CHEM 160 MODULE 3.

PROTEINS

RESOURCE 6: PROTEIN SEQUENCING

DETERMINATION OF THE PRIMARY STRUCTURE OF PROTEINS

Significance of sequence analysis

1. Essential for understanding the protein’s mechanism of action

2. Important for the study of gene structure
3. Recognition of amino acid sequence has led to the chemical synthesis of medically useful
polypeptides

Determination of Amino Acid Composition

1. Break down the polypeptide chain into its constituent amino acids

Acid Hydrolysis

6 N HCl
Proteins free amino acids
o
100 C, 24 hrs

Disadvantage: leads to the destruction of trp, ser, cys, thr;

conversion of asn and gln to asp and glu, respectively

Base Hydrolysis

4 N NaOH
Proteins free amino acids
o
100 C, 10 hrs

Disadvantage: causes racemization

2. Separate the resulting free amino acids

3. Measure the amount of each amino acid

Common Strategy for Sequence Determination

1. Purification of the polypeptide

2. Cleavage of all disulfide bonds
3. Determination of the N-terminal and C-terminal amino acid residues
4. Break the polypeptide into fragments by internal cleavage at specific amino acid residues.
Separate the fragments and determine the amino acid composition of each.
5. Determine the amino acid sequence of each fragment to determine as much of the
sequence as possible.
6. Order the fragments by repeating steps 4 and 5, using a cleavage procedure of different
specificity to generate “overlap peptides.” This process will yield the complete amino acid
sequence.

CHEM 160 Module 3 Resource 6 1/2

Specificity of Cleavage Procedures Used for Sequence Analysis

Terminal Cleavages

Chemical Methods
Method Reagent Specificity
Sanger’s method 2,4-dinitrofluorobenzene Forms a DNP derivative with
(DNFB) N-terminal amino acid and
 amino group of lysine
Edman degradation Phenylisothiocyanate (PITC) Forms a PTH derivative with
N-terminal amino acid
Dansyl chloride treatment Dansyl chloride Forms a sulfonamide
derivative with N-terminal
amino acid
Hydrazinolysis Hydrazine Hydrolyzes all peptide bonds
and releases free C-terminal
amino acid
Enzymatic Methods
Method Specificity
Cleaves peptide bond involving the carboxyl side of N-terminal
Aminopeptidase
amino acids
Cleaves peptide bond involving the amino side of C-terminal
Carboxypeptidase
amino acids

Internal Cleavages

Chemical Method
Reagent Specificity
Cyanogen bromide Cleaves peptide bond involving the carboxyl side of met residues
Enzymatic Methods
Enzyme Specificity
Cleaves peptide bond involving the carboxyl side of basic amino acids
Trypsin
(arg, lys)
Cleaves peptide bond involving the carboxyl side of aromatic amino
Chymotrypsin
acids (phe, trp, tyr) and leu
Cleaves peptide bond involving the amino side of aromatic amino acids
Thermolysin (phe, trp, tyr) and amino acids with bulky non-polar side chains (leu,
ile, val)

Source:

Lecture Booklet in Biochemistry. November 2014. Biochemistry and Agricultural Chemistry Division,
Institute of Chemistry, University of the Philippines Los Baños, College, Laguna. pp. 12-13

CHEM 160 Module 3 Resource 6 2/2