Reginald H.

Garrett
Charles M. Grisham

Chapter 4
Amino Acids
Outline

• What are the structures and properties of amino

acids ?
• What are the acid-base properties of amino acids ?
• What reactions do amino acids undergo ?
• What are the optical and stereochemical properties
of amino acids ?
• What are the spectroscopic properties of amino
acids ?
• How are amino acid mixtures separated and
analyzed ?
4.1 What Are the Structures and
Properties of Amino Acids?
• The common amino acids contain a carbon
atom alpha to a carboxyl carbon.
• This α-carbon has an amino group attached
and an R group (R = H in glycine).
• The are 20 common α-amino acids are used by
the ribosomes to make proteins. These 20
have L chirality at the α-carbon.
• Amino acids join together via peptide bonds.
• Several modified amino acids occur only rarely
in proteins.
• Some amino acids are not found in proteins.
4.1 What Are the Structures and
Properties of Amino Acids?

Figure 4.1 Anatomy of an amino acid. Except for proline

and its derivatives, all of the amino acids commonly found in
proteins possess this type of structure.
4.1 What Are the Structures and
Properties of Amino Acids?

Figure 4.2
Two amino acids
can react with
loss of a water
molecule to form
a covalent bond.
The 20 Common Amino Acids

You should know names, structures, approximate

α-pKa values, 3-letter and 1-letter codes

Classification based on sidechain structure:

• Non-polar amino acids.
• Polar, uncharged amino acids.
• Acidic amino acids.
• Basic amino acids.
Other sidechain structural classifications:
• Aromatic, cyclic, hydroxyl, and thiol amino acids.
The 20 Common Amino Acids

Figure 4.3 Some of the nonpolar (hydrophobic) amino acids.

The 20 Common Amino Acids

Figure 4.3 Some of the nonpolar (hydrophobic) amino acids.

The 20 Common Amino Acids

Figure 4.3 Some of the polar, uncharged amino acids.

The 20 Common Amino Acids

Figure 4.3 Some of the polar, uncharged amino acids.

The 20 Common Amino Acids

Figure 4.3 The acidic amino acids.

The 20 Common Amino Acids

Figure 4.3 The basic amino acids.

Several Amino Acids Occur Rarely in Proteins

We'll see some of these in later chapters

• Selenocysteine in many organisms.
• Pyrrolysine in several archaeal species.
• Hydroxylysine, hydroxyproline – collagen.
• Carboxyglutamate - blood-clotting proteins.
• Pyroglutamate – in bacteriorhodopsin.
• GABA, epinephrine, histamine, serotonin act as
neurotransmitters and hormones.
• Phosphorylated amino acids – a signaling device.
Several Amino Acids Occur Rarely in Proteins

Figure 4.4(a) Some amino acids are less common

Several Amino Acids Occur Rarely in Proteins

Figure 4.4(b) Some amino acids are less common, but

nevertheless found in certain proteins. Hydroxylysine and
hydroxyproline are found in connective-tissue proteins; carboxy-
glutamate is found in blood-clotting proteins; pyroglutamate is
found in bacteriorhodopsin .
Several Amino Acids Occur Rarely in Proteins

Figure 4.4(c) Several amino acids that act as

neurotransmitters and hormones.
4.2 What Are Acid-Base Properties of
Amino Acids?

Amino Acids are Weak Polyprotic Acids

The degree of dissociation depends on the pH
of the medium

• The carboxylic acid group always ionizes first.

• Then the side chain (if ionizable) or the α-amino
group.
• The order of ionization always proceeds from
most acidic to least acidic.
4.2 What Are Acid-Base Properties of
Amino Acids?

The first dissociation is the carboxylic acid

group (using glycine as an example):

+
NH3CH2COOH  +NH3CH2COO- + H+

(+NH3CH2COO-)(H+)
Ka1 = ---------------------------
(+NH3CH2COOH)
4.2 What Are Acid-Base Properties of
Amino Acids?

The second dissociation is the amino group in

the case of glycine:

+
NH3CH2COO-  NH2CH2COO- + H+

(NH2CH2COO-)(H+)
Ka2 = ---------------------------
(+NH3CH2COO-)
4.2 What Are Acid-Base Properties of
Amino Acids?

Figure 4.5 The ionic forms of the amino acids, shown

without consideration of any ionizations on the side chain.
pKa Values of the Amino Acids

You should know these numbers and know

what they mean

• Alpha carboxyl group: pKa = ~2

• Alpha amino group: pKa = ~9

• These numbers are approximate, but entirely

suitable for our purposes.
Isoelectric Point, pI
The isoelectric point is the pH at which there
is zero net charge

Using Gly again:

• Charges in the first ionization: +1 
0
• Charges in the second ionization: 0  -1
• So, in the case of glycine, the pH at which
there is most of the zero net charge form
occurs half way between the first and second
ionizations.
pI = (pKa1 + pKa2)/2 = 5.95
4.2 What Are Acid-Base Properties of
Amino Acids?
4.2 What Are Acid-Base Properties of
Amino Acids?
pKa Values of the Amino Acids

You should know these numbers and know what

they mean

• Arginine, Arg, R: pKa(guanidino group) = 12.5

• Aspartic Acid, Asp, D: pKa = 3.9
• Cysteine, Cys, C: pKa = 8.3
• Glutamic Acid, Glu, E: pKa = 4.3
• Histidine, His, H: pKa = 6.0
• Lysine, Lys, K: pKa = 10.5
• Tyrosine, Tyr, Y: pKa = 10.1
Titrations of polyprotic amino acids

Figure 4.7
Titration of glutamic
acid
Titrations of polyprotic amino acids

Figure 4.7
Titration of lysine.
A Sample Calculation

What is the pH of a glutamic acid solution

if the alpha carboxyl is 1/4 dissociated?
[1]
pH  2  log10
[3]
• pH = 2.2 + (-0.477)
• pH = 1.723

• Note that, when the group is ¼ dissociated,

¼ are dissociated and ¾ are not; thus the
ratio in the log term is ¼ over ¾ or 1/3.
Another Sample Calculation
What is the pH of a lysine solution if the side
chain amino group is 3/4 dissociated?

[3]
pH  10.5  log10
[1]
• pH = 10.5 + (0.477)
• pH = 10.977 = 11.0

• Note that, when the group is ¾ dissociated, ¾

is dissociated and ¼ is not; thus the ratio in
the log term is ¾ over ¼ or 3/1.
Amino Acids as Buffers
From Table 4.1 we can see that Histidine has pKa
values of 1.8, 6.0, and 9.2.

It can provide effective buffering at a pH equal to

any one of these three pKas.
The titration curve has three regions in which pH
is relatively unaffected by addition of acid or
base.
What is the zero net charge form of His ?
In which ionizations does it appear ?
What is the charge on His at pH 4?
Reactions of Amino Acids
Amino acid composition – an amino acid
analysis gives the number of each amino acid in
the peptide or protein.
• Peptide bonds are cleaved by acid hydrolysis
(6M HCl, 110oC, 18-72 hours). Trp is destroyed.
Amino acid sequence – order of the amino acids
in a peptide or protein.
• Edman’s reagent (phenylisothiocyanate) is the
currently preferred reagent. It reacts with a free
α-amino group of an amino acid or peptide to
produce a phenylthiohydantoin (PTH) derivative.
Detecting Amino Acids
Ninhydrin is the classical
reagent for detecting amino
acids.
Reaction requires 2-5 min
at 100oC and is sensitive
at the nanomole level.
O O O
OH CHO
2 + NH2-CH-COOH + CO2 + N
OH CH3
CH3
O O O

Ruhemann’s Purple
Note: The product from Pro is 570 nm
Yellow and absorbs at 440 nm.
N-Terminal Reagents

F
NO2
DNFB - Sanger’s reagent
(dinitrofluorobenzene)
NO2

DNFB

DANSYL choride
(dimethylaminonaphthalenesulfonyl chloride)
N(CH3)2

DANSYL-Cl

SO2Cl
Other Reagents
C-terminal analysis:
Hydrazine NH2-NH2

CH2-SH
Disulfide reduction:
HO C H
Dithiothreitol - Cleland’s Reagent
H C OH

Thiols reactions: CH2-SH

Cleland's
Iodoacetate I-CH2-COOH

5,5’-dithiobis-(2-nitrobenzoic acid) -
Ellman’s reagent
NO2 S S NO2

COOH Ellman's COOH

Edman’s Reaction

Figure 4.8(a) Edman’s reagent reacts with the N-terminal

residue of a peptide or protein and cleaves the peptide
bond forming a cyclic thiazoline derivative that reacts in
weak aqueous acid to form a PTH-amino acid. This
reaction can proceed down the chain cleaving successive
residues. Samples are used to identify each residue.
Oxidation of Cysteine to Cystine

Figure 4.8(b) Cysteine residues react with each other

to form disulfides. This will occur with O2 in air.
Stereochemistry of Amino Acids
• All common AA except glycine are chiral at
the α-carbon atom.
• L-amino acids predominate in nature and are
the only ones used in ribosomal protein
synthesis.
• D,L-nomenclature is based on D- and L-
glyceraldehyde.
• R,S-nomenclature system is more
convenient, since amino acids like isoleucine
and threonine (with two chiral centers) can be
named unambiguously.
Stereochemistry of Amino Acids
Discovery of Optically Active Molecules and
Determination of Absolute Configuration

Emil Fischer deduced the

structure of glucose in
1891. Fischer’s proposed
structure was confirmed by
J. M. Bijvoet in 1951 (by X-
ray diffraction).
Rules for Description of Chiral Centers in
the (R,S) System

Naming a chiral center in the (R,S) system is accomplished by

viewing the molecule from the chiral center to the atom with
the lowest priority. The priorities of the functional groups are:
SH > OH > NH2 > COOH > CHO > CH2OH > CH3
Spectroscopic Properties

• All amino acids absorb in the infrared (bond

vibrations).
• Only Phe, Tyr, and Trp absorb in the UV
(electronic transitions between energy levels).
• Absorbance at 280 nm is a good method for
determining protein concentration.
• NMR spectra are characteristic of each
residue in a protein, and high resolution NMR
measurements can be used to elucidate
three-dimensional structures of proteins.
Separation of Amino Acids

• Mikhail Tswett, a Russian botanist, first

separated colorful plant pigments by
‘chromatography’.
• Many chromatographic methods exist for
separation of amino acid mixtures:
• Ion exchange chromatography.
• High-performance liquid chromatography.
Peptides and Proteins – the Peptide Bond

Figure 4.14 Peptide formation is the creation of an amide

bond between the carboxyl group of one amino acid and
the amino group of another amino acid.
4.7 What is the Fundamental Structural
Pattern in Proteins?

• Proteins are unbranched polymers of amino acids.

• Amino acids join head-to-tail through formation of
covalent peptide bonds.
• Peptide bond formation results in release of water.
• The peptide backbone of a protein consists of the
repeated sequence –N-Cα-Co-.
• “N” is the amide nitrogen of the amino acid.
• “Cα” is the alpha-C of the amino acid.
• “Co” is the carbonyl carbon of the amino acid.
The Peptide Bond

• Is the biochemical nomenclature for the amide

bond between formed two amino acids.
• Is usually found in the trans conformation.
• Has partial (40%) double bond character due to
resonance.
• Is about 0.133 nm long - shorter than a typical
single bond but longer than a double bond.
• Due to the double bond character, the six atoms
of the peptide bond group are always coplanar.
• N partially positive; O partially negative.
The Peptide Bond

Figure 4.15 The trans conformation of the peptide bond.

4.7 What is the Fundamental Structural
Pattern in Proteins?

Figure 4.16(a) The peptide bond has partial double bond

character. One of the postulated resonance forms is shown
here.
4.7 What is the Fundamental Structural
Pattern in Proteins?

Figure 4.16(b) The peptide bond has partial double

bond character. One of the postulated resonance
forms is shown here.
4.7 What is the Fundamental Structural
Pattern in Proteins?

Figure 4.16(c) The peptide bond is best described as a

resonance hybrid of the forms shown on the two previous
slides.
4.7 What is the Fundamental Structural
Pattern in Proteins?

The coplanar relationship of the atoms in the amide group

is highlighted here by an imaginary shaded plane lying
between adjacent α-carbons.
“Peptides”

• These are short polymers of amino acids.

• Each unit is called a residue.
• 2 residues – dipeptide.
• 3 residues – tripeptide.
• 12-20 residues – oligopeptide.
• Many residues – polypeptide.
 “Protein”

One or more polypeptide chains

• One polypeptide chain - a monomeric protein.

• More than one - multimeric protein.
• Homomultimer - one kind of chain.
• Heteromultimer - two or more different chains.
• Hemoglobin, for example, is a heterotetramer.
• It has two alpha chains and two beta chains.
Proteins - Large and Small
Example proteins

• Insulin – Has an A chain of 21 residues and a B

chain of 30 residues - total mol. wt. of 5,733.
• Glutamine synthetase - 12 subunits of 468
residues each - total mol. wt. of 600,000.
• Connectin proteins:
• alpha - MW 2.8 million.
• beta - MW of 2.1 million, with a length of
1000 nm -it can stretch to 3000 nm.
The Sequence of Amino Acids in a Protein

• Is unique for every protein.

• Predicts the folding arrangement (3o structure).
• Is encoded by the nucleotide sequence of DNA.
• So, is a form of genetic information.
• Is read from the amino terminus (#1) to the
carboxyl terminus (#n).
• Is written from the amino terminus (left) to the
carboxyl terminus (right).
End Chapter 4
Amino Acids