Protein Analysis

- Protein isolates are characterized/analyzed after each step of purification

 Determination of protein concentration
 Determination of number of subunits
 molecular weight determination
 Assessment of biological activity (especially for enzymes)
Protein Content Determination
1. BIURET ASSAY
• Reagent used: strongly alkaline solution containing Cu2+
• Forms a purple colored solution upon complex formation of Cu2+ with peptides with at
least three amino acid residues
• Intensity of purple color is directly proportional to the amount of protein
• Purple complex absorbs at 540 nm.

2. BRADFORD ASSAY
• Reagent used: Coomasie Brilliant Blue G-250
• Dye-binding method
• CBB G-250 binds with the protein through hydrophobic and electrostatic interactions
• Bradford reagent is initially red; Protein-Bradford reagent mixture is blue
• Blue color absorbs at 595 nm
3. LOWRY ASSAY

Summary

Protein sequencing is mainly relying on chemical or enzymatic digestion methods to separate

peptides and detect the amount and composition of amino acid residues. This post will introduce the
principles and experimental steps of protein sequencing.

Definition

Currently, the so-called protein sequencing refers to the detection of proteins’ primary structure,
which contains the number of polypeptide chains in proteins. Polypeptides and proteins can be used
equally in many cases. Amino acid sequence of polypeptides is the biological function of proteins.
Sequencing steps

1. Splitting polypeptide chain

Protein molecules should be separated and purified. Several polypeptides are combined together by
non-covalent bond, which is known as oligomeric protein. For example. 8 mol/L urea or 6mol / L
guanidine hydrochloride can be used to deal with tetramer—Hb and dimer—Enolase.

2. Detecting the number of polypeptide in protein molecules

The number of polypeptides can be determined by detecting the relationship between the number of
moles of amino acid residues and protein molecular weight.

3. Breaking disulfide bonds

Several polypeptides chains are linked by disulfide bonds. Disulfide bonds will be reduced to thiol
with excessive & [beta]- mercaptoethanol under the condition of 8mol / L urea or 6mol / L guanidine
hydrochloride. And then it should be protected by alkyl reagents from re-oxidation.

Cleaving and protecting disulfide bonds

A. performic acid: -CH2SO3H
B. Reduction + oxidation: Mercaptoethanol, DTT +iodoacetic acid, -S-CH2-COOH
C. Sulfurous acid decomposition: -R1-S-S-R2 + HSO3-, R1-S- + R2-S-SOH3

4. Detecting the amino acid composition of polypeptide chains and calculating the molecular
ratio of amino acid composition.

5. Sequencing N-terminal and C-terminal of polypeptide chains

Amino acid of polypeptides is divided into two categories: amino-terminal and carboxyl-terminal. The
N-terminal is much more important in the analysis of amino acid sequence of peptide chains than C-
terminal.

6. Polypeptide can be cleaved into several small peptides. More than two methods can be used
to break peptide samples into two or more sets of peptides or peptide fragments and then separate
them.

7. Determining the amino acid sequencing of each peptide

8. Determining the sequence of peptide fragments in polypeptide chains

9. Determining the position of disulfide bonds in the original polypeptide chain

Generally, pepsin will be used to deal with those polypeptide chains with disulfide bonds. And then
2D-electrophoresis technology will be used to separate each peptide fragment. Analyzing the
composition and sequence of peptide fragments, which may contain disulfide bonds. And then
comparing it with other peptide fragments, which are analyzed by other methods, to determine the
position of disulfide bonds.
What is the difference between chemical synthesis and enzymatic synthesis
(IVT)?
The Chemical ligation method ensures the exact sequence that is requested to be made, and certain
modifications and labeling could also be incorporated. With the chemical method, we can make
up to 250 bases long. The chemical method also has a very low yield, so the overall cost is higher
than the alternative method.
The Enzymatic method (IVT) allows very long sequences (up to 3,000 and higher) to be generated
and has a much higher yield. The difference to this method is that the sequence could have three
added bases leading the sequence as result generated by the promoter. Long RNA synthesis is
generated using bacteriophage polymerase such as T7, T3 or SP6. Depending on the complexity
and length of the sequence, if the RNA is generated using polymerase T7 and T3, it could generate
an additional GGG and SP6 could generate an additional GAA as a result of promoter sequence.
For most molecular biology applications, three extra bases on the 5' end shouldn't interfere with
anything considering the much longer entire RNA.