Protein Sequencing: Sanger, Dansyl, Disulphide Bonds, Edman Degradation (33 mins) | Lecture 100/140 | ASRB NET (ICAR-NET) Animal Biochemistry

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BioinformaticsDNA and Protein Sequence Analysis

Sanger sequencing

Sanger used the reagent 2, 4-dinitrofluorobenzene, which forms a yellow dinitrophenyl (DNP) derivative at terminal amino groups without breaking any peptide bonds.

When the protein is subsequently hydrolysed to break its peptide bonds, the N-terminal amino acid retains its DNP label and can be identified when the hydrolysis products are separated by chromatography.

Dansyl chloride

The fluorescent compound 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) reacts with primary amines to yield dansylated polypeptides.

The treatment of a dansylated polypeptide with aqueous acid at high temperature hydrolyses its peptide bonds. This liberates the dansylated N-terminal residue, which can then be separated chromatographically from the other amino acids and identified by its intense yellow fluorescence.

Disulphide bonds

Disulfide bonds can be reductively cleaved by treating them with 2-mercaptoethanol or another mercaptan (a compound that contains an — SH group).

The resulting free sulfhydryl groups are then alkylated, usually by treatment with iodoacetate, to prevent the re-formation of disulfide bonds through oxidation by oxygen.

Edman degradation

In this process (named after its inventor, Pehr Edman), phenylisothiocyanate (PITC; also known as Edman՚s reagent) reacts with the N-terminal amino group of a polypeptide under mildly alkaline conditions to form a phenylthiocarbamyl (PTC) adduct. This product is treated with anhydrous trifluoroacetic acid, which cleaves the N-terminal residue as a thiazolinone derivative but does not hydrolyse other peptide bonds.

Edman degradation therefore releases the N-terminal amino acid residue but leaves intact the rest of the polypeptide chain. The thiazolinone-amino acid is selectively extracted into an organic solvent and is converted to the more stable phenylthiohydantoin (PTH) derivative by treatment with aqueous acid. This PTH-amino acid can later be identified by chromatography.