3.2 Peptides and proteins has a pI of 3.22, considerably lower than that of glycine. Peptides Are Chains of Amino Acids This is due to the presence of two carboxyl groups which, at the average of their pka values (3.22),con Two amino acid molecules can be covalently joined tribute a net charge of -1 that balances the +l con- through a substituted amide linkage, termed a peptide tributed by the amino group. Similarly, the pI of hist- bond, to yield a dipeptide. Such a linkage is formed by dine, with two groups that are positively charged when removal of the elements of water (dehydration) from protonated, is 7.59(the average of the pKa values of the amino and imidazole groups), much higher than that of group of another(Fig. 3-13).Peptide bond formation is an example of a condensation reaction, a common class Finally,as pointed out earlier, under the general of reactions in living cells Under standard biochemical condition of free and open exposure to the aqueous en- conditions, the equilibrium for the reaction shown in Fig vironment, only histidine has an R group (pk,=60) ure 3-13 favors the amino acids over the dipeptide.To providing significant buffering power near the neutral make the reaction thermodynamically more favorable pH usually found in the intracellular and extracellular the carboxyl group must be chemically modified or ac- fluids of most animals and bacteria ( Table 3-1) tivated so that the hydroxyl group can be more readily eliminated. A chemical approach to this problem is out- SUMMARY 3. 1 Amino acids lined later in this chapter. The biological approach to peptide bond formation is a major topic of Chapter 27 Three amino acids can be joined by two peptide a The 20 amino acids commonly found as bonds to form a tripeptide; similarly, amino acids can be residues in proteins contain an a-carboxyl linked to form tetrapeptides, pentapeptides, and so group, an a-amino group, and a distinctive R forth. When a few amino acids are joined in this fash- group substituted on the a-carbon atom. The ion, the structure is called an oligopeptide. When many a-carbon atom of all amino acids except glycin amino acids are joined, the product is called a polypep- is asymmetric, and thus amino acids can exist tide. Proteins may have thousands of amino acid in at least two stereoisomeric forms. Only the residues. Although the terms "protein"and "polypep L stereoisomers, with a configuration related to tide"are sometimes used interchangeably, molecules re- the absolute configuration of the reference ferred to as polypeptides generally have molecular molecule L-glyceraldehyde, are found in weights below 10,000, and those called proteins have higher molecular weights Other less common amino acids also occur Figure 3-14 shows the structure of a pentapeptide either as constituents of proteins(through As already noted, an amino acid unit in a peptide is often modification of common amino acid residues called a residue(the part left over after losing a hydro- after protein synthesis) or as free metabolites gen atom from its amino group and the hydroxyl moi I Amino acids are classified into five types on th ety from its carboxyl group). In a peptide, the amino basis of the polarity and charge(at pH 7 of acid residue at the end with a free a-amino group is the their R groups amino-terminal (or N-terminal residue; the residue and have characteristic titration curves H R Monoamino monocarboxylic amino acids (with H3N一CHC-OH+H-N—CH-COO nonionizable R groups)are diprotic acids CH3NCH(R)COOH) at low pH and exist in several different ionic forms as the ph is increased. Amino acids with ionizable R groups have additional ionic species, depending on the H R2 oH of the medium and the pKa of the r group CH-C-N--CH-CO0 3.2 Peptides and proteins FIGURE 3-13 Formation of a peptide bond by condensation. The ar- We now turn to polymers of amino acids, the peptides amino group of one amino acid (with R group) acts as a nucleophile and proteins. Biologically occurring polypeptides range to displace the hydroxyl group of another amino acid (with R' group), in size from small to very large, consisting of two or forming a peptide bond(shaded in yellow). Amino groups are good three to thousands of linked amino acid residues. Our nucleophiles, but the hydroxyl group is a poor leaving group and is focus is on the fundamental chemical properties of these not readily displaced. At physiological pH, the reaction shown does occur to any appreciable extent
has a pI of 3.22, considerably lower than that of glycine. This is due to the presence of two carboxyl groups, which, at the average of their pKa values (3.22), contribute a net charge of 1 that balances the 1 contributed by the amino group. Similarly, the pI of histidine, with two groups that are positively charged when protonated, is 7.59 (the average of the pKa values of the amino and imidazole groups), much higher than that of glycine. Finally, as pointed out earlier, under the general condition of free and open exposure to the aqueous environment, only histidine has an R group (pKa 6.0) providing significant buffering power near the neutral pH usually found in the intracellular and extracellular fluids of most animals and bacteria (Table 3–1). SUMMARY 3.1 Amino Acids ■ The 20 amino acids commonly found as residues in proteins contain an -carboxyl group, an -amino group, and a distinctive R group substituted on the -carbon atom. The -carbon atom of all amino acids except glycine is asymmetric, and thus amino acids can exist in at least two stereoisomeric forms. Only the L stereoisomers, with a configuration related to the absolute configuration of the reference molecule L-glyceraldehyde, are found in proteins. ■ Other, less common amino acids also occur, either as constituents of proteins (through modification of common amino acid residues after protein synthesis) or as free metabolites. ■ Amino acids are classified into five types on the basis of the polarity and charge (at pH 7) of their R groups. ■ Amino acids vary in their acid-base properties and have characteristic titration curves. Monoamino monocarboxylic amino acids (with nonionizable R groups) are diprotic acids (H3NCH(R)COOH) at low pH and exist in several different ionic forms as the pH is increased. Amino acids with ionizable R groups have additional ionic species, depending on the pH of the medium and the pKa of the R group. 3.2 Peptides and Proteins We now turn to polymers of amino acids, the peptides and proteins. Biologically occurring polypeptides range in size from small to very large, consisting of two or three to thousands of linked amino acid residues. Our focus is on the fundamental chemical properties of these polymers. Peptides Are Chains of Amino Acids Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide. Such a linkage is formed by removal of the elements of water (dehydration) from the -carboxyl group of one amino acid and the -amino group of another (Fig. 3–13). Peptide bond formation is an example of a condensation reaction, a common class of reactions in living cells. Under standard biochemical conditions, the equilibrium for the reaction shown in Figure 3–13 favors the amino acids over the dipeptide. To make the reaction thermodynamically more favorable, the carboxyl group must be chemically modified or activated so that the hydroxyl group can be more readily eliminated. A chemical approach to this problem is outlined later in this chapter. The biological approach to peptide bond formation is a major topic of Chapter 27. Three amino acids can be joined by two peptide bonds to form a tripeptide; similarly, amino acids can be linked to form tetrapeptides, pentapeptides, and so forth. When a few amino acids are joined in this fashion, the structure is called an oligopeptide. When many amino acids are joined, the product is called a polypeptide. Proteins may have thousands of amino acid residues. Although the terms “protein” and “polypeptide” are sometimes used interchangeably, molecules referred to as polypeptides generally have molecular weights below 10,000, and those called proteins have higher molecular weights. Figure 3–14 shows the structure of a pentapeptide. As already noted, an amino acid unit in a peptide is often called a residue (the part left over after losing a hydrogen atom from its amino group and the hydroxyl moiety from its carboxyl group). In a peptide, the amino acid residue at the end with a free -amino group is the amino-terminal (or N-terminal) residue; the residue 3.2 Peptides and Proteins 85 H3N C R1 H C O OH H N H C R2 H COO H H2O 2O H3N C R1 H C O N H C R2 H COO FIGURE 3–13 Formation of a peptide bond by condensation. The - amino group of one amino acid (with R2 group) acts as a nucleophile to displace the hydroxyl group of another amino acid (with R1 group), forming a peptide bond (shaded in yellow). Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily displaced. At physiological pH, the reaction shown does not occur to any appreciable extent. 8885d_c03_085 12/23/03 10:22 AM Page 85 mac111 mac111:reb:
Chapter 3 Amino Acids, Peptides, and Proteins ever, the r groups of some amino acids can ionize (table 3-1), and in a peptide these contribute to the overall CHa CHa acid-base properties of the molecule(Fig. 3-15). Thus he acid-base behavior of a peptide can be predicted H, H CH3 from its free a-amino and a-carboxyl groups as well as 且-c-N C-C-N-C-COO the nature and number of its ionizable R groups Like free amino acids, peptides have characteristic titration curves and a characteristic isoelectric pH (pD Amino- Carboxyl at which they do not move in an electric field. These erminal end properties are exploited in some of the techniques used FIGURE 3-14 The pentapeptide serylglycyltyrosylalanylleucine, or to separate peptides and proteins, as we shall see later Ser-Gly-Tyr-Ala-Leu. Peptides are named beginning with the amino- in the chapter. It should be emphasized that the pk terminal residue, which by convention is placed at the left. The pep- value for an ionizable R group can change somewhat tide bonds are shaded in yel ellow; the R groups are in red. when an amino acid becomes a residue in a peptide. The loss of charge in the a-carboxyl and a-amino groups the interactions with other peptide r groups, and other at the other end, which has a free carboxyl group, is the environmental factors can affect the pka. The pka val carboxyl-terminal (C-terminal) residue ues for R groups listed in Table 3-1 can be a useful guide Although hydrolysis of a peptide bond is an exer to the ph range in which a given group will ionize, but gonic reaction, it occurs slowly because of its high acti- they cannot be strictly applied to peptides vation energy. As a result, the peptide bonds in proteins are quite stable, with an average half-life(t1/2) of about Biologically Active Peptides and Polypeptides 7 years under most intracellular conditions Occur in a Vast Range of Sizes Peptides Can Be Distinguished by Their No generalizations can be made about the molecular lonization behavior weights of biologically active peptides and proteins in re- lation to their functions. Naturally occurring peptides Peptides contain only one free a-amino group and one range in length from two to many thousands of amino free a-carboxyl group, at opposite ends of the chain acid residues. Even the smallest peptides can have bio- (Fig. 3-15). These groups ionize as they do in free amino logically important effects. Consider the commercially acids, although the ionization constants are different be- synthesized dipeptide L-aspartyl-L-phenylalanine methyl cause an oppositely charged group is no longer linked ester, the artificial sweetener better known as aspartame to the a carbon. The a-amino and a-carboxyl groups of or NutraSweet all nonterminal amino acids are covalently joined in the peptide bonds, which do not ionize and thus do not con- tribute to the total acid-base behavior of peptides. How CHO HSN-CH-C-N--CH-C-OCH CH-CH L-Aspartyl-L-phenylalanine methyl ester NH Many small peptides exert their effects at very low CH-CH2-CH2-co0 concentrations. For example, a number of vertebrate hormones(Chapter 23) are small peptides. These in- clude oxytocin (nine amino acid residues, which is se creted by the posterior pituitary and stimulates uterine Gly CH2 contractions; bradykinin (nine residues), which inhibits inflammation of tissues; and thyrotropin-releasing fac tor(three residues), which is formed in the hypothala NH mus and stimulates the release of another hormone H一C] H2-CH2-CH2-NHs thyrotropin, from the anterior pituitary gland. Some extremely toxic mushroom poisons, such as amanitin COO are also small peptides, as are many antibiotics FIGURE 3-15 Alanylglutamylglycyllysine. This tetrapeptide has one Slightly larger are small polypeptides and oligopep- free a-amino group, one free a-carboxyl group, and two ionizable r tides such as the pancreatic hormone insulin, which con- groups. The groups ionized at pH 7.0 are in red. tains two polypeptide chains, one having 30 amino acid
at the other end, which has a free carboxyl group, is the carboxyl-terminal (C-terminal) residue. Although hydrolysis of a peptide bond is an exergonic reaction, it occurs slowly because of its high activation energy. As a result, the peptide bonds in proteins are quite stable, with an average half-life (t1/2) of about 7 years under most intracellular conditions. Peptides Can Be Distinguished by Their Ionization Behavior Peptides contain only one free -amino group and one free -carboxyl group, at opposite ends of the chain (Fig. 3–15). These groups ionize as they do in free amino acids, although the ionization constants are different because an oppositely charged group is no longer linked to the carbon. The -amino and -carboxyl groups of all nonterminal amino acids are covalently joined in the peptide bonds, which do not ionize and thus do not contribute to the total acid-base behavior of peptides. However, the R groups of some amino acids can ionize (Table 3–1), and in a peptide these contribute to the overall acid-base properties of the molecule (Fig. 3–15). Thus the acid-base behavior of a peptide can be predicted from its free -amino and -carboxyl groups as well as the nature and number of its ionizable R groups. Like free amino acids, peptides have characteristic titration curves and a characteristic isoelectric pH (pI) at which they do not move in an electric field. These properties are exploited in some of the techniques used to separate peptides and proteins, as we shall see later in the chapter. It should be emphasized that the pKa value for an ionizable R group can change somewhat when an amino acid becomes a residue in a peptide. The loss of charge in the -carboxyl and -amino groups, the interactions with other peptide R groups, and other environmental factors can affect the pKa. The pKa values for R groups listed in Table 3–1 can be a useful guide to the pH range in which a given group will ionize, but they cannot be strictly applied to peptides. Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes No generalizations can be made about the molecular weights of biologically active peptides and proteins in relation to their functions. Naturally occurring peptides range in length from two to many thousands of amino acid residues. Even the smallest peptides can have biologically important effects. Consider the commercially synthesized dipeptide L-aspartyl-L-phenylalanine methyl ester, the artificial sweetener better known as aspartame or NutraSweet. Many small peptides exert their effects at very low concentrations. For example, a number of vertebrate hormones (Chapter 23) are small peptides. These include oxytocin (nine amino acid residues), which is secreted by the posterior pituitary and stimulates uterine contractions; bradykinin (nine residues), which inhibits inflammation of tissues; and thyrotropin-releasing factor (three residues), which is formed in the hypothalamus and stimulates the release of another hormone, thyrotropin, from the anterior pituitary gland. Some extremely toxic mushroom poisons, such as amanitin, are also small peptides, as are many antibiotics. Slightly larger are small polypeptides and oligopeptides such as the pancreatic hormone insulin, which contains two polypeptide chains, one having 30 amino acid H3N C C COO H2 H C O N H C CH2 H C O OCH3 L-Aspartyl-L-phenylalanine methyl ester (aspartame) 86 Chapter 3 Amino Acids, Peptides, and Proteins H3N C CH2OH H C O N H C H H C O N H C CH2 H C O N H C CH3 H C OH N H C C C CH3 CH3 H H2 COO Amino- Carboxylterminal end terminal end O H FIGURE 3–14 The pentapeptide serylglycyltyrosylalanylleucine, or Ser–Gly–Tyr–Ala–Leu. Peptides are named beginning with the aminoterminal residue, which by convention is placed at the left. The peptide bonds are shaded in yellow; the R groups are in red. Ala C COO NH O C C NH O C C NH O C C N H3 H CH3 H CH2 CH2 COO H2 H CH2 CH2 CH2 CH2 N Lys H3 Gly Glu FIGURE 3–15 Alanylglutamylglycyllysine. This tetrapeptide has one free -amino group, one free -carboxyl group, and two ionizable R groups. The groups ionized at pH 7.0 are in red. 8885d_c03_086 12/23/03 10:22 AM Page 86 mac111 mac111:reb:
3.2 Peptides and proteins residues and the other 21. Glucagon, another pancre- chemical constituents by dividing its molecular weight atic hormone, has 29 residues; it opposes the action of by 110. Although the average molecular weight of the insulin Corticotropin is a 39-residue hormone of the an- 20 common amino acids is about 138, the smaller amino terior pituitary gland that stimulates the adrenal cortex. acids predominate in most proteins. If we take into ac- How long are the polypeptide chains in proteins? As count the proportions in which the various amino acids Table 3-2 shows, lengths vary considerably. Human cyto- occur in proteins ( Table 3-1), the average molecular chrome c has 104 amino acid residues linked in a single weight of protein amino acids is nearer to 128. Because chain; bovine chymotrypsinogen has 245 residues. At a molecule of water(Mr 18)is removed to create each the extreme is titin, a constituent of vertebrate muscle, peptide bond, the average molecular weight of an amino which has nearly 27,000 amino acid residues and a mo- acid residue in a protein is about 128-18=110 lecular weight of about 3, 000, 000. The vast majority of naturally occurring proteins are much smaller than this Polypeptides Have Characteristic containing fewer than 2,000 amino acid residues. Some proteins consist of a single polypeptide chain Amino Acid Compositions but others, called multisubunit proteins, have two or Hydrolysis of peptides or proteins with acid yields a mix more polypeptides associated noncovalently (Table ture of free a-amino acids. When completely hydrolyzed, 3-2). The individual polypeptide chains in a multisub- each type of protein yields a characteristic proportion unit protein may be identical or different. If at least two or mixture of the different amino acids. The 20 common are identical the protein is said to be oligomeric, and amino acids almost never occur in equal amounts in a the identical units(consisting of one or more polypep- protein. Some amino acids may occur only once or not tide chains) are referred to as protomers. Hemoglobin, at all in a given type of protein; others may occur in for example, has four polypeptide subunits: two large numbers. Table 3-3 shows the composition of the identical a chains and two identical B chains, all four amino acid mixtures obtained on complete hydrolysis of held together by noncovalent interactions. Each a sub- bovine cytochrome c and chymotrypsinogen, the inac- unit is paired in an identical way with a B subunit within tive precursor of the digestive enzyme chymotrypsin the structure of this multisubunit protein, so that he- These two proteins, with very different functions, also moglobin can be considered either a tetramer of four differ significantly in the relative numbers of each kind polypeptide subunits or a dimer of aB protomers. of amino acid they contain. A few proteins contain two or more polypeptide Complete hydrolysis alone is not sufficient for an chains linked covalently. For example, the two polypep- exact analysis of amino acid composition, however, be- tide chains of insulin are linked by disulfide bonds In cause some side reactions occur during the procedure such cases, the individual polypeptides are not consid- For example, the amide bonds in the side chains of as ered subunits but are commonly referred to simply as paragine and glutamine are cleaved by acid treatment chains yielding aspartate and glutamate, respectively. The side We can calculate the approximate number of amino chain of tryptophan is almost completely degraded by cid residues in a simple protein containing no other acid hydrolysis, and small amounts of serine, threonine TaBlE 3-2 Molecular Data on Some Proteins Molecular Number of Number of residues polypeptide chains Cytochrome c(human) 13,000 Ribonuclease A(bovine pancreas 13,700 Lysozyme( chicken egg white) 13,930 129 oglobin(equine hea Chymotrypsin(bovine pancreas 241 Chymotrypsinogen(bovine) 22.000 Hemoglobin(human) 4,500 Serum albumin(human) 68500 609 RNA polymerase(E. coll) 450.000 4,158 513,000 4.536 1111314125121 Glutamine synthetase(E co) 619,000 5,628 Titin(human) 2.993.000 26,926
residues and the other 21. Glucagon, another pancreatic hormone, has 29 residues; it opposes the action of insulin. Corticotropin is a 39-residue hormone of the anterior pituitary gland that stimulates the adrenal cortex. How long are the polypeptide chains in proteins? As Table 3–2 shows, lengths vary considerably. Human cytochrome c has 104 amino acid residues linked in a single chain; bovine chymotrypsinogen has 245 residues. At the extreme is titin, a constituent of vertebrate muscle, which has nearly 27,000 amino acid residues and a molecular weight of about 3,000,000. The vast majority of naturally occurring proteins are much smaller than this, containing fewer than 2,000 amino acid residues. Some proteins consist of a single polypeptide chain, but others, called multisubunit proteins, have two or more polypeptides associated noncovalently (Table 3–2). The individual polypeptide chains in a multisubunit protein may be identical or different. If at least two are identical the protein is said to be oligomeric, and the identical units (consisting of one or more polypeptide chains) are referred to as protomers. Hemoglobin, for example, has four polypeptide subunits: two identical chains and two identical chains, all four held together by noncovalent interactions. Each subunit is paired in an identical way with a subunit within the structure of this multisubunit protein, so that hemoglobin can be considered either a tetramer of four polypeptide subunits or a dimer of protomers. A few proteins contain two or more polypeptide chains linked covalently. For example, the two polypeptide chains of insulin are linked by disulfide bonds. In such cases, the individual polypeptides are not considered subunits but are commonly referred to simply as chains. We can calculate the approximate number of amino acid residues in a simple protein containing no other chemical constituents by dividing its molecular weight by 110. Although the average molecular weight of the 20 common amino acids is about 138, the smaller amino acids predominate in most proteins. If we take into account the proportions in which the various amino acids occur in proteins (Table 3–1), the average molecular weight of protein amino acids is nearer to 128. Because a molecule of water (Mr 18) is removed to create each peptide bond, the average molecular weight of an amino acid residue in a protein is about 128 18 110. Polypeptides Have Characteristic Amino Acid Compositions Hydrolysis of peptides or proteins with acid yields a mixture of free -amino acids. When completely hydrolyzed, each type of protein yields a characteristic proportion or mixture of the different amino acids. The 20 common amino acids almost never occur in equal amounts in a protein. Some amino acids may occur only once or not at all in a given type of protein; others may occur in large numbers. Table 3–3 shows the composition of the amino acid mixtures obtained on complete hydrolysis of bovine cytochrome c and chymotrypsinogen, the inactive precursor of the digestive enzyme chymotrypsin. These two proteins, with very different functions, also differ significantly in the relative numbers of each kind of amino acid they contain. Complete hydrolysis alone is not sufficient for an exact analysis of amino acid composition, however, because some side reactions occur during the procedure. For example, the amide bonds in the side chains of asparagine and glutamine are cleaved by acid treatment, yielding aspartate and glutamate, respectively. The side chain of tryptophan is almost completely degraded by acid hydrolysis, and small amounts of serine, threonine, 3.2 Peptides and Proteins 87 TABLE 3–2 Molecular Data on Some Proteins Molecular Number of Number of weight residues polypeptide chains Cytochrome c (human) 13,000 104 1 Ribonuclease A (bovine pancreas) 13,700 124 1 Lysozyme (chicken egg white) 13,930 129 1 Myoglobin (equine heart) 16,890 153 1 Chymotrypsin (bovine pancreas) 21,600 241 3 Chymotrypsinogen (bovine) 22,000 245 1 Hemoglobin (human) 64,500 574 4 Serum albumin (human) 68,500 609 1 Hexokinase (yeast) 102,000 972 2 RNA polymerase (E. coli) 450,000 4,158 5 Apolipoprotein B (human) 513,000 4,536 1 Glutamine synthetase (E. coli) 619,000 5,628 12 Titin (human) 2,993,000 26,926 1 8885d_c03_087 12/23/03 10:22 AM Page 87 mac111 mac111:reb:
Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3-3 Amino Acid Composition of TABLE 3-4 Conjugated Pt Two Proteins Class Prosthetic group Example Number of residues Lipoproteins oer molecu ule of protein β1 Lipoprotein of blood Amino Bovine Glycoproteins Carbohydrates Immunoglobulin G chymotrypsinogen Phosphoproteins Phosphate groups Casein of milk Hemoproteins Heme(iron porphyrin) Hemoglobin Flavoprotein Flavin nucleotides Succinate dehydrogen Metalloproteins Iron Asp 8 Alcohol dehydrogenase GIn 10 Calcium Glu Dinitrogenase Copper Plastocyanin His 94366824418143 32094 metal Anumber of proteins contain more than one pros hetic group. Usually the prosthetic group plays an im- 269 portant role in the proteins biological function There are several levels of protein structure 23 For large macromolecules such as proteins, the tasks of describing and understanding structure are approached lyr at several levels of complexity, arranged in a kind of con- ceptual hierarchy. Four levels of protein structure are 104 commonly defined(Fig. 3-16). A description of all co- valent bonds (mainly peptide bonds and disulfide such as acid hydrolysis, A bonds) linking amino acid residues in a polypeptide er designated Asx(or B). Similarty, when Glu and chain is its primary structure. The most important el ey are together designated Gi(or Z). In addition, Trp is es must be employed to obtain an accurate assessment of ement of primary structure is the sequence of amino acid residues. Secondary structure refers to particu- larly stable arrangements of amino acid residues giving and tyrosine are also lost. When a precise amino acid rise to recurring structural patterns. Tertiary struc composition is required, biochemists use additional pro- ture describes all aspects of the three-dimensional fold- cedures to resolve the ambiguities that remain from acid ng of a polypeptide. When a protein has two or more hydrolysis polypeptide subunits, their arrangement in space is re- ferred to as quaternary structure. Primary structure Some Proteins Contain Chemical Groups is the focus of Section 3.4; the higher levels of structure Other Than Amino acids are discussed in Chapter 4. Many proteins, for example the enzymes ribonuclease A and chymotrypsinogen, contain only amino acid SUMMARY 3.2 Peptides and Proteins esidues and no other chemical constituents; these are considered simple proteins. However, some proteins a Amino acids can be joined covalently through contain permanently associated chemical components peptide bonds to form peptides and proteins in addition to amino acids; these are called conjugated Cells generally contain thousands of different proteins. The non-amino acid part of a conjugated pro- proteins, each with a different biological activity. tein is usually called its prosthetic group. Conjugated u Proteins can be very long polypeptide chains of proteins are classified on the basis of the chemical na- 100 to several thousand amino acid residues ture of their prosthetic groups (Table 3-4); for exam- However, some naturally occurring peptides ple, lipoproteins contain lipids, glycoproteins contain have only a few amino acid residues. Some sugar groups, and metalloproteins contain a specific proteins are composed of several noncovalently
and tyrosine are also lost. When a precise amino acid composition is required, biochemists use additional procedures to resolve the ambiguities that remain from acid hydrolysis. Some Proteins Contain Chemical Groups Other Than Amino Acids Many proteins, for example the enzymes ribonuclease A and chymotrypsinogen, contain only amino acid residues and no other chemical constituents; these are considered simple proteins. However, some proteins contain permanently associated chemical components in addition to amino acids; these are called conjugated proteins. The non–amino acid part of a conjugated protein is usually called its prosthetic group. Conjugated proteins are classified on the basis of the chemical nature of their prosthetic groups (Table 3–4); for example, lipoproteins contain lipids, glycoproteins contain sugar groups, and metalloproteins contain a specific metal. A number of proteins contain more than one prosthetic group. Usually the prosthetic group plays an important role in the protein’s biological function. There Are Several Levels of Protein Structure For large macromolecules such as proteins, the tasks of describing and understanding structure are approached at several levels of complexity, arranged in a kind of conceptual hierarchy. Four levels of protein structure are commonly defined (Fig. 3–16). A description of all covalent bonds (mainly peptide bonds and disulfide bonds) linking amino acid residues in a polypeptide chain is its primary structure. The most important element of primary structure is the sequence of amino acid residues. Secondary structure refers to particularly stable arrangements of amino acid residues giving rise to recurring structural patterns. Tertiary structure describes all aspects of the three-dimensional folding of a polypeptide. When a protein has two or more polypeptide subunits, their arrangement in space is referred to as quaternary structure. Primary structure is the focus of Section 3.4; the higher levels of structure are discussed in Chapter 4. SUMMARY 3.2 Peptides and Proteins ■ Amino acids can be joined covalently through peptide bonds to form peptides and proteins. Cells generally contain thousands of different proteins, each with a different biological activity. ■ Proteins can be very long polypeptide chains of 100 to several thousand amino acid residues. However, some naturally occurring peptides have only a few amino acid residues. Some proteins are composed of several noncovalently 88 Chapter 3 Amino Acids, Peptides, and Proteins *In some common analyses, such as acid hydrolysis, Asp and Asn are not readily distinguished from each other and are together designated Asx (or B). Similarly, when Glu and Gln cannot be distinguished, they are together designated Glx (or Z). In addition, Trp is destroyed. Additional procedures must be employed to obtain an accurate assessment of complete amino acid content. Number of residues per molecule of protein* Amino Bovine Bovine acid cytochrome c chymotrypsinogen Ala 6 22 Arg 2 4 Asn 5 15 Asp 3 8 Cys 2 10 Gln 3 10 Glu 9 5 Gly 14 23 His 3 2 Ile 6 10 Leu 6 19 Lys 18 14 Met 2 2 Phe 4 6 Pro 4 9 Ser 1 28 Thr 8 23 Trp 1 8 Tyr 4 4 Val 3 23 Total 104 245 Amino Acid Composition of Two Proteins TABLE 3–3 TABLE 3–4 Conjugated Proteins Class Prosthetic group Example Lipoproteins Lipids 1-Lipoprotein of blood Glycoproteins Carbohydrates Immunoglobulin G Phosphoproteins Phosphate groups Casein of milk Hemoproteins Heme (iron porphyrin) Hemoglobin Flavoproteins Flavin nucleotides Succinate dehydrogenase Metalloproteins Iron Ferritin Zinc Alcohol dehydrogenase Calcium Calmodulin Molybdenum Dinitrogenase Copper Plastocyanin 8885d_c03_088 12/23/03 10:22 AM Page 88 mac111 mac111:reb:
3.3 Working with Proteins Primary Secondary Tertiary Quaternary structure structure structure tructure Gly val Amino acid residues a helix Polypeptide chain Assembled subunits FIGURE 3-16 Levels of structure in proteins. The primary structure lix is a part of the tertiary structure of the folded polypeptide, which consists of a sequence of amino acids linked together by peptide bonds is itself one of the subunits that make up the qu and includes any disulfide bonds. The resulting polypeptide can be the multisubunit protein, in this case hemoglobin. ry structure of coiled into units of secondary structure, such as an a helix. The he- associated polypeptide chains, called subunits. pare subcellular fractions or to isolate specific or Simple proteins yield only amino acids on ganelles(see Fig. 1-8) hydrolysis; conjugated proteins contain Once the extract or organelle preparation is ready, addition some other component, such as a various methods are available for purifying one or more metal or organic prosthetic group of the proteins it contains. Commonly, the extract is sub- a The sequence of amino acids in a protein is jected to treatments that separate the proteins into dif- characteristic of that protein and is called its ferent fractions based on a property such as size or primary structure. This is one of four generally charge, a process referred to as fractionation. Early recognized levels of protein structure fractionation steps in a purification utilize differences in protein solubility, which is a complex function of pH temperature, salt concentration, and other factors. The solubility of proteins is generally lowered at high salt 3.3 Working with Proteins concentrations, an effect called"salting out. The addi Our understanding of protein structure and function has tion of a salt in the right amount can selectively pre been derived from the study of many individual proteins cipitate some proteins, while others remain in solution. To study a protein in detail, the researcher must be able Ammonium sulfate((NHA2 SO4) is often used for this to separate it from other proteins and must have the purpose because of its high solubility in water. techniques to determine its properties. The necessary A solution containing the protein of interest often methods come from protein chemistry, a discipline as must be further altered before subsequent purification old as biochemistry itself and one that retains a central steps are possible. For example, dialysis is a procedure position in biochemical research. that separates proteins from solvents by taking advan tage of the proteins larger size. The partially purified Proteins Can Be Separated and Purified extract is placed in a bag or tube made of a semper meable membrane. When this is suspended in a much A pure preparation is essential before a proteins prop- larger volume of buffered solution of appropriate ionic erties and activities can be determined. Given that cells strength the membrane allows the exchange of salt and contain thousands of different kinds of proteins, how buffer but not proteins. Thus dialysis retains large pro- can one protein be purified? Methods for separating pro- teins within the membranous bag or tube while allow teins take advantage of properties that vary from one ing the concentration of other solutes in the protein protein to the next, including size, charge, and binding preparation to change until they come into equilibrium properties with the solution outside the membrane Dialysis might The source of a protein is generally tissue or mi- be used, for example, to remove ammonium sulfate from obial cells. The first step in any protein purification the protein preparation. procedure is to break open these cells, releasing their The most powerful methods for fractionating pro proteins into a solution called a crude extract. If nec- teins make use of column chromatography, which essary, differential centrifugation can be used to pre- takes advantage of differences in protein charge, size
associated polypeptide chains, called subunits. Simple proteins yield only amino acids on hydrolysis; conjugated proteins contain in addition some other component, such as a metal or organic prosthetic group. ■ The sequence of amino acids in a protein is characteristic of that protein and is called its primary structure. This is one of four generally recognized levels of protein structure. 3.3 Working with Proteins Our understanding of protein structure and function has been derived from the study of many individual proteins. To study a protein in detail, the researcher must be able to separate it from other proteins and must have the techniques to determine its properties. The necessary methods come from protein chemistry, a discipline as old as biochemistry itself and one that retains a central position in biochemical research. Proteins Can Be Separated and Purified A pure preparation is essential before a protein’s properties and activities can be determined. Given that cells contain thousands of different kinds of proteins, how can one protein be purified? Methods for separating proteins take advantage of properties that vary from one protein to the next, including size, charge, and binding properties. The source of a protein is generally tissue or microbial cells. The first step in any protein purification procedure is to break open these cells, releasing their proteins into a solution called a crude extract. If necessary, differential centrifugation can be used to prepare subcellular fractions or to isolate specific organelles (see Fig. 1–8). Once the extract or organelle preparation is ready, various methods are available for purifying one or more of the proteins it contains. Commonly, the extract is subjected to treatments that separate the proteins into different fractions based on a property such as size or charge, a process referred to as fractionation. Early fractionation steps in a purification utilize differences in protein solubility, which is a complex function of pH, temperature, salt concentration, and other factors. The solubility of proteins is generally lowered at high salt concentrations, an effect called “salting out.” The addition of a salt in the right amount can selectively precipitate some proteins, while others remain in solution. Ammonium sulfate ((NH4)2SO4) is often used for this purpose because of its high solubility in water. A solution containing the protein of interest often must be further altered before subsequent purification steps are possible. For example, dialysis is a procedure that separates proteins from solvents by taking advantage of the proteins’ larger size. The partially purified extract is placed in a bag or tube made of a semipermeable membrane. When this is suspended in a much larger volume of buffered solution of appropriate ionic strength, the membrane allows the exchange of salt and buffer but not proteins. Thus dialysis retains large proteins within the membranous bag or tube while allowing the concentration of other solutes in the protein preparation to change until they come into equilibrium with the solution outside the membrane. Dialysis might be used, for example, to remove ammonium sulfate from the protein preparation. The most powerful methods for fractionating proteins make use of column chromatography, which takes advantage of differences in protein charge, size, 3.3 Working with Proteins 89 Primary structure Secondary structure Tertiary structure Quaternary structure Amino acid residues Lys Lys Gly Gly Leu Val Ala His Helix Polypeptide chain Assembled subunits FIGURE 3–16 Levels of structure in proteins. The primary structure consists of a sequence of amino acids linked together by peptide bonds and includes any disulfide bonds. The resulting polypeptide can be coiled into units of secondary structure, such as an helix. The helix is a part of the tertiary structure of the folded polypeptide, which is itself one of the subunits that make up the quaternary structure of the multisubunit protein, in this case hemoglobin. 8885d_c03_089 12/23/03 11:06 AM Page 89 mac111 mac111:reb: