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Chapter 3 - Amino Acids and Primary Structure of Proteins

Functions of proteins:
1- catalysts - enzymes for metabolic pathways
2- storage and transport - e.g. myoglobin and hemoglobin
3- structural - e.g. actin, myosin
4- mechanical work - movement of flagella and cilia, microtubule movement
during mitosis, muscle contraction
5- decoding information - translation and gene expression
6- hormones and hormone receptors
7- specialized functions - e.g. antibodies

Structure of amino acids

There are 20 common amino acids called α -amino acids because they all have an amino
(NH3+) group and a carboxyl group (COOH) attached to C-2 carbon (α carbon).

At pH of 7, amino group is protonated (-NH3+) and carboxyl group is ionized (COO-). The
amino acid is called a zwitterion.
pKa of a carboxyl group = 1.8 - 2.5
pKa of a amino group = 8.7 - 10.7

The α carbon is chiral or asymmetric ( 4 different groups are attached to the carbon;
exception is glycine.)

Amino acids exist as stereoisomers (same molecular formula, but differ in arrangement of
Designated D(right) or L(left).
Amino acids used in nature are of L configuration.

carboxylate group at top --> points away

side chain at bottom
α amino group orientation determines
NH3+ on left = L
NH3+ on right = D

Can also use RS system of nomenclature.


Structures of 20 common amino acids:

Amino acids are grouped based upon the properties and structures of side chains.

1) aliphatic (R groups consist of carbons and hydrogens)

glycine - R=H smallest a.a. with no chiral center
alanine - R=CH3 methyl group
valine R = branched; hydrophobic; important in protein folding
leucine R= 4 carbon branched side chain
isoleucine R = 2 chiral centers
proline R = ring; puts bends or kinks in proteins; contains a secondary amino

2) aromatic (R groups have phenyl ring)

phenylalanine - very hydrophobic
tyrosine - hydrophobic, but not as much because of polar groups
tryptophan - “

Absorb UV light at 280 nm --> used to estimate [protein]

3) sulfur-containing R groups
methionine - sulfur is internal (hydrophobic)
cysteine - sulfur is terminal --> highly reactive; can form disulfide bonds

4) side chains with alcohols

serine - β -hydroxyl groups --> hydrophilic
threonine - “

5) basic R groups
histidine - hydrophilic side chains - + charged at neutral pH
lysine - “
arginine - strong base

6) acidic R groups and amide derivatives

aspartate - β carboxyl group - confer - charges on proteins
glutamate - γ carboxyl group

asparagine - amide of aspartate - side groups uncharged --> polar

glutamine - amide of glutamate - “

Amide groups can form H bonds with atoms of other polar amino acids.


Ionization of Amino Acids

All amino acids are have a neutral net charge at physiological pH (7.4).

The α carboxyl and α amino groups and any other ionizable groups determine charge.

Each amino acid has 2 or 3 pKa values (7 amino acids have side chains that are ionizable)
(see Table 3.2). This complicates the basic titration curve, so that there are 3 inflection
points rather than 2.

At a given pH, amino acids have different net charges.

Can use titration curves for amino acids to show ionizable groups.
The isoelectric point (pI) is the pH at which the amino acid has no net charge
= zwitterion.
If pH > pI, amino acid would be negatively charged.
If pH < pI, amino acid would be positively charged.
If pH = pI, amino acid would have no charge.

Can use Henderson-Hasselbalch equation to calculate the fraction of group ionized at a

given pH.
a carboxyl group pKa 1.8-2.5
a amino group pKa 8.7-10.7

If pH< pKa, a greater amount of the group is protonated (NH3+ or COOH)

If pH > pKa, there is a greater ionization and a greater amount of unprotonated or anion
form (NH2 or COO-).
If pH = pKa, then [conjugate base] =[weak acid].
For the ionization of the carboxyl group of alanine,

pH = pKa + log [conjugate base]

[weak acid]

7 = 2.4 + log [RCOO-]


4.6 = log [RCOO-]


39810:1 meaning the anion predominates greatly (almost all COOH groups are

For the ionization of the α amino group of alanine,

7 = 9.9 + log [NH2] -NH3+ ---> NH2 = H+



-2.9 = log [NH2]


0.001:1 or 1 in 1000 molecules (undissociated group predominates)

At pI of 6.15, there is no net charge (all of the carboxyl groups are unprotonated, and none
of the amino group is unprotonated).

pI = (pKa1 + pKa2)/2

For R groups that are ionizable, the pI is not simply the average!!!

Peptide Bonds

The primary structure of a protein is the linear sequence of amino acids that are covalently
bonded to form a polypeptide chain.

Formed by condensation reaction in which a molecule of water is removed.

Each amino acid residue is called by replacing -ine or -ate with -yl
glycine ---> glycyl

The peptide bond is a planar bond with no rotation around C-N axis. If is also in the trans
form. Will talk about the consequences later.

Protein Purification Techniques

All work done at 4oC to minimize degradation.

1- preparation of the protein solution

With appropriate buffer, must first disrupt cells by mechanical
homogenization with either detergent and/or enzyme treatment.

Use a centrifuge to separate into pellet and supernatant.

2- fractionation (relies on protein solubility differences)

Use ammonium sulfate (salt) --> interferes with noncovalent bonds between protein
and other molecules. Remember solubility is based upon interactions of molecule
with water molecules via hydrogen bonds.

Different proteins precipitate out at different [salt].

Use centrifuge to remove precipitated protein --> resuspend in buffer.


Use dialysis to change out solvent and get rid of NH4SO4.

3- chromatography
Further fractionates proteins based upon protein’s interaction with matrix.
Most commonly used is column chromatography.
Uses beads or cellulose fibers.
Protein solution is washed through column.
Eluate collected and assayed for protein.

There are three types of column chromatography:

1) ion-exchange (anion or cation)
Separates based upon protein charge.
Elute by changing [salt].
2) gel filtration
Uses porous resin.
Separates based upon protein size.
3) affinity chromatography
Attach ligand to matrix. Can be substrate, antibody, etc.
Eluate using high [ligand] or high [salt].
Results in 1000-10,000 fold purification.

4- electrophoresis

Separates proteins based upon migration in an electric field.

PAGE - polyacrylamide gel electrophoresis

Uses acrylamide as gel matrix.

Separates based upon size and charge (buffer is slightly basic, so most
proteins have negative charge).

SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis

Uses SDS and 2-mercaptoethanol.

Separation based upon size only.

For both, must stain gel to visualize proteins.

Bands can be cut out of gel, protein electroeluted, and purified.


Amino Acid Composition of Proteins

1- amino acid analysis

Gives relative quantities of each amino acid, but nothing about order.

First must boil protein in 6N HCl for 24 hours to break peptide bonds.

Subject hydrolysate to ion-exchange chromatography at different pH’s OR use

phenylisothiocyanate (PITC) to generate derivations and subject result to HPLC.

1- acid hydrolysis converts asparagine to aspartic acid and glutamine to glutamic acid
2- also lose some serine, threonine, and tyrosine.

3- side chain of tryptophan is destroyed.

Amino Acid Residue Sequence

Use Edman degradation

Uses PITC reagent that reacts with the free N-terminus to form a PTC-peptide --->
treat with trifluoroacetic acid ---> last amino acid is cleaved ---> extract with organic
solvent (butyl chloride)---> PTH-amino acid ---> analyzed by chromatography.

Then take remaining peptide, remove next to last amino acid, etc.

Is now automated --> sequenator.

Good for small peptide of < 50 amino acid residues.

If protein is greater than 50 amino acid residues, must use proteases or chemical reagents
to cleave some of the peptide bonds.
1- cyanogen bromide - reacts with methionine residues - cuts on COOH side
2- proteases
trypsin - cleaves to carboxyl side of lysine and arginine
chymotrypsin - cleaves at aromatic and bulks nonpolar side chains
(phenylalanine, tyrosine, tryptophan)
Staphylococcus aureus V8 protease - cleaves to carboxyl side of
glutamate and aspartate

Need to use at least 2 different cleavage techniques to obtain overlapping sequences using
Edman degradation.