Amino Acid Sequences can be determined by Automated Edman Degradation
The amino acid composition of the peptide is determined. The peptide is hydrolyzed into its constituent amino acid by heating it in 6 N HCl at 110°C for 24 hours. Stanford Moore and William Stein showed that amino acids in hydrolysates can be separated by ionexchange chromatography on columns of sulfonated polystyrene and quantitated by reacting them with ninhydrin.
Amino acids treated this way give an intense blue color, except for praline, which gives a yellow color because it contains a secondary amino group. The concentration of amino acid in a solution is proportional to the optical absorbance of the solution after heating it with ninhydrin. This technique can detect a microgram (10nmol) of an amino acid, which is about the amount present in a thumbprint.
As little as a nanogram (10 pmol) of an amino acid can be detected by means of fluorescamine, which reacts with the a-amino group of a highly fluorescent product.The identity of the amino acid is revealed by its elution volume, which in the volume of buffer used to remove the amino acid from the column.
The amino-terminal residue of a protein or peptide can be identified by labeling it with a compound that forms a stable covalent link.
Fluorodinitrobenzene (FDNB) was first used for this purpose by Frederick Sanger. Dabsyl chloride is now commonly used because it forms intensely colored derivatives that can be detected with high sensitivity. It reacts with an uncharged a-NH2 group to form a sulfonamide derivative that is stable under conditions that hydrolyze peptide bonds.
Although the dabsyl method for determining the amino-terminal residue is sensitive and powerful, it cannot be used repeatedly on the same peptide because the peptide is totally degraded in the acid-hydrolysis step. Pehr Edman devised a method for labeling the amino-terminal residue and cleaving it from the peptide without disrupting the peptide bonds between the other amino acid residues. The Edman degradation sequentially removes one residue at a time from the amino end of a peptide. Phenyl isothiocyanate reacts with the uncharged terminal amino group of the peptide to form a phenylthiocarbamoyl derivative. Then, under mildy acidic conditions, a cyclic derivative of the terminal amino acid is liberated, which leaves an intact peptide shortened by one amino acid.
Analyses of protein structures have been markedly accelerated by the development of sequenators, which are automated instruments for the determination of amino acid sequence. In a liquid-phase sequenator, a thin film of protein in a spinning cylindrical cup is subjected to the Edman degradation. The reagents and extracting solvents are passed over the immobilized film of protein, and the released PTH-amino acid is identified by high-pressure liquid chromatography (also called high-performance liquid chromatography, HPLC).
One cycle of the Edman degradation is carried out in less than two hours. By repeated degradations, the amino acid sequence of some fifty residues in a protein can be determined. Gas-phase sequenators can analyze picomole quantities of peptides and proteins. This high sensitivity makes it feasible to analyze the sequence of a protein sample eluted from a single band of an SDS-polyacrylamide gel. display_block('edman'); ?>