Chapter 5 Amino Acids, Peptides, and Proteins
`
`123
`
`i
`HOC
`HN—C—H
`
`R
`Nonionic
`form
`
`°
`-O- ¢
`H.N—C—H
`
`R
`Zwitterionic
`form
`
`figure 5-9
`Nonionic and zwitterionic forms of amino acids. The
`nonionic form does not occur in significant amounts in
`aqueous solutions. The zwitterion predominatesat
`neutral pH.
`
`figure 5-10
`Titration of an amino acid. Shown here is the titration
`curve of 0.1 m glycine at 25 °C. The ionic species pre-
`dominating at key points in the titration are shown above
`the graph. The shaded boxes, centered at about
`pK, = 2.34 and pk, = 9.60, indicate the regions of
`greatest buffering power.
`
`NH,
`px,
`
`———
`CH,
`COOH
`Pkg=A.34
`
`13
`
`NH,
`NH,
`|
`|
`pK,
`ie — CHa
`Coo
`coo-
`
`eee ete
`
`bi
`
`Tr
`
`1
`
`a
`
`al
`
`i
`
`
`
`
`ii
`
`pH
`
`;
`
`
`
`0
`
`0.5
`
`1
`
`1.5
`
`aid
`
`2
`
`OH (equivalents)
`
`Amino Acids Can Act as Acids and Bases
`When an aminoacid is dissolved in water, it exists in solution as the dipolar
`ion, or zwitterion (German for “hybrid ion”), shown in Figure 5-9. A zwit-
`terion can act as either an acid (proton donor):
`
`—COO° + H'
`
`Z2—-O—-]
`
`H|
`
`R—C—COO™ —
`+NH,
`Zwitterion
`
`or a base (proton acceptor):
`
`;
`;
`RaTee +H* =——i
`*NH,
`*NH,
`Zwitterion
`
`Substances having this dual nature are amphoteric and are often called
`ampholytes(from “amphoteric electrolytes”). A simple monoamino mono-
`carboxylic o-amino acid, such as alanine, is a diprotic acid when fully pro-
`tonated—it has two groups, the —COOH group and the —NH3 group, that
`can yield protons:
`
`ELT
`to fe
`R—C—cooH > R—C—COO- — R—C—COO-
`
`Net charge:
`
`“NH,
`+1
`
`‘NH,
`0
`
`NH,
`"ell
`
`Amino Acids Have Characteristic Titration Curves
`Acid-base titration involves the gradual addition or removal of protons
`(Chapter 4). Figure 5-10 showsthetitration curve of the diprotic form of
`glycine. The plot has twodistinct stages, corresponding to deprotonation of
`two different groups on glycine. Each of the two stages resembles in shape
`the titration curve of a monoprotic acid, such as acetic acid (see Fig. 4-15),
`and can be analyzed in the same way. At very lowpH, the predominantionic
`species of glycine is ~H;N—CH,—COOH, the fully protonated form. At the
`midpoint in the first stage of the titration, in which the —COOHgroup of
`glycine loses its proton, equimolar concentrations of the proton-donor
`(*H,N—CH,—COOH) and proton-acceptor (“H;N—CH,—COO ) species
`are present. At the midpointofanytitration, a pointof inflection is reached
`where the pH is equal to the pK,of the protonated group being titrated (see
`Fig. 4-16). For glycine, the pH at the midpointis 2.34, thus its —COOH
`group has a pK, (labeled pk, in Fig. 5-10) of 2.34. (Recall from Chapter 4
`that pH and pK,are simply convenient notations for proton concentration
`and the equilibrium constant for ionization, respectively. The pK, is a mea-
`sure of the tendency of a group to give up a proton, with that tendency de-
`creasing tenfold as the pK,increases by one unit.) Asthetitration proceeds,
`another important point is reached at pH 5.97. Here there is another point
`of inflection, at which removal of thefirst proton is essentially complete and
`
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`

`124
`
`Part [| Structure and Catalysis
`
`removal of the second has just begun. At this pH glycine is present largely
`as the dipolar ion ~H;N—CH;—COO-. Weshall return to the significance of
`this inflection point in the titration curve (pl in Fig. 5-10) shortly.
`The second stage of the titration corresponds to the removal of a pro-
`ton from the —NH3 group ofglycine. The pH at the midpointof this stage
`is 9.60, equal to the pK,(labeled pX; in Fig. 5-10) for the —NH} group. The
`titration is essentially complete at a pH of about 12, at which point the pre-
`dominant form of glycine is HN—CH,—COO-.
`From the titration curve of glycine we can derive several important
`pieces of information. First, it gives a quantitative measure of the pK, of
`each of the two ionizing groups: 2.34 for the —COOH group and 9.60 for the
`—NHj;group. Note that the carboxyl group of glycine is over 100 times
`more acidic (more easily ionized) than the carboxyl group of acetic acid,
`which, as we saw in Chapter 4, has a pK, of 4.76, about average for a car-
`boxyl group attached to an otherwise unsubstituted aliphatic hydrocarbon.
`The perturbed pX,of glycine is caused by repulsion between the departing
`proton and the nearby positively charged amino group on the a-carbon
`atom, as described in Figure 5-11. The opposite charges on the resulting
`zwitterion are stabilizing, nudging the equilibrium farther to the right. Sim-
`ilarly, the pK, of the amino groupin glycine is perturbed downward relative
`to the average pK, of an amino group. This effect is due partly to the elec-
`tronegative oxygen atoms in the carboxyl groups, which tend to pull elec-
`trons toward them regardless of the carboxyl group charge, increasing the
`tendencyof the amino group to give up a proton. Hence, the a-amino group
`has a pK,that is lower than that of an aliphatic amine such as methylamine
`(Fig. 5-11). In short, the pK, of any functional group is greatly affected by
`its chemical environment, a phenomenon sometimes exploited in the active
`i
`isitel
`i
`i
`h
`
`Ssbendontheperturbedpit,values ofproton dononlacernioraroupe of
`
`‘
`:
`specific residues.
`
`a
`
`figure 5-11
`
`Effect ofthe chemical envionment on pKThe pA
`
`values for the ionizable groupsin glycine are lower than
`those for simple, methyl-substituted amino and carboxyl
`groups. These downward perturbations of pK, are due to
`intramolecular interactions. Similar effects can be caused
`by chemical groups that happen to be positioned
`nearby—for example, in the active site of an enzyme.
`
`pK,
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`ieee
`
`|
`|
`|
`
`i
`
`
`
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`|
`|
`CH,—NH, a CH;—NH,
`fe
`|
`|
`Boas
`|
`Ht
`|
`Methylamine |
`The normalpK,for an
`amino group is about 6
`|
`|
`HY
`NH,
`||
`= H—-C—CO0-
`A |
`|
`|
`: hy 7
`aa
`|
`a-Aminoacid (glycine)
`pK, = 9,60
`Electronegative oxygen atoms
`in the carboxyl group pull electrong
`away from the amino group,
`;
`lowering its pK,.
`
` |
`
`|
`
`ii
`
`i
`
`seen
`
`H
`
`Methyl-substituted
`carboxy!and.
`
`and
`arboxyl
`aminogroups
`
`Carboxyl and
`~~ inglycine
`amino groups
`oe
`
`xo
`|
`| CH;,—COOH ~ CH,—CO0O~
`i
`op
`ud
`|
`Ht
`|
`_ Acetic acid |
`|
`The normal pK,for a
`|
`carboxyl group is about4.8.
`|
`|
`
`| we
`|
`COOH — |
`CC
`in)
`ee a
`[|
`a
`|
`fe
`|
`a)
`a-Aminoacid (glycine)
`pK, = 2.34
`Repulsion between the amino
`group and the departing proton
`lowersthe pK, for the carboxyl
`group, and oppositely charged
`groups lower the pK,by stabi-
`lizing the zwitterion.
`
`

`

`Chapter 5 Amino Acids, Peptides, and Proteins
`
`a
`
`The second piece of information provided by the titration curve of
`glycine (Fig. 5-10) is that this amino acid has two regions of buffering
`power (see Fig. 4-17). One of these is the relatively flat portion of the
`curve, extending for approximately one pH unit on either side of thefirst
`pK,of 2.34, indicating that glycine is a good buffer near this pH. The other
`buffering zone is centered around pH 9.60. Note that glycine is not a good
`buffer at the pH ofintracellular fluid or blood, about 7.4. Within the buffer-
`ing ranges of glycine, the Henderson-Hasselbalch equation (Chapter 4) can
`be used to calculate the proportions of proton-donor and proton-acceptor
`species of glycine required to make a buffer at a given pH.
`
`
`
`
`
`
`COOH
`H,N—CH
`CH,
`bu, pi,
`COOH
`
`coo
`H.N—CH
`CH,
`Ou, PKn
`oon
`
`coo
`uii—cH
`bn,
`CH, BRa
`¢oo-
`
`coo
`H.N—CH
`CH,
`CH,
`Coo
`
`
`
`Titration Curves Predict the Electric Charge of Amino Acids
`Another important piece of information derived from the titration curve of
`an amino acid is the relationship betweenits net electric charge and the pH
`of the solution, At pH 5.97, the point of inflection between the two stages in
`its titration curve, glycine is present predominantly asits dipolar form, fully
`ionized but with no net electric charge (Fig. 5-10). The characteristic pH
`at which the net electric charge is zero is called the isoelectric point or
`isoelectric pH,designated pI. For glycine, which has no ionizable group in
`its side chain, the isoelectric pointis simply the arithmetic mean of the two
`pK, values:
`
`iim (pk, + pK,) = 5(2.34 + 9.60) = 5.97
`As is evident in Figure 5-10, glycine has a net negative charge at any pH
`aboveits pI and will thus move toward the positive electrode (the anode)
`when placed in an electric field. At any pH below its pl, glycine has a net
`positive charge and will move toward the negative electrode (the cathode).
`The farther the pH of a glycine solution is from its isoelectric point, the
`greater the net electric charge of the population of glycine molecules.
`At pH 1.0,
`for example, glycine exists almost entirely as the form
`*H,;N—CH,—COOH, with a net positive charge of 1.0. At pH 2.34, where
`there is an equal mixture of *H;N—CH,—COOH and ~H;N—CH,—COO ,
`the average or net positive charge is 0.5. The sign and the magnitude of the
`net charge of any amino acid at any pH can be predicted in the same way.
`
`1
`
`Amino Acids Differ in Their Acid-Base Properties
`The shared properties of many amino acids permit some simplifying gener-
`alizations about their acid-base behaviors.
`All amino acids with a single a-amino group, a single a-carboxyl group,
`and an R group that does notionize have titration curves resembling that of
`glycine (Fig. 5-10). These amino acids have very similar, although not iden-
`tical, pK, values: pK, of the —COOHgroupin the range of 1.8 to 2.4, and
`pK, of the —NHjgroupinthe range of 8.8 to 11.0 (Table 5-1).
`Amino acids with an ionizable R group have more complex titration
`curves, with three stages corresponding to the three possible ionization
`steps; thus they have three pK, values. The additional stage for thetitration
`of the ionizable R group merges to some extent with the other two. The
`titration curves for two amino acids of this type, glutamate andhistidine,
`are shown in Figure 5-12. The isoelectric points reflect the nature of the
`ionizing R groups present. For example, glutamate has a pl of 3.22, consid-
`erably lower than that of glycine. This is due to the presence of two car-
`boxyl groups which, at the average of their pK, values (3.22), contribute a
`net negative 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 pk, values of the
`amino and imidazole groups), much higher than that of glycine.
`
`OH™ (equivalents)
`
`(b)
`
`figure 5-12
`Titration curves for (a) glutamate and (b) histidine. The
`pK, of the R group is designated here as pkg.
`
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`

`126
`
`Part II
`
`Structure and Catalysis
`
`Another important generalization can be made about the acid-base be-
`havior of the 20 standard aminoacids. As pointed outearlier, under the gen-
`eral condition of free and open exposure to the aqueous environment, only
`histidine has an R group (pA, = 6.0) providing significant buffering power
`near the neutral pH usually found in the intracellular and intercellular flu-
`ids of most animals and bacteria. No other amino acid has an ionizable side
`chain with a pK, value near enough to pH 7.0 to be an effective physiologi-
`cal buffer (Table 5-1).
`
`Peptides and Proteins
`We nowturn to polymers of amino acids, the peptides and proteins. Bio-
`logically occurring peptides range in size from small to verylarge, consist-
`ing of two or three to thousands of linked amino acid residues. The focus
`here 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
`a-carboxyl group of one amino acid and the a-amino group of another
`(Fig. 5-13). Peptide bond formation is an example of a condensation reac-
`tion, a commonclass of reactionin living cells. Under standard biochemical
`conditions the reaction shown in Figure 5-13 has an equilibrium that favors
`reactants rather than products. To make the reaction thermodynamically
`more favorable, the carboxyl group must be chemically modified or acti-
`vated so that the hydroxyl group can be more readily eliminated. A chemi-
`cal 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 forma tripep-
`tide; similarly, amino acids can be linked to form tetrapeptides and pen-
`tapeptides. When a few aminoacids 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.
`Figure 5-14 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 a hydroxyl moiety
`from its carboxyl group). In a peptide, the amino acid residue at the end
`with a free w-amino groupis the amino-terminal (or N-terminal) residue;
`the residue at the other end, which has a free carboxyl group, is the car-
`boxyl-terminal (C-terminal) residue.
`Although hydrolysis of a peptide bond is an exergonic reaction, it oc-
`curs slowly because of its high activation energy. As a result, the peptide
`bondsin proteins are quite stable, with a half-life (t,.) of about 7 years un-
`der most intracellular conditions.
`
`as
`a
`H,N—CH—C—OH + H-N—CH—COO"
`
`|0
`
`wo Axo
`yg
`HyN—CH-+G—N--CH—COO”
`
`figure 5-13
`Formation of a peptide bond by condensation. The
`a-amino group of one amino acid (with R® group) acts as
`a nucleophile (see Table 3-4) to displace the hydroxy!
`group of another amino acid (with R? group), forming a
`peptide bond (shadedin gray). 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.
`
`OH
`
`CH, CH,
`‘CH
`Se”
`H CH, H CH,
`a be be
`nieee
`
`H O
`
`H
`
`Oo
`
`Amino-
`terminal end
`
`Carboxyl-
`terminal end
`
`figure 5-14
`The pentapeptide seryiglycyltyrosylalanylleucine, or
`Ser—Gly—Tyr-Ala—Leu. Peptides are named beginning
`with the amino-terminal residue, which by convention
`is placed at the left. The peptide bonds are shaded in
`gray, the R groupsare in red.
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`

`Chapter 5 Amino Acids, Peptides, and Proteins
`
`127
`
`Peptides Can Be Distinguished by Their lonization Behavior
`Peptides contain only one free a-amino group and one free a-carboxyl
`group, one at each endof the chain (Fig. 5-15). These groupsionize as they
`do in free amino acids, although the ionization constants are different be-
`cause the oppositely charged group is absent from the a carbon. The a-
`amino and a-carboxyl groups of all nonterminal amino acids are covalently
`joined in the form of 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 5-1), and in a peptide these
`contribute to the overall acid-base properties of the molecule (Fig. 5-15).
`Thus the acid-base behavior of a peptide can be predicted from its free a-
`amino and a-carboxyl groups as well as the nature and numberofits ioniz-
`able 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 px, value for an ionizable R group can
`change somewhat when an amino acid becomesa residuein a peptide. The
`loss of charge in the e-carboxyl and a-amino groups, interactions with other
`peptide R groups, and other environmental factors can affect the pX,. The
`pK, values for R groupslisted in Table 5-1 can be a useful guide to the pH
`range in which a given groupwill ionize, but they cannotbe strictly applied
`to peptides.
`Biologically Active Peptides and Polypeptides
`Occur in a Vast Range of Sizes
`No generalizations can be made about the molecular weightsof biologically
`active peptides and proteins in relation to their function. Naturally occur-
`ring peptides range in length from two amino acids to many thousands of
`residues. Even the smallest peptides can have biologically important ef-
`fects. Consider the commercially synthesized dipeptide L-aspartyl-L-phenyl-
`alanine methyl ester, the artificial sweetener better known as aspartame or
`NutraSweet.
`Manysmall peptides exert their effects at very low concentrations. For
`example, a number of vertebrate hormones (Chapter 23) are small pep-
`tides. 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 aminoacid residues and the other 21. Glucagon, another pancre-
`atic hormone,has 29 residues; it opposes the action of insulin. Corticotropin
`is a 39-residue hormoneof the anterior pituitary gland thatstimulates the
`adrenal cortex.
`How long are the polypeptide chains in proteins? As Table 5-2 shows,
`lengths vary considerably. Human cytochromec has 104 aminoacid residues
`linked in a single chain; bovine chymotrypsinogen has 245 residues. At the
`extremeis 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 polypeptides are much smaller than this,
`containing less than 2,000 amino acid residues.
`Someproteins consist of a single polypeptide chain, but others, called
`multisubunit proteins, have two or more polypeptides associated
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`figure 5-15
`Alanylglutamylglycyllysine. This tetrapeptide has one free
`a-amino group, one free a-carboxyl group, and two joniz-
`able R groups. The groups ionized at pH 7.0 are in red.
`
`CcoO™
`on
`H,N—CH—C—N—CH—C—OCH,
`H
`
`L-Aspartyl-L-phenylalanine methyl ester
`(aspartame)
`
`‘ileal
`O=C
`
`|N
`
`H
`
`CH—CH,—CH,—COO
`O=C
`
`O=C
`
`NH
`
`|e
`
`e—CH, —CH,—CH,—NHg
`coo
`
`Glu
`
`Gly
`
`Lys
`
`

`

`128
`
`Part 11 Structure and Catalysis
`
`table Sagres colfccnal
`| Molecular Data on SomeProteins
`
`Numberof i
`Molecular
`Numberof
`polypeptide ©
`
`weight
`residues
`chains
`|
`13,000
`104
`i
`|
`13,700
`124
`|
`|
`13,930
`129
`1
`16,890
`153
`1
`21,600
`241
`3
`22,000
`245
`1
`64,500
`574
`4
`68,500
`609
`1
`102,000
`972
`2
`450,000
`4,158
`5
`513,000
`4,536
`1
`619,000
`5,628
`12
`1
`2,993,000
`26,926
`
`i
`
`|
`t
`
`_ Cytochrome c (human)
`_ Ribonuclease A (bovine pancreas)
`| Lysozyme (egg white)
`| Myoglobin (equine heart)
`| Chymotrypsin (bovine pancreas)
`Chymotrypsinogen (bovine)
`_ Hemoglobin (human)
`| Serum albumin (human)
`_ Hexokinase (yeast)
`| RNA polymerase (E. coli)
`| Apolipoprotein B (human)
`| Glutamine synthetase (E. coli)
`| Titin (human)
`
`noncovalently (Table 5-2). The individual polypeptide chains in a multi-
`subunit protein may be identical or different. If at least two are identical the
`protein is said to be oligomeric, and 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 6 chains, all four held together by noncovalent interactions. Each
`e subunit is paired in an identical way with a 8 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 a6 protomers.
`A few proteins contain two or more polypeptide chains linked cova-
`lently. For example, the two polypeptide chains of insulin are linked by
`disulfide bonds. In such cases, the individual polypeptides are not consid-
`ered 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 group by dividing its molecu-
`lar weight by 110. Although the average molecular weight of the 20 stan-
`dard aminoacids is about 138, the smaller amino acids predominate in most
`proteins; if we take into accountthe proportions in which the various amino
`acids occur in proteins (Table 5-1), the average molecular weight is nearer
`to 128. Because a molecule of water (M, 18) is removed to create each pep-
`tide bond, the average molecular weight of an amino acid residue in a pro-
`tein is about 128 — 18 = 110.
`
`Polypeptides Have Characteristic Amino Acid Compositions
`Hydrolysis of peptides or proteins with acid yields a mixture of free a-amino
`acids. When completely hydrolyzed, each type of protein yields a charac-
`teristic proportion or mixture of the different amino acids. The 20 standard
`amino acids almost never occur in equal amounts in a protein. Some amino
`acids may occur only once per molecule or notatall in a given type of pro-
`tein; others may occur in large numbers. Table 5-3 shows the composition
`of the amino acid mixtures obtained on complete hydrolysis of bovine cy-
`tochrome 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
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`they contain.
`
`22
`4
`15
`8
`10
`10
`5
`23
`2
`10
`19
`14
`
`2 6 9
`
`28
`23
`
`8 4
`
`23
`245
`
`*Note that standard procedures for the acid hydrolysis of
`proteins convert Asn and Gin to Asp and Glu, respectively.
`In addition, Trp is destroyed. Special procedures must be
`employed to determine the amounts of these aminoacids.
`
`table 5-3
`
`Amino Acid Composition of Two Proteins*
`Numberof residues
`per molecule of protein
`Bovine
`Bovine
`cytochrome c
`chymotrypsinogen
`
`Amino
`acid
`
`Ala
`
`Arg
`Asn
`
`Asp
`Cys
`Gln
`Glu
`
`Gly
`His
`lle
`Leu
`
`Lys
`Met
`Phe
`Pro
`Ser
`Thr
`
`Trp
`Tyr
`Val
`Total
`
`aOPRrPOOrARANAAAWHKEWOWNWOLD
`
`1
`
`1
`
`104
`
`

`

`Chapter 5 Amino Acids, Peptides, and Proteins
`
`129
`
`Some Proteins Contain Chemical Groups Other Than Amino Acids
`Manyproteins, for example the enzymes ribonuclease and chymotrypsino-
`gen, contain only amino acid residues and no other chemical groups; these
`are considered simple proteins. However, some proteins contain perma-
`nently associated chemical components in addition to amino acids; these
`are called conjugated proteins. The non—aminoacid 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 5-4); for example, lipoproteins contain lipids, glycoproteins con-
`tain sugar groups, and metalloproteins contain a specific metal. A num-
`ber of proteins contain more than one prosthetic group. Usually the pros-
`thetic group plays an importantrole in the protein’s biological function.
`
`Conjugated Proteins
`Class
`
`Prosthetic group(s)
`
`Example
`
`Lipoproteins
`Glycoproteins
`Phosphoproteins
`Hemoproteins
`Flavoproteins
`Metalloproteins
`
`Lipids
`Carbohydrates
`Phosphate groups
`Heme (iron porphyrin)
`Flavin nucleotides
`lron
`Zinc
`Calcium
`
`Molybdenum
`Copper
`
`8,-Lipoprotein of blood
`Immunoglobulin G
`Casein of milk
`
`Hemoglobin
`Succinate dehydrogenase
`Ferritin
`
`Alcohol dehydrogenase
`Calmodulin
`
`Dinitrogenase
`Plastocyanin
`
`There Are Several Levels of Protein Structure
`For large macromolecules such as proteins, the tasks of describing and un-
`derstanding structure are approached at several
`levels of complexity,
`arranged in a kind of conceptual hierarchy. Four levels of protein structure
`are commonly defined (Fig. 5-16). 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
`structurerefers 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. Whena pro-
`tein has two or more polypeptide subunits, their arrangementin space is re-
`ferred to as quaternary structure.
`
`figure 5-16
`Levels of structure in proteins. The primary structure
`consists of a sequence of aminoacidslinked together by
`peptide bondsand includes any disulfide bonds. The
`resulting polypeptide can be coiled into units of sec-
`ondary structure, such as an @ helix. The helix is a part
`of the tertiary structure of the folded polypeptide, whichis
`itself one of the subunits that make up the quaternary
`structure of the multisubunit protein, in this case hemo-
`globin.
`
`Primary
`structure
`
`Secondary
`structure
`
`eS nl ne
`
`Tertiary
`structure
`
`Quaternary
`structure
`
`
`
`
`
`Polypeptide chain
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`Assembled subunits
`
`=
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`

`130
`
`Part II Structure and Catalysis
`
`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 any detail it
`must be separated from all other proteins, and techniques must be available
`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 of a protein is essential before its properties, armino acid
`composition, and sequence can be determined. Given that cells contain
`thousands ofdifferent 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. For example, many proteins bind to other biomol-
`ecules with great specificity, and such proteins can be separated on the ba-
`sis of their binding properties.
`The sourceof a protein is generally tissue or microbial cells. The first
`step in any protein purification procedure is to break open thesecells, re-
`leasing 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. 2-20).
`Once the extract or organelle preparation is ready, various methodsare
`available for purifying one or moreof the proteins it contains. Commonly,
`the extract is subjected to treatments that separate the proteins into dif-
`ferent fractions based on some property such as size or charge, a process
`referred to as fractionation. Early fractionation steps in a purification uti-
`lize differences in protein solubility, which is a complex function of pH, tem-
`perature, 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 amounts can selectively precipitate some
`proteins, while others remain in solution. Ammonium sulfate ((NH4)250,)
`is often used for this purpose becauseofits high solubility in water.
`A solution containing the protein of interest often must be further al-
`tered before subsequent purification steps are possible. For example, dial-
`ysis is a procedure that separates proteins from solvents by taking advan-
`tage 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 larger volume ofbuffered solution of appropriate ionic strength, the
`membrane allows the exchangeof salt and buffer but not proteins. Thus
`dialysis retains large proteins within the membranous bag or tube whileal-
`lowing the concentration of other solutes in the protein preparation to
`change until they come into equilibrium with the solution outside the mem-
`brane. Dialysis might be used, for example, to remove ammonium sulfate
`from the protein preparation.
`The most powerful methodsfor fractionating proteins make useof col-
`umn chromatography, which takes advantage of differences in protein
`charge, size, binding affinity, and other properties (Fig. 5-17). A porous
`solid material with appropriate chemical properties (the stationary phase)
`is held in a column, and a buffered solution (the mobile phase) percolates
`through it. The protein-containing solution is layered on the top of the col-
`umn, then also percolates through the solid matrix as an ever-expanding
`band within the larger mobile phase (Fig. 5-17b). Individual proteins mi-
`grate faster or more slowly through the column depending on their proper-
`ties. For example, in cation-exchange chromatography (Fig. 5-18a), the
`solid matrix has negatively charged groups. In the mobile phase, proteins
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`

`Chapter 5 Amino Acids, Peptides, and Proteins
`
`131
`
`Ny PY
`a
`
`4
`
`figure 5-17
`Column chromatography. (a) The standard elements of a
`chromatographic column. A solid, porous material is sup-
`ported inside a column generally made of some form of
`plastic. The solid material (matrix) makes up the sta-
`tionary phase through whichflows a solution, 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. (b) The protein solu-
`tion to be separated is layered on top of the column and
`allowed to percolate into the solid matrix. Additional solu-
`tion is added on top. The protein solution forms a band
`within the mobile phasethatis initially the depth of the
`protein solution applied to the column. As proteins
`migrate through the column, they are retarded to different
`degreesby their different interactions with the matrix
`material. The overall protein band thus widensasit
`moves through the column. Individual types of proteins
`(such as A, B, and C, shownin blue, red, and green)
`gradually separate from each other, forming bands within
`the broader protein band. Separation improves (resolu-
`tion increases) as the length of the column increases.
`However, eachindividual protein band also broadens
`with time due to diffusional spreading, a process that
`decreasesresolution. In this example, protein A is well
`separated from B and C, but diffusional spreading pre-
`vents complete separation of B and C under these
`conditions.
`
`Proteins
`
`
`(a)
`
`[
`
`.
`Reservoir
`
`Solution
`(mobile
`phase)
`
`
`Solid
`porous
`
`matrix
`(stationary
`
`phase)
`
`Porous
`
`support
`
`— Effluent
`
`é6
`
`with a net positive charge migrate through the matrix more, slowly than
`those with a net negative charge, because the migration of the formeris re-
`tarded moreby interaction with the stationary phase. The two types of pro-
`tein can separate into two distinct bands. The expansion of the protein
`band in the mobile phase (the protein solution) is caused both by separa-
`tion 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 length. As the length of time spent on the column increases, the res-
`olution can decline as a result of diffusional spreading within each protein
`band.
`Figure 5-18 shows two other variations of column chromatographyin
`addition to ion exchange.
`A modern refinement in chromatographic methods is HPLC,or high-
`performance liquid chromatography. H