《生物化学原理》英文版 chapter 3 AMINO ACIDS, PEPTIDES AND PROTEINS

Chapter 3 Amino Acids, Peptides, and Proteins binding affinity, and other properties(Fig. 3-17. a length And as the length of time spent on the column porous solid material with appropriate chemical prop- increases, the resolution can decline as a result of dif- erties (the stationary phase) is held in a column, and a fusional spreading within each protein band buffered solution (the mobile phase) percolates through Figure 3-18 shows two other variations of column it. The protein-containing solution, layered on the top chromatography in addition to ion exchange. Size of the column, percolates through the solid matrix as an exclusion chromatography separates proteins ac- ever-expanding band within the larger mobile phase cording to size. In this method, large proteins emerge (Fig. 3-17. Individual proteins migrate faster or more from the column sooner than small ones-a somewhat slowly through the column depending on their proper- counterintuitive result. The solid phase consists of ties. For example, in cation-exchange chromatogra- beads with engineered pores or cavities of a particula phy (Fig. 3-18a), the solid matrix has negatively size Large proteins cannot enter the cavities, and so harged groups. In the mobile phase, proteins with a net take a short (and rapid) path through the column positive charge migrate through the matrix more slowly around the beads. Small proteins enter the cavities, and than those with a net negative charge because the mi- migrate through the column more slowly as a result ( fig. gration of the former is retarded more by interaction 3-18b). Affinity chromatography is based on the with the stationary phase. The two types of protein can binding affinity of a protein. The beads in the column eparate into two distinct bands. The expansion of the have a covalently attached chemical group. a protein protein band in the mobile phase(the protein solution) with affinity for this particular chemical group will bind is caused both by separation of proteins with different to the beads in the column, and its migration will be re- properties and by diffusional spreading. As the length tarded as a result(Fig. 3-18c) of the column increases, the resolution of two types of A modern refinement in chromatographic method protein with different net charges generally improves HPLC, or high-performance liquid chromatogra- However, the rate at which the protein solution can flow phy. HPLC makes use of high-pressure pumps that through the column usually decreases with colu speed the movement of the protein molecules down the column, as well as higher-quality chromatographic ma- terials that can withstand the crushing force of the pres- surized flow. By reducing the transit time on the col umn, HPLC can limit diffusional spreading of protein bands and thus greatly improve resolution. The approach to purification of a protein that has not previously been isolated is guided both by estab- lished precedents and by common sense. In most cases several different methods must be used sequentially to purify a protein completely. The choice of method is Reservoir FIGURE 3-17 Column chromatography. The standard elements of a chromatographic column include a solid, porous material supported inside a column, generally made of plastic or glass. The solid material (matrix)makes up the stationary phase through which flows a solu- tion, the mobile phase. The solution that passes out of the column at the bottom(the effluent) is constantly replaced by solution supplied from a reservoir at the top. The protein solution to be separated is lay ered on top of the column and allowed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a band within the mobile phase that is initially the depth of the pro- tein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different inter- Porous actions with the matrix material. The overall protein band thus widens Proteins as it moves through the column. Individual types of proteins(such as A, B, and C, shown in blue, red, and green) gradually separate from each other, forming bands within the broader protein band. Separa tion improves (resolution increases) as the length of the column in creases.However, each individual protein band also broadens with time due to diffusional spreading, a process that decreases resolution. In this example, protein A is well separated from B and C, but diffu sional spreading prevents complete separation of B and C under thes conditions

length. And as the length of time spent on the column increases, the resolution can decline as a result of dif￾fusional spreading within each protein band. Figure 3–18 shows two other variations of column chromatography in addition to ion exchange. Size￾exclusion chromatography separates proteins ac￾cording to size. In this method, large proteins emerge from the column sooner than small ones—a somewhat counterintuitive result. The solid phase consists of beads with engineered pores or cavities of a particular size. Large proteins cannot enter the cavities, and so take a short (and rapid) path through the column, around the beads. Small proteins enter the cavities, and migrate through the column more slowly as a result (Fig. 3–18b). Affinity chromatography is based on the binding affinity of a protein. The beads in the column have a covalently attached chemical group. A protein with affinity for this particular chemical group will bind to the beads in the column, and its migration will be re￾tarded as a result (Fig. 3–18c). A modern refinement in chromatographic methods is HPLC, or high-performance liquid chromatogra￾phy. HPLC makes use of high-pressure pumps that speed the movement of the protein molecules down the column, as well as higher-quality chromatographic ma￾terials that can withstand the crushing force of the pres￾surized flow. By reducing the transit time on the col￾umn, HPLC can limit diffusional spreading of protein bands and thus greatly improve resolution. The approach to purification of a protein that has not previously been isolated is guided both by estab￾lished precedents and by common sense. In most cases, several different methods must be used sequentially to purify a protein completely. The choice of method is 90 Chapter 3 Amino Acids, Peptides, and Proteins Solid porous matrix (stationary phase) Porous support Effluent Reservoir Protein sample (mobile phase) Proteins A B C FIGURE 3–17 Column chromatography. The standard elements of a chromatographic column include a solid, porous material supported inside a column, generally made of plastic or glass. The solid material (matrix) makes up the stationary phase through which flows a solu￾tion, the mobile phase. The solution that passes out of the column at the bottom (the effluent) is constantly replaced by solution supplied from a reservoir at the top. The protein solution to be separated is lay￾ered on top of the column and allowed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a band within the mobile phase that is initially the depth of the pro￾tein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different inter￾actions with the matrix material. The overall protein band thus widens as it moves through the column. Individual types of proteins (such as A, B, and C, shown in blue, red, and green) gradually separate from each other, forming bands within the broader protein band. Separa￾tion improves (resolution increases) as the length of the column in￾creases. However, each individual protein band also broadens with time due to diffusional spreading, a process that decreases resolution. In this example, protein A is well separated from B and C, but diffu￾sional spreading prevents complete separation of B and C under these conditions. binding affinity, and other properties (Fig. 3–17). A porous solid material with appropriate chemical prop￾erties (the stationary phase) is held in a column, and a buffered solution (the mobile phase) percolates through it. The protein-containing solution, layered on the top of the column, percolates through the solid matrix as an ever-expanding band within the larger mobile phase (Fig. 3–17). Individual proteins migrate faster or more slowly through the column depending on their proper￾ties. For example, in cation-exchange chromatogra￾phy (Fig. 3–18a), the solid matrix has negatively charged groups. In the mobile phase, proteins with a net positive charge migrate through the matrix more slowly than those with a net negative charge, because the mi￾gration of the former is retarded more by interaction with the stationary phase. The two types of protein can separate into two distinct bands. The expansion of the protein band in the mobile phase (the protein solution) is caused both by separation of proteins with different properties and by diffusional spreading. As the length of the column increases, the resolution of two types of protein with different net charges generally improves. However, the rate at which the protein solution can flow through the column usually decreases with column 8885d_c03_090 12/23/03 10:23 AM Page 90 mac111 mac111:reb:

e Large net positive charge O Net positive charge O Net negative charge O Large net negative charge 8 Polymer beads with natively charge functional groups Protein mixture is added 0 cross-linked polymer.L Protein mixture is added to column containing cation exchangers. Protein molecules se 8 in the earlier fractions. 1234 Proteins move through the column at rates determined by their net charge at the ph being used. With cation exchangers proteins with a more ne of FIGURE 3-18 Three chromatographic methods used in protein purifi- cation(a) lon-exchange chromatography exploits differences in the ign and magnitude of the net electric charges of proteins at a given H. The column matrix is a synthetic polymer containing bound charged groups; those with bound anionic groups are called cation exchangers, and those with bound cationic groups are called anion changers. lon-exchange chromatography on a cation exchanger is shown here. The affinity of each protein for the charged groups on the Solution column is affected by the pH (which determines the ionization state ligand of the molecule) and the concentration of competing free salt ions in he surrounding solution. Separation can be optimized by gradually changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient. (b) Size-exclusion chromatography, also called gel filtration, separates proteins according to size. The column matrix is a cross-linked polymer with pores of selected size Larger proteins migrate faster than smaller ones, because they are too large to enter the pores in the beads and hence take a more direct ute through the column. The smaller proteins enter the pores and are slowed by their more labyrinthine path through the column 30 (c) Affinity chromatography separates proteins by their binding speci ficities. The proteins retained on the column are those that bind specifically to a ligand cross linked to the beads. (In biochemistry, the term"ligand"is used to refer to a group or molecule that binds to a macromolecule such as a protein. After proteins that do not bind to the ligand are washed through the column, the bound protein of protein of interest. particular interest is eluted (washed out of the column) by a solutio are washed through is eluted by ligand solution

Protein mixture is added to column containing cation exchangers. (a) 1 2 34 5 6 Large net positive charge Net positive charge Net negative charge Large net negative charge Proteins move through the column at rates determined by their net charge at the pH being used. With cation exchangers, proteins with a more negative net charge move faster and elute earlier. Polymer beads with negatively charged functional groups FIGURE 3–18 Three chromatographic methods used in protein purifi￾cation. (a) Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH. The column matrix is a synthetic polymer containing bound charged groups; those with bound anionic groups are called cation exchangers, and those with bound cationic groups are called anion exchangers. Ion-exchange chromatography on a cation exchanger is shown here. The affinity of each protein for the charged groups on the column is affected by the pH (which determines the ionization state of the molecule) and the concentration of competing free salt ions in the surrounding solution. Separation can be optimized by gradually changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient. (b) Size-exclusion chromatography, also called gel filtration, separates proteins according to size. The column matrix is a cross-linked polymer with pores of selected size. Larger proteins migrate faster than smaller ones, because they are too large to enter the pores in the beads and hence take a more direct route through the column. The smaller proteins enter the pores and are slowed by their more labyrinthine path through the column. (c) Affinity chromatography separates proteins by their binding speci￾ficities. The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads. (In biochemistry, the term “ligand” is used to refer to a group or molecule that binds to a macromolecule such as a protein.) After proteins that do not bind to the ligand are washed through the column, the bound protein of particular interest is eluted (washed out of the column) by a solution containing free ligand. Protein molecules separate by size; larger molecules pass more freely, appearing in the earlier fractions. 1 2 34 5 6 Protein mixture is added to column containing cross-linked polymer. Porous polymer beads (b) Unwanted proteins are washed through column. Protein of interest is eluted by ligand solution. Protein of interest Ligand Protein mixture is added to column containing a polymer-bound ligand specific for protein of interest. Mixture of proteins 3 4 5 6 7 8 Solution of ligand 1 2 3 4 5 (c) 8885d_c03_091 12/23/03 10:23 AM Page 91 mac111 mac111:reb:

Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3-5 A Purification Table for a Hypothetical Enz Fraction volume Specific activity Procedure or step (mg) (units/ mg 1. Crude cellular extract 1.400 10,000 100.000 10 2. Precipitation with ammonium sulfate 3,000 000 32 3. lon-exchange chromatography 80.000 4. Size-exclusion chromatography 100 60.000 5. Affinity chromatography 45.000 15000 Note: All data represent the status of the sample after the designated procedure has been camied out Activity and specific activity are defined on page 94 somewhat empirical, and many protocols may be tried ticle molecule, V to the electrical potential. Electro- before the most effective one is found. Trial and error phoretic mobility is also equal to the net charge of can often be minimized by basing the procedure on pu- the molecule, Z, divided by the frictional coefficient, f rification techniques developed for similar proteins which reflects in part a proteins shape. Thus Published purification protocols are available for many V Z thousands of proteins. Common sense dictates that in- expensive procedures such as salting out be used first, The migration of a protein in a gel during electro- when the total volume and the number of contaminants phoresis is therefore a function of its size and its shape re greatest. Chromatographic methods are often im- practical at early stages, because the amount of chro- An electrophoretic method commonly employed for matographic medium needed increases with sample estimation of purity and molecular weight makes use of size. As each purification step is completed, the sample the detergent sodium dodecyl sulfate (SDs) size generally becomes smaller (Table 3-5), making it feasible to use more sophisticated (and expensive) O-(CH)CHs chromatographic procedures at later stages Proteins Can Be Separated and Characterized Sodium dodecyl sulfate by Electrophoresis SDS binds to most proteins in amounts roughly propor Another important technique for the separation of pro- tional to the molecular weight of the protein, about one teins is based on the migration of charged proteins in molecule of SDS for every two amino acid residues. The an electric field, a process called electrophoresis. bound SDS contributes a large net negative charge, ren- These procedures are not generally used to purify pro- dering the intrinsic charge of the protein insignificant teins in large amounts, because simpler alternatives are and conferring on each protein a similar charge-to-mass usually available and electrophoretic methods often ratio. In addition, the native conformation of a protein adversely affect the structure and thus the function of is altered when Sds is bound, and most proteins assume proteins. Electrophoresis is, however, especially useful a similar shape. Electrophoresis in the presence of SDS as an analytical method. Its advantage is that proteins therefore separates proteins almost exclusively on the an be visualized as well as separated, permitting a basis of mass(molecular weight), with smaller polypep- esearcher to estimate quickly the number of different tides migrating more rapidly. After electrophoresis, the proteins in a mixture or the degree of purity of a par- proteins are visualized by adding a dye such as ticular protein preparation. Also, electrophoresis allows Coomassie blue, which binds to proteins but not to the determination of crucial properties of a protein such as gel itself (Fig. 3-19b). Thus, a researcher can monitor its isoelectric point and approximate molecular weight the progress of a protein purification procedure as the Electrophoresis of proteins is generally carried out number of protein bands visible on the gel decreases af- in gels made up of the cross-linked polymer polyacryl- ter each new fractionation step. When compared with amide(Fig. 3-19). The polyacrylamide gel acts as a mo- the positions to which proteins of known moleculal lecular sieve, slowing the migration of proteins approx- weight migrate in the gel, the position of an unidenti imately in proportion to their charge-to-mass ratio. fied protein can provide an excellent measure of its mo- Migration may also be affected by protein shape. In elec- lecular weight (Fig. 3-20). If the protein has two ormore trophoresis, the force moving the macromolecule is the different subunits, the subunits will generally be sepa- electrical potential, E. The electrophoretic mobility of rated by the SDs treatment and a separate band will ap- the molecule, u, is the ratio of the velocity of the par- pear for each. 8 SDS Gel Electrophoresis

92 Chapter 3 Amino Acids, Peptides, and Proteins somewhat empirical, and many protocols may be tried before the most effective one is found. Trial and error can often be minimized by basing the procedure on pu￾rification techniques developed for similar proteins. Published purification protocols are available for many thousands of proteins. Common sense dictates that in￾expensive procedures such as salting out be used first, when the total volume and the number of contaminants are greatest. Chromatographic methods are often im￾practical at early stages, because the amount of chro￾matographic medium needed increases with sample size. As each purification step is completed, the sample size generally becomes smaller (Table 3–5), making it feasible to use more sophisticated (and expensive) chromatographic procedures at later stages. Proteins Can Be Separated and Characterized by Electrophoresis Another important technique for the separation of pro￾teins is based on the migration of charged proteins in an electric field, a process called electrophoresis. These procedures are not generally used to purify pro￾teins in large amounts, because simpler alternatives are usually available and electrophoretic methods often adversely affect the structure and thus the function of proteins. Electrophoresis is, however, especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of different proteins in a mixture or the degree of purity of a par￾ticular protein preparation. Also, electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer polyacryl￾amide (Fig. 3–19). The polyacrylamide gel acts as a mo￾lecular sieve, slowing the migration of proteins approx￾imately in proportion to their charge-to-mass ratio. Migration may also be affected by protein shape. In elec￾trophoresis, the force moving the macromolecule is the electrical potential, E. The electrophoretic mobility of the molecule,
, is the ratio of the velocity of the par￾ticle molecule, V, to the electrical potential. Electro￾phoretic mobility is also equal to the net charge of the molecule, Z, divided by the frictional coefficient, f, which reflects in part a protein’s shape. Thus:

E V
Z f The migration of a protein in a gel during electro￾phoresis is therefore a function of its size and its shape. An electrophoretic method commonly employed for estimation of purity and molecular weight makes use of the detergent sodium dodecyl sulfate (SDS). SDS binds to most proteins in amounts roughly propor￾tional to the molecular weight of the protein, about one molecule of SDS for every two amino acid residues. The bound SDS contributes a large net negative charge, ren￾dering the intrinsic charge of the protein insignificant and conferring on each protein a similar charge-to-mass ratio. In addition, the native conformation of a protein is altered when SDS is bound, and most proteins assume a similar shape. Electrophoresis in the presence of SDS therefore separates proteins almost exclusively on the basis of mass (molecular weight), with smaller polypep￾tides migrating more rapidly. After electrophoresis, the proteins are visualized by adding a dye such as Coomassie blue, which binds to proteins but not to the gel itself (Fig. 3–19b). Thus, a researcher can monitor the progress of a protein purification procedure as the number of protein bands visible on the gel decreases af￾ter each new fractionation step. When compared with the positions to which proteins of known molecular weight migrate in the gel, the position of an unidenti￾fied protein can provide an excellent measure of its mo￾lecular weight (Fig. 3–20). If the protein has two or more different subunits, the subunits will generally be sepa￾rated by the SDS treatment and a separate band will ap￾pear for each. SDS Gel Electrophoresis (CH2)11CH3 O Na S O O O Sodium dodecyl sulfate (SDS) TABLE 3–5 A Purification Table for a Hypothetical Enzyme Fraction volume Total protein Activity Specific activity Procedure or step (ml) (mg) (units) (units/mg) 1. Crude cellular extract 1,400 10,000 100,000 10 2. Precipitation with ammonium sulfate 280 3,000 96,000 32 3. Ion-exchange chromatography 90 400 80,000 200 4. Size-exclusion chromatography 80 100 60,000 600 5. Affinity chromatography 6 3 45,000 15,000 Note: All data represent the status of the sample after the designated procedure has been carried out. Activity and specific activity are defined on page 94. 8885d_c03_092 12/23/03 10:23 AM Page 92 mac111 mac111:reb:

3.3 Working with Proteins Sample Well 二二2 FIGURE 3-19 Electrophoresis. (a) Different samples are loaded in tein (or protein subunit): smaller proteins move through the gel more wells or depressions at the top of the polyacrylamide gel. The proteins rapidly than larger proteins and therefore are found nearer the bottom nove into the gel when an electric field is applied. The gel minimizes of the gel. This gel illustrates the purification of the enzyme RNa poly- convection currents caused by small temperature gradients, as well as merase from E. coli. The first lane shows the proteins present in the protein movements other than those induced by the electric field rude cellular extract. Successive lanes(left to right) show the proteins (b)Proteins can be visualized after electrophoresis by treating the gel present after each purification step. The purified protein contain with a stain such as Coomassie blue, which binds to the proteins but subunits, as seen in the last lane on the right. not to the gel itself. Each band on the gel represents a different pro- Isoelectric focusing is a procedure used to de- ture is applied, each protein migrates until it reaches termine the isoelectric point (pl of a protein (Fi the ph that matches its pI (Table 3-6). Proteins with 3-21). A ph gradient is established by allowing a mix- different isoelectric points are thus distributed differ- ture of low molecular weight organic acids and bases ently throughout the gel. (ampholytes; p 81) to distribute themselves in an elec Combining isoelectric focusing and SDs electropho- tric field generated across the gel. When a protein mix- resis sequentially in a process called two-dimensional ⊙「L FIGURE 3-20 Estimating the molecular weight of a protein. The electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight, Mr.(a)Standard proteins of Myosin 200,000 known molecular weight are subjected to electrophoresis (lane 1) These marker proteins can be used to estimate the molecular weight of an unknown protein(lane 2).(b)A plot of log M, of the B-Galactosidase 116, 250 Glycogen phosphorylase b 97, 400 marker proteins versus relative migration during electrophoresis is linear, which allows the molecular weight of the unknown protein to be read from the graph Ovalbumin 45.000 Carbonic anhydrase 31,000 Unknown Soybean trypsin inhibitor 21,500 Lysozyme 14, 400

Sample Well Direction of migration + – (a) (b) FIGURE 3–19 Electrophoresis. (a) Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection currents caused by small temperature gradients, as well as protein movements other than those induced by the electric field. (b) Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to the proteins but not to the gel itself. Each band on the gel represents a different pro￾tein (or protein subunit); smaller proteins move through the gel more rapidly than larger proteins and therefore are found nearer the bottom of the gel. This gel illustrates the purification of the enzyme RNA poly￾merase from E. coli. The first lane shows the proteins present in the crude cellular extract. Successive lanes (left to right) show the proteins present after each purification step. The purified protein contains four subunits, as seen in the last lane on the right. 200,000 116,250 97,400 66,200 45,000 31,000 21,500 14,400 Mr standards Unknown protein Myosin b-Galactosidase Glycogen phosphorylase b Bovine serum albumin Ovalbumin Carbonic anhydrase Soybean trypsin inhibitor Lysozyme – + 1 2 (a) log Mr Relative migration Unknown protein (b) FIGURE 3–20 Estimating the molecular weight of a protein. The electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight, Mr. (a) Standard proteins of known molecular weight are subjected to electrophoresis (lane 1). These marker proteins can be used to estimate the molecular weight of an unknown protein (lane 2). (b) A plot of log Mr of the marker proteins versus relative migration during electrophoresis is linear, which allows the molecular weight of the unknown protein to be read from the graph. Isoelectric focusing is a procedure used to de￾termine the isoelectric point (pI) of a protein (Fig. 3–21). A pH gradient is established by allowing a mix￾ture of low molecular weight organic acids and bases (ampholytes; p. 81) to distribute themselves in an elec￾tric field generated across the gel. When a protein mix￾ture is applied, each protein migrates until it reaches the pH that matches its pI (Table 3–6). Proteins with different isoelectric points are thus distributed differ￾ently throughout the gel. Combining isoelectric focusing and SDS electropho￾resis sequentially in a process called two-dimensional 3.3 Working with Proteins 93 8885d_c03_093 1/16/04 6:48 AM Page 93 mac76 mac76:385_reb:

Chapter 3 Amino Acids, Peptides, and An ampholyte incorporated FIGURE 3-21 Isoelectric focusing. This technique separates proteins according to their isoelectric points. A stable ph grader is established in the gel by the addition of appropriate ampholytes. A protein mixture is placed in a well on the gel. With an A stable ph gradient Protein solution is After staining, proteins applied electric field, proteins enter the gel is established in th gel after application field is reapplied. distributed along pH of an electric field gradient according to equivalent to its pl. Remember that when their pI values pl, the net charge of a protein is zero electrophoresis permits the resolution of complex 25 to 38C. Also, very high substrate concentrations are mixtures of proteins(Fig. 3-22). This is a more sensi- generally used so that the initial reaction rate, measured tive analytical method than either electrophoreti experimentally, is proportional to enzyme concentration method alone. Two-dimensional electrophoresis sepa-(Chapter 6) rates proteins of identical molecular weight that differ By international agreement, 1.0 unit of enzyme ac in pl, or proteins with similar pl values but different mo- tivity is defined as the amount of enzyme causing trans lecular weights formation of 1.0 umol of substrate per minute at 25C under optimal conditions of measurement. The term Unseparated Proteins Can Be Quantified activity refers to the total units of enzyme in a solu tion. The specific activity is the number of enzyme To purify a protein, it is essential to have a way of de- units per milligram of total protein(Fig. 3-23). The spe- tecting and quantifying that protein in the presence of cific activity is a measure of enzyme purity: it increases many other proteins at each stage of the procedure during purification of an enzyme and becomes maximal Often, purification must proceed in the absence of any and constant when the enzyme is pure (Table 3-5) information about the size and physical properties of the protein or about the fraction of the total protein mass it represents in the extract. For proteins that are en- Table 3-6 The Isoelectric Points zymes, the amount in a given solution or tissue extract of some proteins can be measured, or assayed, in terms of the catalytic effect the enzyme produces-that is, the increase in Protein p the rate at which its substrate is converted to reaction products when the enzyme is present. For this purpose Pepsin 1.0 one must know (i) the overall equation of the reaction Egg album 4.6 catalyzed, (2) an analytical procedure for determining Serum albl 4.9 the disappearance of the substrate or the appearance of a reaction product, 3) whether the enzyme requires co- 5.2 factors such as metal ions or coenzymes, (4) the de- Hemoglobin 6.8 pendence of the enzyme activity on substrate concen- Myoglobin tration, (5) the optimum pH, and (6) a temperature Chymotrypsinogen 9.5 zone in which the enzyme is stable and has high activ Cytochrom ity. Enzymes are usually assayed at their optimum pH Lysozyme 11.0 and at some convenient temperature within the range

pH 9 pH 3 – + – + – + An ampholyte solution is incorporated into a gel. Decreasing pH A stable pH gradient is established in the gel after application of an electric field. Protein solution is added and electric field is reapplied. After staining, proteins are shown to be distributed along pH gradient according to their pI values. FIGURE 3–21 Isoelectric focusing. This technique separates proteins according to their isoelectric points. A stable pH gradient is established in the gel by the addition of appropriate ampholytes. A protein mixture is placed in a well on the gel. With an applied electric field, proteins enter the gel and migrate until each reaches a pH equivalent to its pI. Remember that when pH
pI, the net charge of a protein is zero. electrophoresis permits the resolution of complex mixtures of proteins (Fig. 3–22). This is a more sensi￾tive analytical method than either electrophoretic method alone. Two-dimensional electrophoresis sepa￾rates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but different mo￾lecular weights. Unseparated Proteins Can Be Quantified To purify a protein, it is essential to have a way of de￾tecting and quantifying that protein in the presence of many other proteins at each stage of the procedure. Often, purification must proceed in the absence of any information about the size and physical properties of the protein or about the fraction of the total protein mass it represents in the extract. For proteins that are en￾zymes, the amount in a given solution or tissue extract can be measured, or assayed, in terms of the catalytic effect the enzyme produces—that is, the increase in the rate at which its substrate is converted to reaction products when the enzyme is present. For this purpose one must know (1) the overall equation of the reaction catalyzed, (2) an analytical procedure for determining the disappearance of the substrate or the appearance of a reaction product, (3) whether the enzyme requires co￾factors such as metal ions or coenzymes, (4) the de￾pendence of the enzyme activity on substrate concen￾tration, (5) the optimum pH, and (6) a temperature zone in which the enzyme is stable and has high activ￾ity. Enzymes are usually assayed at their optimum pH and at some convenient temperature within the range 25 to 38 C. Also, very high substrate concentrations are generally used so that the initial reaction rate, measured experimentally, is proportional to enzyme concentration (Chapter 6). By international agreement, 1.0 unit of enzyme ac￾tivity is defined as the amount of enzyme causing trans￾formation of 1.0
mol of substrate per minute at 25 C under optimal conditions of measurement. The term activity refers to the total units of enzyme in a solu￾tion. The specific activity is the number of enzyme units per milligram of total protein (Fig. 3–23). The spe￾cific activity is a measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure (Table 3–5). 94 Chapter 3 Amino Acids, Peptides, and Proteins Protein pI Pepsin 1.0 Egg albumin 4.6 Serum albumin 4.9 Urease 5.0 -Lactoglobulin 5.2 Hemoglobin 6.8 Myoglobin 7.0 Chymotrypsinogen 9.5 Cytochrome c 10.7 Lysozyme 11.0 The Isoelectric Points of Some Proteins TABLE 3–6 8885d_c03_094 12/23/03 10:24 AM Page 94 mac111 mac111:reb: