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CHMI 2227E
Biochemistry I
Amino acids:
-Structure
-General
chemical properties
CHMI 2227 - E.R. Gauthier, Ph.D.
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Proteins: Building blocks of life!


Proteins are extremely complex molecules;
Proteins are involved in a vast variety of biological
structures and phenomena:






Silk
Spider web
Horns
Antibiotics
Toxins





Enzymes
Antibodies
Hormones
Membrane transporters
Haemoglobin
The building block for all proteins are amino
acids.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Amino acids
Ca

COOH
An amino acid is a molecule in
which a carbon atom (Ca) is
bonded simultaneously to a
carboxylic acid (COOH) and an
amine group (NH2);

Hundreds of amino acids exist in
nature;

However, only 20 amino acids are
used by the cell to make proteins.
H C NH2
R

Each amino acid differs from all
the others by the nature of the R
group.

Each R group differs from the
others in several ways:



Size
Charge
Water solubility
CHMI 2227 - E.R. Gauthier, Ph.D.
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General properties of amino acids.

Note that the Ca is
bonded to 4 different
groups and is chiral;

Two stereoisomers are
distinguished:



L-amino acids
D-amino acids
Only the L-amino acids
are found in proteins.
L = amino group is on the left in the
Fisher representation.
 L and D: arbitrary. Has nothing to
do with the direction of rotation of
polarised light by the amino acid.4
CHMI 2227 - E.R. Gauthier, Ph.D.

General properties of amino acids.

Every amino acids has at least two
chemical groups that can be ionized:
 Carboxylic Acid
 Amino
Net charge: +1
Acidic pH
(COOH)
(NH2)
Net charge: 0
pKa 1
(pH 2-2.5)
pKa 2
(pH 9-10.5)
Zwitterion
CHMI 2227 - E.R. Gauthier, Ph.D.
Net charge: -1
Basic pH
5
Amino acid titration
1. simplest scenario

pI = isoelectric point = pH
where 100% zwitterion

pI = pKa1 + pKa2
2

The fraction of COOH or
NH2 group that is charged at
any given pH can be
determined with the
Henderson-Hasselbach
equation:
Buffer!
Buffer!
pH = pKa + log AHA
CHMI 2227 - E.R. Gauthier, Ph.D.
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The twenty amino acids:
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
1. Hydrophobic amino acids


All these amino acids are NOT soluble in water.
Note: glycine is NOT optically active. Why?
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
1. Hydrophobic amino acids

Proline is the only cyclic amino acid;
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
2. Aromatic amino acids
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
2. Aromatic amino acids

Tyr, Trp and Phe are very
useful:

Their aromatic ring absorb
UV light at 260-280 nm.

Form the basis of protein
detection at 280 nm.
Absorbance
max
(nm)
Molar
Absorbance
(M-1 cm-1)
Phenylalanine
257.4
197
Tyrosine
274.6
1420
Tryptophan
279.8
5690
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
3. polar, uncharged amino acids
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
Cysteine and cystine
In biochem:
-oxidation: lose H
-Reduction: gain H
Disulfide bond
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
Tyr, Ser, Thr
Ser
PO4-3
ATP
Protein
kinase
Protein
phosphatase
ADP
H2O
OP=O
Phospho-Ser O
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
4. Negatively charged amino acids
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
4. Negatively charged amino acids
100% D
pH
9.5
pKa2
100% C
pKa1
A
pKaR
B
4.1
pKa2
C
pKaR
D
2.1
What is the pI of glutamate?
pKa1
100% B
100% A
NaOH
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
5. Positively charged amino acids
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
5. Positively charged amino acids: His
pKa2
pKaR
CHMI 2227 - E.R. Gauthier, Ph.D.
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Structure of the 20 amino acids
pKa values of amino acids
CHMI 2227 - E.R. Gauthier, Ph.D.
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Analysis of amino acids
1. Ion exchange chromatography
pH << pI
pH >> pI
+
+
Elution

Different types of ion exchange resins exist:

Cation exchange: negatively charged/separation of cations
 Anion exchange: positively changed/separation of anions.

Obviously, the type of resin to be used will depend on the charge of
the amino acid on interest, itself dependent on the pH of the solution.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Analysis of amino acids
2. Detection of amino acids: ninhydrin reagent
O
COOH
OH
OH
C
NH3+
R-HC=O
CO2
O
O
R
N
O
Ninhydrin
2
Amino acid
O
O
Purple!!

While Trp, Phe and Tyr can be detected by their A260-280nm, the other amino acids
cannot;

Ninhydrin reacts with the amine group of amino acids, generating a purple product
(yellow in the case of Pro).

The ninhydrin reaction allows one to detect and quantify (A570nm) the amino acids
contained in the fractions of CHMI
the IEX
2227column.
- E.R. Gauthier, Ph.D.
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