AMINO ACIDS AND PROTEINS

PROF. LARA LELLI – I.I.S. BENEDETTO CASTELLI

 What are -amino acids?
COOH Carboxyl group
At pH 7, COO
-
both groups
H2N C H Amino group are ionized HN C H
+ 3

R R

Side group / chain: variable properties

If Cis asymmetric (attached to four different types of atoms or groups of atoms),

amino acids can exist as 2 enantiomers (optical isomers).

COO - COO -

H NH
+ 3 H3N+ H
R R
Enantiomer (R) Enantiomer (S)
 Let’s review stereo-descriptor D / L
CHO CHO
L-glyceraldehyde: D-glyceraldehyde:
causes the plane of causes the plane of
polarization to rotate H H polarization to rotate to
to the left (-) HOH2C CH2OHthe right (+, dexter side)
OH HO
CHO CHO

(S) Enantiomer HO H H OH (R) Enantiomer

CH2OH CH2OH

Monosaccharides with the Monosaccharides with the

absolute configuration (S) absolute configuration (R)
at the last stereocentre are at the last stereocentre are
assigned the stereo- assigned the stereo-
descriptor L. descriptor D.

L-glucosio D-glucosio

While the optical rotation of glyceraldehyde is (+) for R and (−) for S, this is not true for all
monosaccharides (e.g. D-(-)-fructose, called Levulose).
The stereochemical rotation can only be determined by the chemical structure, whereas
the optical rotation can only be determined empirically.
 Let’s apply D/L to amino acids
CHO CHO
L-glyceraldehyde: D-glyceraldehide:
causes the plane of causes the plane of
polarization to rotate H H polarization to rotate to
to the left (-) (Laevus) HOH2C CH2OH the right (+) (Dexter)
OH HO
CHO CHO

(S) Enantiomer HO H H OH
(R) Enantiomer

CH2OH CH2OH

Amino acids with the Amino acids with the

absolute configuration COO- COO- absolute
(S) at the C are configuration (R) at
assigned the stereo- HN
+ 3
H H NH
+
the C are assigned
descriptor L. the stereo-descriptor
3
D.
R R
As for sugars, there is no correspondence between the optical rotation of the amino acid
and its absolute configuration: e.g. []25°C D-Serine = - 13°
The stereochemical rotation can only be determined by the chemical structure, whereas
the optical rotation can only be determined empirically.
 IN ALL LIVING ORGANISMS:

NH3+ OH
H H

-OOC R (CHOH)n-CHO
HOCH2
CHO

COO- (CHOH)n

(S) H
+ 3
N H H OH (R)
R CH2OH
L- Amino acids D - Monosaccharides

Omochirality makes plain that organisms evolved from a common ancestor

 NATURAL AMINO ACIDS (12 + 8 + 2)

(Selenocysteine, 1986)

(Pyrrolisine, 2002
archaebacteria)
Unusual
Essential: an essential amino acid is an amino acid that cannot be synthesized de novo (from raw materials) by the human organism.
Why are amino acids ionized at pH 7?

COO
-

HN C H
+ 3

R
 Ka
HA + H2O A- + H3O+
 1) NEUTRAL SIDE CHAIN (EITHER NONPOLAR OR POLAR)

COOH COO- pKa2 = 9-10 COO-

pKa1 = 1.8-2.8

H3N+ H H3N+ H H2N H

R R R

H2A+ HA A-

2) ACIDIC SIDE CHAINS

COOH pKa1 = 1.88 COO- pKa2 = 3.65 COO- pKa3 = 9.68 COO-

H3N+ H H3N+ H H3N+ H H2N H

CH2-COOH CH2-COOH CH2-COO- CH2-COO-

H3A+ H2A HA- A2-

3) BASIC SIDE CHAINS

COOH pKa1 = 2.18 COO- pKa2 = 8.95 COO- pKa3 = 10.53 COO-

H3N+ H H3N+ H H2N H H2N H

(CH2)4-NH3+ (CH2)4-NH3+ (CH2)4-NH3+ (CH2)4-NH2

H3A2+ H2A+ HA A-
1) NEUTRAL SIDE CHAIN

COOH COO- pKa2 = 9-10 COO-

pKa1 = 1.8-2.8

H3N+ H H3N+ H H2N H

R R R

H2A+ HA A-

pH= pI

The isoelectronic point or isoionic point is the pH

at which the amino acid is neutral, i.e. the zwitterion form is dominant.

The isoelectronic point will be halfway between

(or the average of)
the two pKas:

pI = 1/2 (pKa1 + pKa2)

For the simplest amino acid, Glycine, pKa1= 2.34 and pKa2 = 9.6, pI = 5.97
 2) ACIDIC SIDE CHAINS

COOH pKa1 = 1.88 COO- pKa2 = 3.65 COO- pKa3 = 9.68 COO-

H3N+ H H3N+ H H3N+ H H2N H

CH2-COOH CH2-COOH CH2-COO- CH2-COO-

H3A+ H2A HA- A2-

pH= pI

For Aspartic acid the neutral form is dominant between pH 1.88 and 3.65,
pI is halfway between the two values:

pI = 1/2 (pKa1 + pKa3)

pI = 2.77
 3) BASIC SIDE CHAINS
COOH pKa1 = 2.18 COO- pKa2 = 8.95 COO- pKa3 = 10.53 COO-

H3N+ H H3N+ H H2N H H2N H

(CH2)4-NH3+ (CH2)4-NH3+ (CH2)4-NH3+ (CH2)4-NH2

H3A2+ H2A+ HA A-

pH= pI

For Lysine, the neutral form is dominant between pH 8.95 and 10.53
pI is halfway between the two values

pI = 1/2 (pKa2 + pKa3)

pI = 9.74
 ISOELECTRONIC POINT AND ELECTROPHORESIS
1) NEUTRAL SIDE CHAIN

COOH COO- pKa2 = 9-10 COO-

pKa1 = 1.8-2.8

H3N+ H H3N+ H H2N H

R R R

H2A+ HA A-

pH= pI

At pI the amino acid is neutral and so it does not migrate in an electric field.
If pH is higher than pI, the a.a. is negatively charged and it migrates towards the anode,
if pH is lower than pI, it migrates towards the cathode.
 TITRATION CURVE OF ALANINE (R = -CH3)

1) NEUTRAL SIDE CHAIN

COOH COO- pKa2 = 9-10 COO-

pKa1 = 1.8-2.8

H3N+ H H3N+ H H2N H

R R R

H2A+ HA A-
 DISTRIBUTION DIAGRAM OF ALANINE
1

0.9

0.8
Alfa = relative fraction 0.7

0.6
 = CX / Ctot alfa 2 (H2A+)

alfa
0.5 punto isoelettrico
alfa 1 (HA)
0.4 alfa 0 (A-)
0.3

0.2

In the pH range 5-8 the 0.1

predominant species is HA 0
0 2 4 6 8 10 12 14
pH

1) NEUTRAL SIDE CHAIN

COOH COO- pKa2 = 9-10 COO-

pKa1 = 1.8-2.8

H3N+ H H3N+ H H2N H

R R R

H2A+ HA A-