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Amino Acids
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Chapter Outline
v Amino acids: Tetrahedral alpha () carbon with H, amino group, carboxyl group, and side chain v Amino acids in proteins joined by peptide bonds v Classification Nonpolar: Ala, val, leu, ile, pro, met, phe, trp Polar uncharged: Gly, ser, thr, tyr, asn, gln , cys Acidic: Asp, glu Basic: His, lys, arg v Modified amino acids: Hydroxylysine, hydroxproline, thyroxin, methyl histidine, methyl arginine, methyl lysine, carboxyglutamic acid, pyroglutamic acid, aminoadipic acid v Small molecules derived from amino acids: GABA, histamine, serotonin, alanine, epinephrine, penicillamine, ornithine, citrulline, betaine, homocysteine, homoserine v Ionic properties of amino acids Carboxyl group: pKas range from 2.0 to 2.4 Amino group: pK as range from 9.0 to 9.8 Side chains - Acidic pKas for asp, glu - Basic pKas for his, arg, lys v Amino acid chemistry Carboxyl chemistry Amino chemistry Side chain chemistry v Chirality: asymmetric carbon has four different groups attached Optically active compounds - Dextrorotatory (+), clockwise rotation - Levorotatory (-), counterclockwise rotation Asymmetric carbons - One asymmetric carbon: Mirror image isomers or enantiomers - More than one asymmetric carbon: enantiomers and diastereomers D,L System: Typically naturally occurring amino acids: L-amino acids; related to Lglyceraldehyde (R,S) System v Ultraviolet spectral properties of amino acids: Only phe, try, trp absorb UV light above 250 nm v Separation of amino acids Ion exchange - Anion exchangers: Matrix is positively charged so anions bind - Cation exchangers: Matrix is negatively charged so cations bind
Chapter Objectives
It is imperative to learn the structures and one letter codes for the amino acids. Amino acids are key molecules, both as components of proteins and as intermediates to important biomolecules. The structures are shown in Figure 4.3 of Biochemistry. Figure 4.3 is presented on the following two pages. Classification of Amino Acids Here is an alternative classification scheme. Acidic amino acids and their amides: aspartic acid, asparagine, glutamic acid, glutamine. Basic amino acids: histidine, lysine, arginine. Aromatic amino acids: phenylalanine, tyrosine, tryptophan. Sulfur containing amino acids: cysteine, methionine. Imido acid: proline. Hydrophobic side chains: glycine, alanine, valine, leucine, isoleucine. Hydroxylic amino acids: serine, threonine, (tyrosine). To memorize the structures of the 20 amino acids, here are some aids: The side chain of glycine is H, that of alanine is a methyl group, CH3. The side chain of valine is just the methyl side chain of alanine with two methyl groups substituting for two Hs. (In effect it is just dimethylalanine.) The side chain of leucine is just valine with a -CH2- group between the valine side chain and the carbon (or, leucine is alanine with the valine side chain attached). Isoleucine is an isomer of leucine with one of the methyl groups exchanged with a H on the carbon attached to the carbon. Several of the amino acids have alanine as a base. Phenylalanine has a phenyl group (i.e., benzene ring) attached to alanine. Tyrosine is hydroxylated phenylalanine. Serine has a hydroxyl group substituting for a H on the methyl group of alanine. Cysteine can be thought of as the sulfur variety of serine i.e., with a sulfhydryl group in place of the hydroxyl group of serine. Threonine, a hydroxylic amino acid, can be thought of as methylated serine. The acidic amino acids and their amides are quite easy to remember. Aspartic acid and glutamic acid have carboxylic acid groups attached to -CH2- and (-CH2-)2, respectively. The amides of these amino acids, namely asparagine and glutamine, have ammonia attached to the side chain carboxyl groups in amide linkage. The basic amino acids all have six carbons, counting the carboxyl carbon and the carbon. Lysine's side chain is a linear chain of four -CH2- groups with a terminal amino group. Arginine has a linear side chain of three -CH2- groups to which a guanidinium group is attached. (The sixth carbon is the central C of the guanidinium group.) Guanidinium is like urea but with a nitrogen group replacing the carbonyl oxygen. Histidine has an imidazole ring attached to alanine. The ring contains two nitrogens and three carbons, a five-membered ring. An easy way to remember the structure of proline is to know that it derives from glutamic acid. The carboxyl side chain forms a C-N bond to the amino group to form this imido acid. (In the process, the carbon is reduced so the side chain carboxyl oxygens do not show up in proline.)
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Figure 4.3 The amino acids that are the building blocks of most proteins can be classified as (a) nonpolar (hydrophobic), (b) polar, neutral, (c) acidic, or (d) basic. Also shown are the one-letter and three-letter codes used to denote amino acids. For each amino acid, the ball-and-stick (left) and space-filling (right) models show only the side chain.
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Aspartate COO+H 3N
Leucine COO+
Isoleucine COO+
Methionine COO+
Threonine COO+
C H H
C H CH2 COO-
H3 N C H CH2 H3 C CH CH3
H3 N C H H3 C C H CH2 CH3
H3N
H3 N C H H C OH CH3
2. Without reference to the text, give the one-letter and three-letter abbreviations for asparagine, arginine, cysteine, lysine, proline, tyrosine, and tryptophan. Answer: For many of the amino acids the l letter code is the first letter of the amino acid. Problems arise when two amino acids start with the same letter. Whenever this occurs, the amino acid with the lowest molecular weight is assigned the letter. The other amino acid is given a phonetic letter or the closest unused letter in the alphabet. The amino acids assigned their first letters are: Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Threonine, Proline, Cysteine, Methionine and Histidine. Aspartic acid is D, an easy way to remember it is to pronounce it as "asparDic" acid. Glutamic acid is E, E follows D, glutamic acid follows aspartic acid in chemical structure. E is also the closest unused letter to G. F is for phenylalanine, (Fenylalanine). R is for arginine, remembered by pronouncing arginine (Rginine). N is for asparagine; remember to stress the N sound. Glutamine follows asparagine, its l letter code is the next unused consonant after N, namely Q. Lysine is K, the closest unused letter to L. Tyrosine is Y; Y is the dominant sound in tyrosine. Tryptophan is W; remember "double ring, double you". 3. Write equations for the ionic dissociations of alanine, glutamate, histidine, lysine, and phenylalanine. Answer: Alanine dissociation: COOH
+
COO+
COONH 2 C H CH3
H3N
C H CH 3
H3 N C H CH3
Glutamate Dissociation:
COOH
+H N 3
COO+H N 3
COO+H N 3
C H CH 2 CH 2 COOH
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COO+
COO+
COOH2N C H CH2 N
H3 N C H CH2 +HN
H3 N C H CH2 N NH COO-
+H N 3
+H N 3
+H
3N
COO+H3 N C H CH2
4. How is the pK a of the a-NH3+ group affected by the presence on an amino acid of the a-COOH? Answer: The pKa on an isolated amino group is around 10 (as for example, the side chain amino group of lysine whose pKa = 10.5 (see Table 4.1). In general, the pKas of amino groups in the amino acids are around 9.5. Thus, the proximity of the carboxyl group lowers the pK a of the amino group. 5. Draw an appropriate titration curve for aspartic acid, labeling the axis and indicating the equivalence points and the pK a values. Answer: For a titration curve the independent variable is the amount of acid or base used to titrate the amino acid. The dependent variable is pH.
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The equivalence point is a point on the titration curve reached after a molar equivalent of acid or base is added to the amino acid. For aspartic acid there are actually three equivalence points at around 3.0, 6.2 and 12.2. 6. Calculate the concentrations of all ionic species in a 0.25 M solution of histidine at pH 2, pH 6.4, and pH 9.3. Answer: The pKas for histidine are 1.8, 6.0, and 9.2. At pH 2.0, we can ignore the two higher pKas. At this pH, histidine's side chain and the -amino group are protonated and thus both are positively charged. So, the ionic species of histidine at pH = 2.0 depend critically on the ionization state of the carboxyl group. When the carboxyl group is in the carboxylate form, histidine will be in the 1+ ionic form. When the carboxyl group is protonated, and hence uncharged, histidine will carry a 2+ charge. For the -carboxyl group:
pH = pK a + log [COO ] or, [ COOH]
[COO ] = 10 pH pK a = 102 .0 1.8 = 1.58 or [COO ] = 1.58 [COOH] and, [COOH] [COO ] + [COOH] = 0.25M Thus, 1.58 [COOH] + [COOH] = 2.58[ COOH] = 0.25M and, .25M [COOH] = = 0.097M and, 2.58 [COO ] = 1.58 [COOH] = 0.153M So, at pH = 2.0, [His 2+ ] = 0.097M and [His1+ ] = 0.153M
The concentration of uncharged histidine and negatively charged histidine are both small but can be estimated as follows. The concentration of uncharged histidine will be approximately equal to the concentration of the unprotonated side-chain species. Using the HendersonHasselbalch equation with pKa = 6.0, pH = 2.0, and [His 1+] = 0.153 M, we find that
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or,
pH 6.0
The concentration of negatively charged histidine is calculated using the Henderson-Hasselbalch equation with pK a = 9.2, pH = 2.0, and [His 0] = 1.53 x 10-5 M. In this case
pH = 9.2 + log [His1-] [His0 ] [His1- ] [His0 ] or,
At pH = 2.0 we have:
At pH = 6.4, we can assume that the carboxyl group is fully unprotonated and carries a -1 charge while the -amino group is fully protonated and carries a +1 charge. Thus, the ionic species of histidine depends critically on the ionic state of the imidazole side chain. When the imidazole group is protonated, histidine carries a net +1 charge. When it is unprotonated, histidine is uncharged. So, using [His 0 ] pH = 6.0 + log and [His 0 ] + [His1 + ] = 0.25M , [His1 + ]
we find that the ionic species of histidine at pH = 6.4 are : [His 0 ] = 0.179M and [His1+ ] = 0.071M [His2+] and [His1-] are calculated using the Henderson-Hasselbalch equation with pH = 6.4, pKa = 1.8 and 9.2 and [His 1+] = 0.071 M and [His0] = 0.179 M. We find [His2+] = 2.8 x 10 -6 M and [His1-] = 2.25 x 10-4 M.
At pH = 6.4 we have:
At pH = 9.3, the carboxyl group is unprotonated and negatively charged, the imidazole group is unprotonated and uncharged, and only the protonation state of the -amino group needs to be determined. Histidine, at pH = 9.3 will be portioned between the His0 and His 1- states. From
pH = 9.2 + log [His1- ]
0
[His ] we find that the ionic species of histidine at pH = 9.3 are : [His0 ] = 0.111M, [His 1- ] = 0.139M and [His2 + ] = 1.75 10-12 M, [His + ] = 5.5 10-5 M
7. Calculate the pH at which the carboxyl group of glutamic acid is two-thirds dissociated. Answer: The pKa of carboxyl group of glutamic acid is 4.3.
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When the - carboxyl group is two - thirds dissociated : [C OO ] [C OO ] + [C OOH] Thus, [C OOH] = 1
[C OO ] 2 Upon subs tituting into the Henderson - Hasselbalch equation we find : pH = 4.3 + log pH = 4.6 [ C OO ] [C OOH] = 4.3 + log [C OO ] 1 2 [ C OO ] = 4.3 + log 2
8. Calculate the pH at which the -amino group of lysine is 20% dissociated. Answer: For the lysine side chain, pK a = 10.5. Using the Henderson - Hasselbalch equation [ N H2 ] pH = pK N H + + log 3 + [ N H3 ] When the - amino group is 20% d issociated:
[N H2 ] + [ N H+ ] 3 Thus, [ N H2 ] = [ N H2 ]
+ = .2 (20%) or, [N H2 ] = .2 [ N H2 ] + [ N H3 ]
1 + [ N H3 ] 4 Upon subs tituting into the Henderson - Hasselbalch equation we find : 1 [ N H+ ] 3 [ N H2 ] 4 pH = 10.5 + log = 10 .5 + log = 10.5 log 4 pH = 9.9
+ [ N H3 ] + [ N H3 ]
9. Calculate the pH of a 0.3 M solution of (a) leucine hydrochloride, (b) sodium leucinate, and (c) isoelectric leucine. Answer: The solution 0.3 M leucine HCl is composed of 0.3 M leucine and 0.3 M HCl. Thus, we expect it to have an acidic pH and only the carboxyl group of leucine is involved in proton equilibrium. The pKa of the carboxyl of leucine is 2.4. Thus,
pH = pK a + log [ A] [ HA] = 2.4 + log [ COO ] [ COOH] (1) and,
[COO ] + [ COOH ] = 0.3M (2) For charge neutrality : [Cl - ] + [COO - ] = [H + ] + [NH + ] 3
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pH = 2.4 + log
Recognizing that log[H+ ] = -pH we see that pH = 2.4 log 0.3 pH or pH = 2.4 log 0.3 = 1.46 2
Note: If we did not make the simplification that [H+]<0.3 we would have had to solve a quadratic and would have found that pH = 1.49. (b) sodium leucinate Here we assume the solution is basic; therefore, only the amino group is involved. mathematical solution is similar to that given for (a) with pKa = 9.6. [ NH 2 ] [ NH 2 ] pH = pK a + log = 9.6 + log (1) and, + + [ NH 3 ] [ NH 3 ]
+ [ NH 2 ] +[ NH 3 ] = 0.3M (2)
The
For charge neutrality : [OH - ] + [COO - ] = [NH+ ] + [Na + ] 3 If we recognize that [COO - ] = [Na + ] (because Na + and the amino acid are in equimolar amounts), the charge neutrality equation may be simplified to : [OH - ] = [NH+ ] and combined with (1) and (2) we have : 3 pH = 9.6 + log 0.3 -[OH - ] [OH- ] 0.3 [OH - ]
If we make the assu mption that [OH- ] << 0.3 pH = 9.6 + log = 9.6 + log 0.3 log[OH- ]
Recognizing that pOH log[OH - ] and that pH + pOH =14 we have pH = 9.6 + log 0.3 + pOH = 9.3 + log 0.3 + 14 - pH or pH = 9.6 +14 + log 0.3 = 11.5 2
(c) isoelectric leucine Isoelectric leucine is a leucine solution at a pH at which there is no net charge on the molecule. For this to occur the carboxyl group must be unprotonated to the same extent as the amino group is protonated. This must occur at a pH half way between the pKas of the two groups
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[ NH2 ] [NH + ] 3
and recog nizing that [COOH] = [Amino acid] total - [COO ] and that [NH 2 ] = [Amino acid] total - [NH + ] 3 upon substituting, solving for [ COO ] and [ NH + ], and setting these terms 3 equal at pH = pI we find that pI = pKCOOH + pKNH +
3
or
10. Quantitative measurements of optical activity are usually expressed in terms of the specific rotation, []D25, defined as Measured rotation in degrees 100 25 [ ]D = (Optical path in dm) (conc. in g/ml) For any measurement of optical rotation, the wavelength of the light used and the temperature must both be specified. In this case, D refers to the "D line" of sodium at 589 nm and 25 refers to a measurement temperature of 25C. Calculate the concentration of a solution of L-arginine that rotates the incident light by 0.35 in an optical path length of 1 dm (decimeter). Answer:
[ ] 25 = D [Arg] = Measured rotation in degrees 100 (Optical path in dm) (conc. in g/ml) 0.35 100 [ ] 25 1dm D 0.35100 = 2.8 g/ml 12.5 1dm
11. Absolute configurations of the amino acids are referenced to D- and Lglyceraldehyde on the basis of chemical transformations that can conv ert the molecule of interest to either of these reference isomeric structures. In such reactions, the stereochemical consequences for the asymmetric centers must be understood for each reaction step. Propose a sequence of reactions that would demonstrate that L(-)-serine is stereochemically related to L(-)-glyceraldehyde. Answer: The aldehyde group of L(-)-glyceraldehyde is converted to L-glyceric acid by oxidation and L-glyceric acid is converted to 2-hydroxypropanoic acid. 2-Hydroxypropanoic acid is converted to 2-bromopropanoic acid by nucleophilic substitution using concentrated HBr. The reaction results in alteration in stereochemistry. 2-Bromopropanoic acid is subsequently converted to L-(-)-alanine by ammonolysis and alteration in stereochemistry. Finally, L-(-)alanine is convert serine. The reaction sequence is shown below.
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acid
CHO HO C H HO
COOH C H
CH2OH
CH2OH
COOH H C Br H2 N
COOH C H H2N
COOH C H
CH3
L -(-)-Alanine
CH2OH
L -(-)-Serine
12. Describe the stereochemical aspects of the structure of cystine, the structure that is a disulfide-linked pair of cysteines. Answer: Cystine (dicysteine) has two chiral carbons, the two a-carbons of the cysteine moieties. Since each chiral center can exist in two forms there are 4 stereoisomers of cystine. However, it is not possible to distinguish the difference between L-cysteine/D-cysteine and D-cysteine/Lcysteine dimers. So three distinct isomers are formed. H H H H
-OOC C NH3+ CH 2 S S CH 2 +H3N C COOH L-cysteineL-cysteine +H3N +H3N C COOCH 2 S S CH 2 C COOH L-cysteineD-cysteine -OOC -OOC C NH3+ CH 2 S S CH 2 C NH3+ H D-cysteineL-cysteine -OOC +H3N C COOCH 2 S S CH 2 C NH3+ H D-cysteineD-cysteine
13. Draw a simple mechanism for the reaction of a cysteine sulfhydryl group with iodoacetamide. Answer:
O I CH2 C NH2 SCH2 +H3 N C COOH +H3N NH2
I-
14. Describe the expected elution pattern for a mixture of aspartate, histidine, isoleucine, valine, and arginine on a column of Dowex-50.
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COO - H C C NH3+
Since moving from highest to lowest priority involves counterclockwise movement the -carbon is in the S configuration.
For the -carbon, the priority is OH > C > CH 3 > H. The orientation of the C -C bond was already defined for the -carbon as being into the plane therefore it must be out of the plane for the -carbon, as must be the bond to -CH3. Thus, bonds to OH and H are into the plane. By rotating the molecule such that H is behind the -carbon, we find the OH to the left, CH3 below and C above:
C HO C
H CH3
Since moving from highest to lowest priority involves clockwise movement the -carbon is in the R configuration. The -carbon is in the R configuration.
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H C NH3 + +H3 N C H
CH3 CH3 CH3 CH3 L-Threonine D-Threonine L-Allothreonine D-A llothreonine By inspection we see that L-threonine is (2S, 3R) threonine D-threonine is (2R,3S) threonine L-allothreonine is (2S,3S) threonine D-allothreonine is (2R,3R) threonine
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8. DTNB (Ellman's reagent), iodoacetate, and N-ethylmaleimide are three agents that are used to modify which amino acid? What is the reactive group on the amino acid with which all three agents react? 9. Although there are twenty common amino acids, there are hundreds of different amino acids found in nature, some of which derive from the common amino acids by simple chemical (biochemical) modification. Name one common modification of amino acids found in nature. 10a. Of the twenty common amino acids which amino acid has a pKa around neutrality? b. What amino acid lacks an amino group?
Answers
1. peptide; amino acids; amino group; carboxyl group; water; N -terminal (or amino ); Cterminal (or carboxyl); polypeptide; protein; hydrolysis. 2. Nonpolar or hydrophobic; neutral polar; acidic; basic. 3. a./neutral polar; b./acidic; c./nonpolar; d./neutral polar, acidic, and basic; e./basic; f./neutral polar, acidic, and basic; g./basic; h./nonpolar; i./nonpolar; j./neutral polar. 4. a. glycine; b. tyrosine; c. methionine; d. cysteine; e. leucine. 5. Four; two; enantiomers (or mirror image isomers); mirror; L. 6. Phenylalanine, tyrosine, and tryptophan. 7. ion exchange, HPLC 8. cysteine; sulfhydryl group. 9. Hydroxylation, methylation, carboxylation, phosphorylation. 10. a./Histidine; b./proline.
Additional Problems
1. Sketch the titration curve of histidine. 2. In the protein hemoglobin, a number of salt bridges (ionic bonds) are broken in going from oxygenated to deoxygenated hemoglobin. One in particular involves an aspartic acid: histidine ionic interaction. (a). At pH = 7.0, would you expect this salt bridge to form? Explain. (b). Under certain conditions (low oxygen tension) the salt bridge does form. Explain how the influence of aspartic acid on histidine is similar to the influence of the carboxyl group on the pKa of the amino group (and vice versa). 3. How might the presence of calcium ions affect the apparent pKas of the side chains of aspartic acid and glutamic acid? 4a. The ultraviolet spectra of proteins are largely determined by what three amino acids? b. In working with nucleic acids it is often useful to measure the optical densities at 260 nm and 280 nm. Nucleic acids (DNA and RNA) absorb more strongly at 260 nm than 280 nm such that the ratio A 260 : A 280 ranges from 1.6 to 2.0. What might happen to the A 260 : A 280 ratio of a solution of nucleic acid contaminated with protein?
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Abbreviated Answers
1. Histidine is the only amino acid with a pKa around neutrality. The pKas of histidine are 1.8, 6.0, and 9.2. The titration curve will be similar to the one shown in the answer to problem 5 but with an additional plateau around pH = 6.0. 2. (a) The pKas of the side chains of aspartic acid and histidine are 3.9 and 6.0 respectively. At pH = 7.0, the side chain of aspartic acid is unprotonated and therefore negatively charged, whereas the side chain of histidine is also unprotonated and therefore uncharged. We might not expect this interaction to occur. (b) The fact that the salt bridge forms indicates that the two amino acid side chains are close together in the three dimensional structure of the protein. The proximity of the negatively charged aspartic acid must therefore raise the pK a of the histidine side group. 3. Calcium (Ca2+) may bind to carboxylate ions. Therefore, the presence of calcium may lower the apparent pK as of aspartic acid and glutamic acid. 4. a./Phe, Tyr, and Trp.; b./ A260 : A 280 ratio may be lower. c./The molar absorptivity for Trp at 280 nm and 260 nm are approximately 4,000 and 3,000 respectively. The molar absorptivity for Tyr at 280 nm and 260 nm are approximately 1,000 and 500 respectively. The A260 : A 280 ratio is given by: (2 3,000) + 500 Ratio = = 0.72 (2 4,000) +1, 000 d./Phe. The A260 : A 280 ratio would be much greater that 1. 5. Here is a start that is by no means original. "I think that I shall never see a ...." Try composing one with the nucleoside one-letter code (A,T,G and C) if you really want a challenge.
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Summary
Amino acids are the building blocks of proteins. There are 20 amino acids commonly found in proteins. The general structure of an amino acid is based on a central -carbon, to which is attached a carboxyl group, a hydrogen, an amino group, and a variable side chain or R group. Amino acids can be classified according to the nature of their R groups into three classes. The nonpolar, hydrophobic amino acids include alanine, valine, leucine and isoleucine in addition to proline, methionine, phenylalanine and tryptophan. Polar, uncharged amino acids include glycine, serine, threonine, tyrosine, asparagine, glutamine, and cysteine. The acidic amino acids are aspartic acid and glutamic acid and the basic amino acids include histidine, lysine, and arginine. The amino acids are weak polyprotic acids. The carboxyl group is a rather strong carboxylic acid with pK a around 2.0, whereas the amino group is a base with pK a around 9.5. At neutral pH, the carboxyl group is unprotonated and negatively charged whereas the amino group is protonated and positively charged. Considering only these two groups at pH = 7.0, amino acids are zwitterionic. Several of the side chains have titratable groups including aspartic acid, glutamic acid, cysteine, tyrosine, arginine, histidine, and lysine. In proteins, amino acids are joined by peptide (or amide) bonds. The carboxyl group of one amino acid is joined to the amino group of another amino acid, with elimination of the elements of water. In addition to the twenty common amino acids, there are a large number of modified amino acids found in nature. Examples include hydroxylation of lysine and proline, methylation of lysine, carboxylation of glutamic acid, phosphorylation of serine, threonine and tyrosine. Metabolites derived from amino acids play key roles in cells. Knowing the structures of ornithine, citrulline, aspartic acid and arginine will serve as a strong basis for understanding the urea cycle. Transamination of glutamic acid and aspartic acid produce -ketoglutarate and oxaloacetate, respectively, two citric acid cycle intermediates. The same reaction with alanine produces pyruvate. A great many small molecules acting as neurotransmitters, hormones, antibiotics, and other roles are directly derived from amino acids. The amino acids can participate in a number of useful chemical reactions, some specific to either the carboxyl group or the amino group common to all amino acids. Other chemistries are specific for side groups. For example, the sulfhydryl group of cysteine can be modified with Nethylmaleimide or iodoacetate. It will react with DTNB in a reaction producing a colored compound, which can be readily quantitated. The amino group of lysine, hydroxyl groups of serine, tyrosine and threonine and the carboxyl groups of aspartic and glutamic acid can participate in specific reactions. The -carbons of all of the amino acids except glycine are chiral. Thus amino acids can exist as stereoisomers. In nature, the L amino acids predominate. We will consider stereochemistry again when sugar structures are discussed. There are two general methods for separating and analyzing amino acid mixtures. In one method, the ionic properties of the amino acids are exploited in ion exchange. Ion exchange involves a stationary charged species in equilibrium with a counter ion. Conditions are established such that the amino acids can displace the counter ion and bind to the stationary phase. The degree to which amino acids bind to the stationary phase depends on their intrinsic charge and on the concentration of counter ion with which they compete. Removal of amino acids from the stationary phase can be accomplished either by changing the pH of the solution (and thus changing the charge on the amino acids) or increasing the concentration of counter ion. The conditions under which a particular amino acid is displaced from the stationary phase are characteristic of the amino acid. The other technique for separating amino acids is to react the amino terminus with a hydrophobic group and to separate the derivatized amino acids by reverse phase chromatography. In reverse phase chromatography, derivatized amino acids are bound to a stationary, hydrophobic phase and subsequently eluted with increasing concentrations of a polar substance (e.g., methanol).
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