasematrices available commerciallycontain ligands such as NAD+ or ATP analogs. AmongpH elute at different salt concentrations at another pH.the most powerful and widely applicable affinity matri-ces are those used for the purification of suitably modi-HydrophobicInteractionChromatographyfied recombinant proteins.These include a Ni2+matrixHydrophobicinteractionchromatography separatesthat binds proteins with an attached polyhistidine“tagproteins based on their tendency to associate with a sta-and a glutathionematrix that binds a recombinant pro-tionaryphasematrix coated with hydrophobicgroupstein linked to glutathione S-transferase.(eg, phenyl Sepharose, octyl Sepharose).Proteins withexposed hydrophobic surfaces adhere tothe matrix viahydrophobic interactions that are enhanced by a mobilePeptidesArePurifiedbyReversed-Phasephase of high ionic strength,Nonadherent proteins areHigh-PressureChromatographyfirstwashedaway.ThepolarityofthemobilephaseisThe stationary phase matrices used in classic columnthen decreased by gradually lowering the salt concentra-chromatography are spongy materials whose compress-tion. If the interaction between protein and stationaryibility limits flow of the mobile phase.High-pressure liqphase is particularly strong, ethanol or glycerol may beuidchromatography(HPLC)employsincompressibleadded to the mobile phase to decrease its polarity andsilica or alumina microbeads as the stationary phase andfurther weaken hydrophobic interactions.pressures of up to a few thousand psi.Incompressiblematricespermitbothhighflowratesandenhancedreso-AffinityChromatographylution.HPLC can resolve complex mixtures of lipids orAffinity chromatography exploits the high selectivity ofpeptides whose properties differ only slightly. Reversed-phase HPLC exploits a hydrophobic stationary phase ofmost proteins for their ligands. Enzymes may be puri-

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE / 23 A B C Figure 4–2. Size-exclusion chromatography. A: A mixture of large molecules (diamonds) and small molecules (circles) are applied to the top of a gel filtration column. B: Upon entering the column, the small molecules enter pores in the sta￾tionary phase matrix from which the large molecules are excluded. C: As the mo￾bile phase flows down the column, the large, excluded molecules flow with it while the small molecules, which are temporarily sheltered from the flow when in￾side the pores, lag farther and farther behind. the mobile phase. Proteins elute in inverse order of the strength of their interactions with the stationary phase. Since the net charge on a protein is determined by the pH (see Chapter 3), sequential elution of proteins may be achieved by changing the pH of the mobile phase. Alternatively, a protein can be subjected to con￾secutive rounds of ion exchange chromatography, each at a different pH, such that proteins that co-elute at one pH elute at different salt concentrations at another pH. Hydrophobic Interaction Chromatography Hydrophobic interaction chromatography separates proteins based on their tendency to associate with a sta￾tionary phase matrix coated with hydrophobic groups (eg, phenyl Sepharose, octyl Sepharose). Proteins with exposed hydrophobic surfaces adhere to the matrix via hydrophobic interactions that are enhanced by a mobile phase of high ionic strength. Nonadherent proteins are first washed away. The polarity of the mobile phase is then decreased by gradually lowering the salt concentra￾tion. If the interaction between protein and stationary phase is particularly strong, ethanol or glycerol may be added to the mobile phase to decrease its polarity and further weaken hydrophobic interactions. Affinity Chromatography Affinity chromatography exploits the high selectivity of most proteins for their ligands. Enzymes may be puri￾fied by affinity chromatography using immobilized sub￾strates, products, coenzymes, or inhibitors. In theory, only proteins that interact with the immobilized ligand adhere. Bound proteins are then eluted either by compe￾tition with soluble ligand or, less selectively, by disrupt￾ing protein-ligand interactions using urea, guanidine hydrochloride, mildly acidic pH, or high salt concentra￾tions. Stationary phase matrices available commercially contain ligands such as NAD+ or ATP analogs. Among the most powerful and widely applicable affinity matri￾ces are those used for the purification of suitably modi￾fied recombinant proteins. These include a Ni2+ matrix that binds proteins with an attached polyhistidine “tag” and a glutathione matrix that binds a recombinant pro￾tein linked to glutathione S-transferase. Peptides Are Purified by Reversed-Phase High-Pressure Chromatography The stationary phase matrices used in classic column chromatography are spongy materials whose compress￾ibility limits flow of the mobile phase. High-pressure liq￾uid chromatography (HPLC) employs incompressible silica or alumina microbeads as the stationary phase and pressures of up to a few thousand psi. Incompressible matrices permit both high flow rates and enhanced reso￾lution. HPLC can resolve complex mixtures of lipids or peptides whose properties differ only slightly. Reversed￾phase HPLC exploits a hydrophobic stationary phase of ch04.qxd 2/13/2003 2:02 PM Page 23

24/CHAPTER4aliphaticpolymers 3-18 carbon atoms in length.Peptidethrough the acrylamide matrix determines the rate ofmixtures are eluted using a gradient of a water-misciblemigration. Since large complexes encounter greater re-organic solvent such as acetonitrile or methanol.sistance, polypeptides separate based on their relativemolecular mass (M,).Individual polypeptides trappedin the acrylamide gel are visualized by staining withProteinPurityisAssessedbidyes such as Coomassie blue (Figure 4-4).PolyacrylamideGel Electrophoresis(PAGE)IsoelectricFocusing (IEF)The most widely used method for determining the pu-Ionic buffers called ampholytes and an applied electricrity of a protein is SDS-PAGEpolyacrylamide gelfield are used to generate a pH gradient within a poly-electrophoresis (PAGE)in the presence of the anionicacrylamide matrix.Applied proteins migrate until theydetergent sodium dodecyl sulfate (SDS).Electrophore-reach the region of the matrix where the pH matchessis separates charged biomolecules based on the rates attheir isoelectric point (pl), the pH at which a peptide'swhich they migrate in an applied electrical field. Fornetchargeis zero.IEFisused inconjunction withSDS-SDS-PAGE,acrylamide is polymerized and cross-PAGE for two-dimensional electrophoresis, which sepa-linked to forma porous matrix.SDS denatures andratespolypeptidesbasedonplinonedimensionandbinds to proteins ata ratio of one molecule of SDS perbased on M, in the second (Figure 4-5). Two-dimen-rwo peptide bonds.When used in conjunction with 2-sional electrophoresis is particularly well suited for sepa-mercaptoethanol or dithiothreitol to reduce and breakrating the components of complex mixtures of proteins.disulfide bonds (Figure 4-3), SDS separates the com-ponent polypeptides of multimeric proteins.The largenumber of anionic SDS molecules, each bearing aSANGERWASTHEFIRSTTODETERMINEcharge of -1, on each polypeptide overwhelms theTHESEQUENCEOFAPOLYPEPTIDEcharge contributions of the amino acid functionalgroups.Since the charge-to-mass ratio of each SDS-Mature insulin consists of the2l-residue Achain andpolypeptide complex is approximately equal, the physi-the30-residueBchain linked by disulfidebonds.Fred-erick Sanger reduced the disulfide bonds (Figure 4-3),cal resistance each peptide encounters as it movessECHDNHHN11173QO=SH48HCOOHCHOHNH34HNSO,-HN29HSFigure4-4.Useof SDS-PAGEtoobserve successivepurificationofarecombinantprotein.ThegelwasFigure4-3.Oxidativecleavageofadjacentpolypep-stainedwithCoomassieblue.Shownareproteinstan-tide chains linked by disulfide bonds (shaded) by per-dards (lane S) of the indicated mass, crude cell extract(E), high-speed supernatant liquid (H), and the DEAE-formicacid (left)orreductivecleavagebyβ-mercap-toethanol (right) forms two peptides that containSepharose fraction (D).The recombinant protein hasacysteic acid residues or cysteinyl residues,respectively.massofabout45kDa

24 / CHAPTER 4 aliphatic polymers 3–18 carbon atoms in length. Peptide mixtures are eluted using a gradient of a water-miscible organic solvent such as acetonitrile or methanol. Protein Purity Is Assessed by Polyacrylamide Gel Electrophoresis (PAGE) The most widely used method for determining the pu￾rity of a protein is SDS-PAGE—polyacrylamide gel electrophoresis (PAGE) in the presence of the anionic detergent sodium dodecyl sulfate (SDS). Electrophore￾sis separates charged biomolecules based on the rates at which they migrate in an applied electrical field. For SDS-PAGE, acrylamide is polymerized and cross￾linked to form a porous matrix. SDS denatures and binds to proteins at a ratio of one molecule of SDS per two peptide bonds. When used in conjunction with 2- mercaptoethanol or dithiothreitol to reduce and break disulfide bonds (Figure 4 –3), SDS separates the com￾ponent polypeptides of multimeric proteins. The large number of anionic SDS molecules, each bearing a charge of −1, on each polypeptide overwhelms the charge contributions of the amino acid functional groups. Since the charge-to-mass ratio of each SDS￾polypeptide complex is approximately equal, the physi￾cal resistance each peptide encounters as it moves through the acrylamide matrix determines the rate of migration. Since large complexes encounter greater re￾sistance, polypeptides separate based on their relative molecular mass (Mr). Individual polypeptides trapped in the acrylamide gel are visualized by staining with dyes such as Coomassie blue (Figure 4–4). Isoelectric Focusing (IEF) Ionic buffers called ampholytes and an applied electric field are used to generate a pH gradient within a poly￾acrylamide matrix. Applied proteins migrate until they reach the region of the matrix where the pH matches their isoelectric point (pI), the pH at which a peptide’s net charge is zero. IEF is used in conjunction with SDS￾PAGE for two-dimensional electrophoresis, which sepa￾rates polypeptides based on pI in one dimension and based on Mr in the second (Figure 4–5). Two-dimen￾sional electrophoresis is particularly well suited for sepa￾rating the components of complex mixtures of proteins. SANGER WAS THE FIRST TO DETERMINE THE SEQUENCE OF A POLYPEPTIDE Mature insulin consists of the 21-residue A chain and the 30-residue B chain linked by disulfide bonds. Fred￾erick Sanger reduced the disulfide bonds (Figure 4–3), NH HN NH HN HN NH HN NH H SO − HS O O O O O O O O 2 O HCOOH SH C2H5 OH S S H H H Figure 4–3. Oxidative cleavage of adjacent polypep￾tide chains linked by disulfide bonds (shaded) by per￾formic acid (left) or reductive cleavage by β-mercap￾toethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively. Figure 4–4. Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie blue. Shown are protein stan￾dards (lane S) of the indicated mass, crude cell extract (E), high-speed supernatant liquid (H), and the DEAE￾Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa. ch04.qxd 2/13/2003 2:02 PM Page 24

25PROTEINS:DETERMINATIONOEPRIMARYSTRUCTURE1pH= 3pH=10IEFSDSPAGEFigure4-5.Two-dimensional IEF-SDS-PAGE.Thegel was stained with Coomassie blue. A crude bacter-ial extract was first subjected to isoelectricfocusing(IEF) in a pH 3-10 gradient. The IEF gel was thenplacedhorizontallyonthetopofanSDSgel,andtheproteins then further resolvedbySDS-PAGE.Noticethe greatly improved resolution of distinct polypep-tides relativetoordinarySDS-PAGE gel (Figure 4-4).separated the A and B chains,and cleaved each chainLargePolypeptidesAreFirstCleaved Intointo smaller peptides using trypsin, chymotrypsin, andSmallerSegmentspepsin. The resulting peptides were then isolated andWhile the first 20-30 residues of a peptide can readilytreated with acid to hydrolyze peptide bonds and gener.be determined by the Edman method,most polypepate peptides withas few as two or three amino acids.tides contain several hundred amino acids. Conse-Each peptide was reacted with 1-fluoro-2,4-dinitroben-quently,mostpolypeptides mustfirstbecleaved intozene (Sanger's reagent), which derivatizes the exposedsmallerpeptidespriortoEdman sequencing.Cleavageα-amino group of amino terminal residues.The aminoalsomaybenecessarytocircumventposttranslationalacid content of each peptidewas then determined.modifications that render a protein's α-amino groupWhile the -amino group of lysine also reacts with"blocked",or unreactive with the Edman reagent.Sanger's reagent, amino-terminal lysines can be distin-It usually is necessary to generate several peptidesguished from those at other positions because they reactusing morethan one method of cleavage.This reflectswith 2 mol of Sanger's reagent.Working backwards toboth inconsistency in the spacing of chemically or enzy-larger fragments enabled Sanger to determine thecommatically susceptible cleavage sites and the need for setsplete sequence of insulin,an accomplishment for whichof peptides whose sequences overlap so one can inferhereceivedaNobel Prizein1958.the sequence of the polypeptide from which they derive(Figure 4-7).Reagents for the chemical or enzymaticTHEEDMANREACTIONENABLEScleavage of proteins include cyanogen bromide (CNBr),PEPTIDES&PROTEINStrypsin,and Stapbylococcus aureus V8protease(TableTOBESEQUENCED4-i). Following cleavage, the resulting peptides are pu-rified by reversed-phase HPLCor occasionally byPehr Edman introduced phenylisothiocyanate (Edman'sSDS-PAGE-and sequenced.reagent) to selectively label the amino-terminal residueof a peptide.In contrast to Sanger'sreagent,theMOLECULARBIOLOGYHASphenylthiohydantoin (PTH) derivative can be removedundermild conditions togeneratea newaminoterminalREVOLUTIONIZEDTHEDETERMINATIONresidue (Figure 4-6). Successive rounds of derivatizationOFPRIMARYSTRUCTUREwith Edman's reagent can therefore be used to sequenceKnowledge of DNA sequences permits deduction ofmany residues of a single sample of peptide. Edman sequencinghas been automated, using a thinfilm or solidthe primary structures of polypeptides. DNA sequencmatrix to immobilize the peptide and HPLC to identifying requires onlyminuteamounts of DNA and canPTH amino acids.Modern gas-phase sequencers canreadily yield the sequence of hundreds of nucleotides.To clone and sequence the DNA that encodes a partic-analyze as little as a few picomoles of peptide

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE / 25 IEF SDS PAGE pH = 3 pH = 10 Figure 4–5. Two-dimensional IEF-SDS-PAGE. The gel was stained with Coomassie blue. A crude bacter￾ial extract was first subjected to isoelectric focusing (IEF) in a pH 3–10 gradient. The IEF gel was then placed horizontally on the top of an SDS gel, and the proteins then further resolved by SDS-PAGE. Notice the greatly improved resolution of distinct polypep￾tides relative to ordinary SDS￾PAGE gel (Figure 4–4). separated the A and B chains, and cleaved each chain into smaller peptides using trypsin, chymotrypsin, and pepsin. The resulting peptides were then isolated and treated with acid to hydrolyze peptide bonds and gener￾ate peptides with as few as two or three amino acids. Each peptide was reacted with 1-fluoro-2,4-dinitroben￾zene (Sanger’s reagent), which derivatizes the exposed α-amino group of amino terminal residues. The amino acid content of each peptide was then determined. While the ε-amino group of lysine also reacts with Sanger’s reagent, amino-terminal lysines can be distin￾guished from those at other positions because they react with 2 mol of Sanger’s reagent. Working backwards to larger fragments enabled Sanger to determine the com￾plete sequence of insulin, an accomplishment for which he received a Nobel Prize in 1958. THE EDMAN REACTION ENABLES PEPTIDES & PROTEINS TO BE SEQUENCED Pehr Edman introduced phenylisothiocyanate (Edman’s reagent) to selectively label the amino-terminal residue of a peptide. In contrast to Sanger’s reagent, the phenylthiohydantoin (PTH) derivative can be removed under mild conditions to generate a new amino terminal residue (Figure 4–6). Successive rounds of derivatization with Edman’s reagent can therefore be used to sequence many residues of a single sample of peptide. Edman se￾quencing has been automated, using a thin film or solid matrix to immobilize the peptide and HPLC to identify PTH amino acids. Modern gas-phase sequencers can analyze as little as a few picomoles of peptide. Large Polypeptides Are First Cleaved Into Smaller Segments While the first 20–30 residues of a peptide can readily be determined by the Edman method, most polypep￾tides contain several hundred amino acids. Conse￾quently, most polypeptides must first be cleaved into smaller peptides prior to Edman sequencing. Cleavage also may be necessary to circumvent posttranslational modifications that render a protein’s α-amino group “blocked”, or unreactive with the Edman reagent. It usually is necessary to generate several peptides using more than one method of cleavage. This reflects both inconsistency in the spacing of chemically or enzy￾matically susceptible cleavage sites and the need for sets of peptides whose sequences overlap so one can infer the sequence of the polypeptide from which they derive (Figure 4–7). Reagents for the chemical or enzymatic cleavage of proteins include cyanogen bromide (CNBr), trypsin, and Staphylococcus aureus V8 protease (Table 4–1). Following cleavage, the resulting peptides are pu￾rified by reversed-phase HPLC—or occasionally by SDS-PAGE—and sequenced. MOLECULAR BIOLOGY HAS REVOLUTIONIZED THE DETERMINATION OF PRIMARY STRUCTURE Knowledge of DNA sequences permits deduction of the primary structures of polypeptides. DNA sequenc￾ing requires only minute amounts of DNA and can readily yield the sequence of hundreds of nucleotides. To clone and sequence the DNA that encodes a partic￾ch04.qxd 2/13/2003 2:02 PM Page 25