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The extra functional groups are shownin red. Desmosine is formed from four residues oflysine, whose carbon backbones are shaded in gray.Selenocysteine is derived from serine. (b) Ornithineand citrulline are intermediates in the biosynthesisof arginine and in the urea cycle. Note that twosystems are used to number carbons in the namingof these amino acids.

The a, /3, y system used fory-carboxyglutamate begins at the a carbon (see Fig.5-2) and extends into the R group. The a-carboxylgroup is not included. In contrast, the numberingsystem used to identify the modified carbon in4-hydroxyproline, 5-hydroxylysine, and 6-iV-methyllysine includes the a-carboxyl carbon, which is designated carbon 1 (or C-l).H+R-C—COOHSubstances having this dual nature are amphoteric and are oftencalled ampholytes, from "amphoteric electrolytes." A simplemonoamino monocarboxylic a-amino acid, such as alanine, is actuallya diprotic acid when it is fully protonated, that is, when both its carboxyl group and amino group have accepted protons. In this form it hastwo groups that can ionize to yield protons, as indicated in the following equation:HIR-C-COOHIPTHIR-C-COO"+INH,H-HR-C-COO~NH2Amino Acids Have Characteristic Titration CurvesTitration involves the gradual addition or removal of protons.

Figure5-9 shows the titration curve of the diprotic form of glycine. Eachmolecule of added base results in the net removal of one proton fromChapter 5 Amino Acids and Peptidesone molecule of amino acid. The plot has two distinct stages, each corresponding to the removal of one proton from glycine. Each of the twostages resembles in shape the titration curve of a monoprotic acid,such as acetic acid (see Fig. 4-10), and can be analyzed in the sameway.

At very low pH, the predominant ionic species of glycine is+H3N—CH2—COOH, the fully protonated form. At the midpoint inthe first stage of the titration, in which the —COOH group of glycineloses its proton, equimolar concentrations of proton-donor(+H3N—CH2—COOH) and proton-acceptor (+H3N—CH2—COO") species are present. At the midpoint of a titration (see Fig. 4-11), the pH isequal to the pKa of the protonated group being titrated.

For glycine, thepH at the midpoint is 2.34, thus its —COOH group has a pKa of 2.34.[Recall that pH and pKa are simply convenient notations for protonconcentration and the equilibrium constant for ionization, respectively(Chapter 4). The pKa is a measure of the tendency of a group to give upa proton, with that tendency decreasing tenfold as the pKa increases byone unit.] As the titration proceeds, another important point is reachedat pH 5.97.

Here there is a point of inflection, at which removal of thefirst proton is essentially complete, and removal of the second has justbegun. At this pH the glycine is present largely as the dipolar ion+H3N—CH2—COO~. We shall return to the significance of this inflection point in the titration curve shortly.The second stage of the titration corresponds to the removal of aproton from the — NH 3 group of glycine. The pH at the midpoint of thisstage is 9.60, equal to the pKa for the —NH3 group. The titration iscomplete at a pH of about 12, at which point the predominant form ofglycine is H2N—CH2—COO-.From the titration curve of glycine we can derive several importantpieces of information. First, it gives a quantitative measure of the pKaof each of the two ionizing groups, 2.34 for the —COOH group and 9.60for the —NH3 group.

Note that the carboxyl group of glycine is over100 times more acidic (more easily ionized) than the carboxyl group ofacetic acid, which has a pKa of 4.76. This effect is caused by the nearbypositively charged amino group on the a-carbon atom, as described inFigure 5-10.The second piece of information given by the titration curve of glycine (Fig. 5-9) is that this amino acid has two regions of bufferingpower (see Fig. 4—12).

One of these is the relatively flat portion of thecurve centered about the first pKa of 2.34, indicating that glycine is agood buffer near this pH. The other buffering zone extends for -1.2 pHunits centered around pH 9.60. Note also that glycine is not a goodbuffer at the pH of intracellular fluid or blood, about 7.4.The Henderson-Hasselbalch equation (Chapter 4) can be used tocalculate the proportions of proton-donor and proton-acceptor speciesof glycine required to make a buffer at a given pH within the bufferingranges of glycine; it also makes it possible to solve other kinds of bufferproblems involving amino acids (see Box 4-2).+NH 3a-Amino acid (glycine)+NH 3H-C-COOH ^ = ^ H-C-COO" + HHNH3NH2NH3CH2CH2COOHCOO"pK2CH2COO"pH0.511.5OH~ (equivalents)Figure 5-9 The titration curve of 0.1 M glycineat 25 °C.

The ionic species predominating at keypoints in the titration are shown above the graph.The shaded boxes, centered about pifi = 2.34 andpK2 = 9.60, indicate the regions of greatest buffering power.Figure 5-10 (a) Interactions between the a-aminoand a-carboxyl groups in an a-amino acid. Thenearby positive charge of the - N H 3 + group makesionization of the carboxyl group more likely (i.e.,lowers the pKa for —COOH).

This is due to a stabilizing interaction between opposite charges on thezwitterion and a repulsive interaction between thepositive charges of the amino group and the departing proton, (b) The normal pKa for a carboxylgroup is approximately 4.76, as for acetic acid.Acetic acid+= 2.34CH3—COOH ^ = ± CH3—COO"1iH(a)119(b)pKa = 4.76ind CatalysisThe Titration Curve Predicts theElectric Charge of Amino AcidsAnother important piece of information derived from the titrationcurve of an amino acid is the relationship between its net electriccharge and the pH of the solution. At pH 5.97, the point of inflectionbetween the two stages in its titration curve, glycine is present as itsdipolar form, fully ionized but with no net electric charge (Fig.

5-9).This characteristic pH is called the isoelectric point or isoelectricpH, designated pi or pH r . For an amino acid such as glycine, which hasno ionizable group in the side chain, the isoelectric point is the arithmetic mean of the two pKa values:pi = | ( p ^ + VK2)which in the case of glycine ispi = 7^(2.34 + 9.60) = 5.97As is evident in Figure 5-9, glycine has a net negative charge at anypH above its pi and will thus move toward the positive electrode (theanode) when placed in an electric field.

At any pH below its pi, glycinehas 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 entirely as theform +H3N—CH2—COOH, with a net positive charge of 1.0. At pH2.34, where there is an equal mixture of +H3N—CH2—COOH and+H3N—CH2—COO~, the average or net positive charge is 0.5. Thesign and the magnitude of the net charge of any amino acid at any pHcan be predicted in the same way.This information has practical importance. For a solution containing a mixture of amino acids, the different amino acids can be separated on the basis of the direction and relative rate of their migrationwhen placed in an electric field at a known pH.Amino Acids Differ in Their Acid-Base PropertiesThe shared properties of many amino acids permit some simplifyinggeneralizations about the acid-base behavior of different classes ofamino acids.All amino acids with a single a-amino group, a single a-carboxylgroup, and an R group that does not ionize have titration curves resembling that of glycine (Fig.

5-9). This group of amino acids is characterized by having very similar, although not identical, values for pKx (thepK of the —COOH group) in the range of 1.8 to 2.4 and for pK2 (of the—NH^ group) in the range of 8.8 to 11.0 (Table 5-1).Amino acids with an ionizable R group (Table 5-1) have more complex titration curves with three stages corresponding to the three possible ionization steps; thus they have three pKa values.

The third stagefor the titration of the ionizable R group merges to some extent withthe others. The titration curves of two representatives of this group,glutamate and histidine, are shown in Figure 5-11. The isoelectricpoints of amino acids in this class reflect the type of ionizing R groupspresent. For example, glutamate has a pi of 3.22, considerably lowerthan that of glycine. This is a result of the presence of two carboxylCOOHCOOH 3 N-CHFigure 5-11 The titration curves of (a) glutamateand (b) histidine. The pKa of the R group is designated pKR.pH3.01.02.0OH" (equivalents)(a)COO"COOHH3N-CHCOO"H3N—CHH,N—CHHNC^CHNH-N"HCH2H 2 N-CHHNCH11 -;CHCHCOO"NH,CH<(** HCN\ CH-NHPK2H10 -piiT2 = 9.17^ y ^8 -pKR = 6.0pH6yS4 _f!21111.02.0OH" (equivalents)(b)13.0122Part II Structure and Catalysisgroups which, at the average of their pKa values (3.22), contribute anet negative charge of - 1 that balances the +1 contributed by theamino group.

Similarly, the pi of histidine, with two groups that arepositively charged when protonated, is 7.59 (the average of the ipKavalues of the amino and imidazole groups), much higher than that ofglycine.Another important generalization can be made about the acidbase behavior of the 20 standard amino acids. 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 powernear the neutral pH usually found in the intracellular and intercellular fluids of most animals and bacteria. All the other amino acids havepKa values too far away from pH 7 to be effective physiological buffers(Table 5-1), although in the interior of proteins the pKa values ofamino acid side chains are often altered.Ion-Exchange Chromatography SeparatesAmino Acids by Electric ChargeIon-exchange chromatography is the most widely used method for separating, identifying, and quantifying the amounts of each amino acidin a mixture.

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