- amino acid sequence is read from N-terminal to C-terminal on a polypeptide
- chemical strategy for determining amino acid sequence of a protein has six
1. If the protein contains more than one polypeptide chain, he chains are
separated and purified.
2. Intrachain disulfide cross-bridges between cysteine residues in the
polypeptide chain are cleaved. If the disulfides are interchain linkages,
then step 2 precedes the first step.
3. The N-terminal and C-terminal residues are identified.
4. Each polypeptide chain is cleaved into smaller fragments, and the amino
acid composition and sequence of each fragment are determined.
5. Step 4 is repeated, using a different cleavage procedure to generate a
different and therefore overlapping set of peptide fragments.
6. The overall amino acid sequence of the protein is reconstructed from the
sequences in overlapping fragments.
Step 1 – Separation of Polypeptide Chains
- if protein is heteromultimer, it must be dissociated into its polypeptide chains
- polypeptide chains must be separated from one another and sequenced
individually by size/charge
- some heteromultimers are linked by interchain disulfide bridges – crosslinks
must be cleaved before dissociation and isolation of chains
Step 2 – Cleavage of Disulfide Bridges
- disulfide bridges must be cleaved in a way so that the original or new disulfide
bridges do not form
- oxidation of a disulfide by performic acid forms two equivalents of cysteic acid
– electrostatic repulsion prevents disulfide recombination
- sulfhydryl compounds also reduce disulfide bridges to two cysteine side chains
– may create other disulfide bonds – alkylating agents prevent recombination
by modifying the SH groups and blocking disulfide bridge formation
Step 3A – N-terminal Analysis
- the amino acid at the N-terminal can be identified by Edman degradation –
identifies the sequence starting at the N-terminal
- phenylisothiocyanate (Edman reagent) combines with