How Are Proteins Isolated and Purified from

Cells?

• The thousands of proteins in cells can be

separated and purified on the basis of size
and electrical charge
• Proteins tend to be least soluble at their
isoelectric point
• Increasing ionic strength at first increases
the solubility of proteins (salting-in), then
decreases it (salting-out)

How Is the Amino Acid Analysis of Proteins

Performed?

• Acid hydrolysis liberates the amino acids

of a protein
• Note that some amino acids are partially or
completely destroyed by acid hydrolysis
• Chromatographic methods are used to
separate the amino acids
• The amino acid compositions of different
proteins are different
 How is the Primary Structure of a Protein
Determined?

• The sequence of amino acids in a protein

is distinctive
• Both chemical and enzymatic
methodologies are used in protein
sequencing

Frederick Sanger was the first - in 1953, he

sequenced the two chains of insulin.

• Sanger's results established that all of the

molecules of a given protein have the
same sequence.
• Proteins can be sequenced in two ways:
- real amino acid sequencing
- sequencing the corresponding DNA in
the gene
 Determining the Sequence
An Eight Step Strategy

• 1. If more than one polypeptide chain,

separate.
• 2. Cleave (reduce) disulfide bridges
• 3. Determine composition of each chain
• 4. Determine N- and C-terminal
residues

Determining the Sequence

An Eight Step Strategy

• 5. Cleave each chain into smaller

fragments and determine the
sequence of each chain
• 6. Repeat step 5, using a different
cleavage procedure to generate a
different set of fragments.
 Determining the Sequence
An Eight Step Strategy

• 7. Reconstruct the sequence of the

protein from the sequences of
overlapping fragments
• 8. Determine the positions of the
disulfide crosslinks

Step 1:
Separation of chains

• Subunit interactions depend on weak

forces
• Separation is achieved with:
- extreme pH
- 8M urea
- 6M guanidine HCl
- high salt concentration (usually
ammonium sulfate)
 Step 2:
Cleavage of Disulfide bridges

• Performic acid oxidation

• Sulfhydryl reducing agents
- mercaptoethanol
- dithiothreitol or dithioerythritol
- to prevent recombination, follow with an
alkylating agent like iodoacetate

Step 3A:
Identify N- and C-terminal residues

• N-terminal analysis:
– Edman's reagent
– phenylisothiocyanate
– derivatives are phenylthiohydantoins
– or PTH derivatives
 Step 3B: :
Identify N- and C-terminal residues

• C-terminal analysis
– Enzymatic analysis (carboxypeptidase)
– Carboxypeptidase A cleaves any residue
except Pro, Arg, and Lys
– Carboxypeptidase B (hog pancreas) only
works on Arg and Lys

Steps 4 and 5:

Fragmentation of the chains

• Enzymatic fragmentation
– trypsin, chymotrypsin, clostripain,
staphylococcal protease
• Chemical fragmentation
– cyanogen bromide
 Enzymatic Fragmentation
• Trypsin - cleavage on the C-side of Lys, Arg
• Chymotrypsin - C-side of Phe, Tyr, Trp
• Clostripain - like trypsin, but attacks Arg
more than Lys
• Staphylococcal protease
– C-side of Glu, Asp in phosphate buffer
– specific for Glu in acetate or bicarbonate
buffer

Chemical Fragmentation
Cyanogen bromide

• CNBr acts only on methionine residues

• CNBr is useful because proteins usually
have only a few Met residues
 Reconstructing the Sequence

Compare cleavage by trypsin and staphylococcal

protease on a typical peptide:
• Trypsin cleavage:
A-E-F-S-G-I-T-P-K L-V-G-K
• Staphylococcal protease:
F-S-G-I-T-P-K L-V-G-K-A-E

Reconstructing the Sequence

L-V-G-K A-E-F-S-G-I-T-P-K L-V-G-K-A-E

F-S-G-I-T-P-K

• Correct sequence:
L-V-G-K-A-E-F-S-G-I-T-P-K
 Amino Acid Sequence Can Be Determined
by Mass Spectrometry

• Mass spectrometry separates particles on

the basis of mass-to-charge ratio
• Fragments of proteins can be generated in
various ways
• MS can also separate these fragments

What is the Nature of Amino Acid

Sequences?

• Sequences and composition reflect the

function of the protein
• Membrane proteins have more
hydrophobic residues, whereas fibrous
proteins may have atypical sequences
• Homologous proteins from different
organisms have homologous sequences
• e.g., cytochrome c is highly conserved