Peptides

Codons

Codons

http://libgallery.cshl.edu/items/show/51962

Amino acids react with each other to

form di-, tri-, oligo-, and poly-peptides

A polypeptide has an amino terminus

and a carboxyl terminus

N-terminal

C-terminal
5

Name
the AA
residues

Each protein has

a unique size,
composition and
sequence of
amino acids

Molecular weight (Dalton)

Peptide hydrolysis
Acidic hydrolysis
Basic hydrolysis
Enzymatic hydrolysis

Acidic and Basic Hydrolysis

H2SO4

(4M) or HCl (6M), boil for 20 hours,

get all L-amino acid, but tryptophan has
been destroyed.

Basic, NaOH (5M), boil 10-20 hours, many

kinds of amino acid has been destroyed,

produce D- L- amino acid, but tryptophan
is stable
10

Enzymatic Hydrolysis

Protease: partial hydrolysis, amino acids are

intact
Trypsin
Chymotrypsin
Peptin

11

How can the amino acid sequence

of a protein be identified?
Genomic Method

Biochemical Method

Sequence the gene

coding for Insulin

Isolate Insulin to
homogeneity

Sequence the mRNA

for Insulin in pancreatic
cells

Determine the amino

acid sequence of
Insulin using a direct
chemical method
12

Frederick Sanger:
Noble Prize in Chemistry, 1958, 1980

13

Insulin- the first

protein to be
sequenced
How many cysteines?

How amino acids

link together to
form insulin?

14

15

Step 1: Isolate and purify the protein of

interest
Step 2: Reduce disufide bonds with DTT:
(Dithiothreitol)

Step 3: Find out how many polypeptides

the protein has
16

Step 3: React amino-terminal

residue with FDNB

17

The amino terminal residue

becomes a DNP-derivative

Concentrated HCl hydrolyzes all

the peptide bonds
18

Now we have a labeled amino acid

How is that helpful?

The phenolic ring absorbs light at
270 nm. Allowing for detection
after chromatography
19

Other common reagents for labeling of

amino acids

20

21

In amino acid chromatography, more than

one physical property is used to separate
molecules one from the other

Net electric charge

Hydrophobicity

22

23

Polystyrene Sulphonate

Negatively charged at a wide pH range

Hydrophobic backbone
Amino acids separate based on their
partitioning between the hydrophilic,
uncharged mobile phase, and the
hydrophobic, charged, stationary phase
24

Polystyrene Sulphonate
Negatively charged amino acids flow through
Positive amino acids interact with the resin
and therefore move more slowly
Hydrophilic amino acids have lower affinity for
the resin than hydrophobic ones
If net charge is the same, hydrophilic amino
acids will elute earlier from the column
compared to hydrophobic ones
25

Absorbance, O.D.

It is possible to resolve most of the 20

amino acids in one run

Elution time, min.

26

Absorbance, O.D.

Separations are highly reproducible

Elution time, min.

27

This procedure allows identification

of N-terminal amino acid of a polypeptide

28

Sequencing by Edman Degradation

Invented by Edman Pehr
Requires an isolated single polypeptide
The peptide is immobilized by its carboxylterminus to a solid support
The maximal practical length for sequencing
by this method is ~50 residues

29

Step 4: Edman
Degradation

React aminoterminus with

phenylisothiocyanate
Alkaline conditions
(PITC)
30

PITC-adduct

PITC-adduct

31

Proteolytic cleavage of polypeptides

32

Proteolytic cleavage of polypeptides

33

The action of
Cyanogen
Bromide

34

Peptide Mapping

35

36

A nonapeptide was determined to have the following amino acid

composition: (Lys)2, (Gly) 2, (Phe) 2, His, Leu, Met. The native
peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB)
and then hydrolyzed; 2,4-dinitrophenylhistidine was identified
by HPLC. When the native peptide was exposed to cyanogen
bromide (CNBr), an octapeptide and free glycine were
recovered. Incubation of the native peptide with trypsin gave a
pentapeptide, a tripeptide, and free Lys. 2,4Dinitrophenylhistidine was recovered from the pentapeptide, and
2,4-dinitrophenylphenylalanine was recovered from the
tripeptide. Digestion with the enzyme pepsin produced a
dipeptide, a tripeptide, and a tetrapeptide. The tetrapeptide was
composed of (Lys) 2, Phe, and Gly. The native sequence was
determined to be: