Amino Acids, Peptides and Proteins

Main source: Campbell, M. K and Farrell, S. O.

(2012). Biochemistry. 7th Edition. United States:
Brooks/Cole, Cengage Learning.
 PROTEINS
• Most complex of all organic molecules.

• Proteins consist of long chains (polypeptides) of variable

subunits of amino acids.

• The building blocks of proteins are amino acids.

• Amino acids are the monomer units of proteins, proteins are

polymers of amino acids.

• There are 20 different kinds of amino acids found in proteins

(known as ‘standard’ or ‘common’ amino acids).
 PROTEINS

• Amino acids have a general structure in common

• Every amino acid consists of a central carbon atom

linking four subunits:
• the amino group (NH2 / NH3+)
• the acid/carboxyl group (COOH / COO-)
• a hydrogen atom (H)
• the variable side chain (referred to as R groups)
 Amino Acids
• Amino acid: a compound that contains
both an amino group and a carboxyl
group
• -Amino acid has an amino group
attached to the carbon adjacent to
the carboxyl group
• -carbon also bound to side chain
group, R
• R gives identity to amino acid
• Two steroisomers of amino acids
are designated L- or D-. Based on
similarity to glyceraldehdye
(Figure 3.2)
 Amino Acids
• The common amino acids have an asymmetric center except for glycine.

• All common amino acids are α-amino acids, i.e. a primary amino group
and a carboxylic acid group substituent on the same carbon atom

• Amino acids are chiral objects meaning that they cannot be

superimposed on their mirror images.

• Amino acids can exist in two forms which are:

• L (amino acids found in proteins are of the L form)
• D (some D amino acids occur in nature eg. bacterial cells walls, some
antibiotics)

• These two stereoisomers are nonsuperimposable mirror images of each

other.
The general structure of an amino acid
 Fig. 3-1a, p.59
FIGURE 3.1 The general formula of amino acids, showing the ionic forms that predominate at pH 7.
Figure 4-1
Figure 4-2
Configuration of amino acids

10
FIGURE 3.2 Stereochemistry of alanine Fig. 3-2a, p.59
Structures and properties of the individual
amino acids
• A classification scheme for amino acids can be
based on the properties of their side chains.

• Two particularly important criteria are the:

(1) nature of the sidechain whether they are:
• polar, or
• nonpolar
(2) presence of an acidic or basic group in the
side chain (for polar)
Classification of Amino Acids

• Hydrophobic: water-fearing, nonpolar side

chains
• Alkyl side chain

• Hydrophilic: water-loving side chains

• Polar, neutral side chains
• Anionic
• Cationic

13
Structures and properties of the individual
amino acids
• Based on the two criteria, amino acids can be
classified as:

(a) nonpolar (hydrophobic)

(b) polar uncharged
(c) polar charged
-acidic (negatively charged)
-basic (positively charged)
Amino acids with nonpolar side chains

Nonpolar side chains

•This group consists of:

• Alanine
• Valine
• Leucine
• Isoleucine
• Proline
• Phenylalanine
• Tyrptophan
• Methionine
 Nonpolar side chains

*
aliphatic cyclic structure

* *

* Each side chain is an aliphatic hydrocarbon group (no benzene ring)

Fig. 3-3a1, p.60
 Nonpolar side chains

*
contains a sulfur atom contains indole ring (aromatic)

*
contains cyclic group (aromatic)

(a) nonpolar (hydrophobic)

Fig. 3-3b1, p.60
Nonpolar side chains
Amino acids with uncharged polar side chains

Uncharged polar side chains

•A group of amino acids with polar side chains that are
electrically neutral (uncharged) at neutral pH.

•This group consists of:

• Serine
• Threonine
• Tyrosine
• Cysteine
• Glutamine
• Asparagine
 Uncharged polar side chains

Asn and Gln have amide groups derived from carboxyl groups; can be
considered as derivatives of Glutamic acid and Aspartic acid
(b) polar, uncharged
Fig. 3-3a2, p.60
Uncharged polar side chains

(b) polar, uncharged

Fig. 3-3a2, p.60
 Uncharged polar side chains

(b) polar, uncharged

Hydroxyl group is a phenol (stronger acid than an aliphatic alcohol ) Fig. 3-3b2, p.60
 Uncharged polar side chains

Has a thiol group (-SH), which can react with other Cys
thiol groups to form disulfide (-S-S-) bridges in proteins in
oxidation reaction
(b) polar, uncharged (d) Basic
Fig. 3-3b2, p.60
Amino acids with charged (carboxyl groups -
acidic) polar side chains
Charged (carboxyl groups - acidic) polar side chains
•Amino acids that have carboxyl groups in their side chains
in addition to the one present in all amino acids.

•A carboxyl group can lose a proton, forming a carboxylate

anion.

•The side chain of each of these amino acids is negatively

charged at neutral pH

•This group consists of:

• Glutamic acid
• Aspartic acid
Charged (carboxyl groups - acidic) polar side
chains

negatively charged at neutral pH

(c) Acidic
Fig. 3-3a3, p.60
Amino acids with charged (basic) polar side
chains
Charged (basic) polar side chains
•Side chains of these amino acids are positively
charged at or near neutral pH

•The side chains of these amino acids has a

nitrogen-containing group that can exist in either a
protonated or deprotonated form

•This group consists of:

• Lysine
• Arginine
• Histidine
Charged (basic) polar side chains

(b) polar, uncharged (d) Basic

Fig. 3-3b2, p.60
Charged (basic) polar side chains

(d) Basic

Fig. 3-3b3, p.60

Table 3-1, p.62
Amino acid structure and properties
• With the exception of glycine, all protein-derived
amino acids have at least one stereocenter (the -
carbon) and are chiral (stereoisomers)
• the vast majority of -amino acids have the L-
configuration at the -carbon (Proline is usually D)

• Side-chain carbons in other amino acids designated

with Greek symbols, starting at a carbon (  …etc)

• Amino acids can be referred to by three-letter or one-

letter codes. Table 3.1 (KNOW THESE)
Individual Amino Acids
• Group A: Nonpolar side chains - Ala, Val, Leu, Ile,
Pro. Phe, Trp, Met.
• Ala, Val, Leu, Ile, Pro- contain aliphatic hydrocarbon
group. Pro has cyclic structure.
• Phe- hydrocarbon aromatic ring.
• Trp- Indole ring side chain, aromatic.
• Met- Sulfur atom in side chain.
Amino Acids (cont’d)
• Group B: Neutral Polar side chains - Ser, Thr, Tyr,
Cys, Glu, Asn

• Ser, Thr- Side chain is polar hydroxyl group

• Tyr- hydroxyl group bonded to aromatic hydrocarbon

group

• Cys- Side chain contains thiol group (-SH)

• Gln, Asn- contain amide bonds in side chain

Amino Acids (cont’d)
• Group C: Acidic Side Chains: Glu, Asp

• Both have a carboxyl group in side chain

• Can lose a proton, forming a carboxylate ion

• These amino acids are negatively charged at neutral

pH
Amino Acids (cont’d)
• Group D: Basic side chains: His, Lys, Arg

• Side chains are positively charged at pH 7

• Arg - side chain is a guanidino group

• His - side chain is an imidazole group

• Lys - side chain NH3 group is attached to an aliphatic

hydrocarbon chain
Amino acid summary
Important structural features:

1) All 20 are -amino acids

2) For 19 of the 20, the -amino group is primary; for proline,

it is secondary

3) With the exception of glycine, the -carbon of each is a

stereocenter

4) Isoleucine and threonine contain a second stereocenter

5) 3- and 1-letter codes in Table 3.1.

Uncommon Amino Acids
• Each derived from a
common amino acid
by a modification

• hydroxylysine and
hydroxyproline are
found only in a few
connective-tissue
proteins, such as
collagen

• thyroxine is found
only in the thyroid
gland
Ionization of Amino Acids
• In amino acid,
carboxyl group (-)
and amino group
(+) are charged at
neutral pH.
• In free amino
acids -carboxyl,
and a-amino
groups have
titratable protons.
Some side chains
do as well
Ionization of Amino Acids
• Remember, amino acids without charged groups on side chain
exist in neutral solution as zwitterions with no net charge
 Acid–base properties of amino acids

An amino acid can never exist as an uncharged compound

Acidity: -COOH Groups
• The average pKa of an -carboxyl group is 2.19

• the greater acidity of the amino acid carboxyl group is

due to the electron-withdrawing inductive effect of the -
NH3+ group
Basicity
• -NH3+ groups: The average value of pKa for an -NH3+ group
is 9.47

Guanidine Group
• The side chain of arginine is a considerably stronger base
than an aliphatic amine
• basicity of the guanido group is attributed to the large
resonance stabilization of the protonated form relative to
the neutral form

Imidazole Group
• The side chain imidazole group of histidine is a heterocyclic
aromatic amine
Ionization vs pH
• Given the value of pKa of each functional group, we
can calculate the ratio of each acid to its conjugate
base as a function of pH

• Consider the ionization of an -COOH

pKa = 2.00
- +
COOH + H2 O COO + H3 O

• writing the acid ionization constant and rearranging

terms gives
[ H 3 O + ] [ -COO - ] [ -COO - ] Ka
Ka = or =
[ -COO H] [ -COO H] [ H 3 O+ ]
Ionization vs pH (cont’d)
• substituting the value of Ka (1 x 10-2) for the hydrogen
ion concentration at pH 7.0 (1.0 x 10-7) gives

[ -COO - ] Ka -2
1.00 x 10 5
= = = 1.00 x 10
[ -COO H] [ H 3 O+ ] -7
1.00 x 10

• at pH 7.0, the -carboxyl group is virtually 100% in

the ionized or conjugate base form, and has a net
charge of -1

• we can repeat this calculation at any pH and

determine the ratio of [-COO-] to [-COOH] and the
net charge on the -carboxyl at that pH
Ionization vs pH (cont’d)
• We can also calculate the ratio of acid to conjugate
base for an -NH3+ group; for this calculation,
assume a value 10.0 for pKa

+ pKa = 10.00
NH3 + H2 O NH2 + H3 O+

• writing the acid ionization constant and rearranging

gives [ -NH ] 2 Ka
=
[ -NH 3 + ] [H 3 O+ ]
Ionization vs pH

• substituting values for Ka of an -NH3+ group and

the hydrogen ion concentration at pH 7.0 gives

[ -NH 2 ] Ka - 10
1.00 x 10 -3
= = = 1.00 x 10
[ -NH 3 ]
+ [H 3 O+ ] -7
1.00 x 10

• at pH 7.0, the ratio of -NH2 to -NH3 + is

approximately 1 to 1000

• at this pH, an -amino group is 99.9% in the acid

or protonated form and has a charge of +1
Henderson-Hasselbalch Equation
• We have calculated the ratio of acid to conjugate
base for an -carboxyl group and an -amino group
at pH 7.0

• We can do this for any weak acid and its conjugate

base at any pH using the Henderson-Hasselbalch
equation
[conjugate base]
pH = pK a + log
[weak acid]
Isoelectric pH
• Isoelectric pH, pI: the pH at which the majority of molecules
of a compound in solution have no net charge

• the pI for glycine, for example, falls midway between the pKa
values for the carboxyl and amino groups

pI = 1 ( p K a COOH + p Ka N H 3 + )
2

= 1 (2.35 + 9.78) = 6.06

2
• Isoelectric pH values for the 20 protein-derived amino acids are
given in Table 3.2
Table 3-2, p.67
The isoelectric point (pI) of an amino acid is the pH at
which it has no net charge
 (sulfhydrl)

(phenol)

50
 pKa Values of Common Amino Acids
• Amino acids and their titratable groups have characteristic
pKa values

• pKa values of side-chain groups, -COOH and -NH3+

depend on the chemical nature of the group.

• Amino acids, peptides and proteins have different pKa values

so it is possible that they have different charges at a given
pH.

• This is useful in separating molecules in an electric field

(electrophoresis).

• Electrophoresis is used in determining important properties of

proteins and nucleic acids.
 pI Value of Amino Acids
• Isoelectric pH or isoelectric point (pI) is the pH at
which a molecule has no net charge.

• At its pI, a molecule will not migrate in an electric

field.

• Equation for calculation of pI value:

pI = (pKa1 + pKa2 ) / 2
Titration of Amino Acids
• When an amino acid is titrated, the titration curve represents the reaction of
each functional group with the hydroxide ion

Self-study
Titration of alanine with NaOH

Self-study
Titration of histidine with NaOH

Self-study
Electrophoresis
• Electrophoresis: the process of separating
compounds on the basis of their electric charge
• electrophoresis of amino acids can be carried out
using paper, starch, agar, certain plastics, and
cellulose acetate as solid supports
• in paper electrophoresis, a paper strip saturated with
an aqueous buffer of predetermined pH serves as a
bridge between two electrode vessels
A mixture of amino acids can be separated by
electrophoresis on the basis of their pI values

Ninhydrin is used to detect the individual amino acids

57
 Reaction of ninhydrin with amino acids

Self-study
A mixture of amino acids can also be separated
on the basis of polarity

59
Ion-exchange chromatography can be used to perform
preparative separation of amino acids

Negatively charged resin binds selectively to positively charged

amino acids
 Ion-Exchange Chromatography

• Cations bind most strongly to cation-exchange resins.

• Anions bind most strongly to anion-exchange resins.

• An amino acid analyzer is an instrument that automates

ion-exchange chromatography.
High Performance Liquid Chromatography
(HPLC) – Schematic Diagram
High Performance Liquid Chromatography
(HPLC) – Schematic Diagram
High Performance Liquid Chromatography
(HPLC) – Sample Injection
High Performance Liquid Chromatography
(HPLC) – Real System
High Performance Liquid Chromatography
(HPLC) – Real System
High Performance Liquid Chromatography
(HPLC) – Real System
The Peptide Bond

• Peptides are formed by linking the carboxyl

group (-COOH) of one amino acid to the
amino group (-NH3+) of another amino acid in
a covalent (amide) bond.

• Water is eliminated in the process and the

linked amino acid residues remain after water is
eliminated.
Peptides and proteins are polymers of amino acids linked
together by amide (peptide) bonds
Formation of a Peptide
Formation of a Peptide

FIGURE 3.8 Formation of a peptide bond

Fig. 3-8a, p.69

Formation of a Peptide

FIGURE 3.8 Formation of peptide bond

A peptide bond has 40% double-bond character

73
 The Peptide Bond

• Proteins consist of polypeptide chains

• the number of amino acids in a protein is usually

100 or more.

• Amide is another term/name for a compound

formed by the reaction between an amino group
and a carboxyl group.
The Peptide Bond

FIGURE 3.9 A small peptide showing the direction of the peptide

chain (N-terminal to C-terminal).
Fig. 3-9, p.69
 The Peptide Bond

Page 82
Because amino acids have two functional groups, amide bond
formation with a mixture of two amino acids affords four products
Geometry of Peptide Bond
• the four atoms of a peptide bond and the two alpha
carbons joined to it lie in a plane with bond angles of
120°about C and N

• to account for this geometry, a peptide bond is most

accurately represented as a hybrid of two contributing
structures (resonance structures)

• the hybrid has considerable C-N double bond

character and rotation about the peptide bond is
restricted
Resonance Structures of Peptide Bond
Peptides
• peptide: the name given to a short polymer of amino acids
joined by peptide bonds; they are classified by the number of
amino acids in the chain

• dipeptide: a molecule containing two amino acids joined by a

peptide bond

• tripeptide: a molecule containing three amino acids joined by

peptide bonds

• polypeptide: a macromolecule containing many amino acids

joined by peptide bonds

• protein: a biological macromolecule of molecular weight 5000

g/mol or greater, consisting of one or more polypeptide chains
Peptides with Physiological Activity

(found in muscle tissue)

Peptides with Physiological Activity (cont’d)

(as hormones)
Sequencing Proteins
Further Reading for Further Understanding!
Garrett, R.H. and Grisham, C.M. (2013). Biochemistry, 5th Edition.
USA: Brooks/Cole, Cengage Learning.

• N-terminal amino acid?

• C-terminal amino acid?
• What is in between?

83
The peptide or protein can be partially hydrolyzed using
endopeptidases
• Trypsin: C-side of Arg and Lys
• Chymotrypsin: C-side of Phe, Tyr, Trp
• Elastase: C-side of Gly and Ala
• No cleavage for any endopeptidase if Pro on either side

Example of trypsin hydrolysis:

84
85
Cyanogen bromide causes the hydrolysis of the amide
bond on the C-side of a methionine residue

86
Mechanism for cleavage of a peptide bond by cyanogen
bromide

87
The end of ‘Amino Acids and
Peptides’ chapter
Nonpolar Side Chains

89
Polar, Neutral Side Chains

90
Polar, Acidic Side Chains

91
Basic, Polar Side Chains

92
Table 4-1 part 1
Table 4-1 part 2
Table 4-1 part 3
Table 4-1 part 4