04-26-2014, 07:44 PM
Edman Degradation
Protein sequencing is the way by which we can determine the correct amino acid sequence in the protein. One of the best methods for this is the Edman degradation technique, developed by Pehr Edman, Swedish biochemist, during the middle of the 20th century. The basic principle behind this technique lies in the cyclical derivatization and cleavage of the labeled terminal amino acid residues. It was first done manually, but it was automated later on - machines were built for this process resulting in the less time needed for the procedure to be performed.
One of the main limitations of the Edman degradation sequencing method is that it works only on short polypeptides (up to 50 amino acids long), because the process cannot be performed fully on the longer ones. This limitation is solved by producing more small peptides form the larger one by cleaving it with trypsin, e.g. (it cleaves only peptide bonds that are made by the carboxyl groups of arginine and lysine).
Once smaller peptides are generated, the process can be initiated with the derivatization of amino acids. The first derivative formed from the terminal amino acid is phenylthiocarbamoyl (under alkaline conditions), the second derivative is thiazolinone, which is then cleaved (under acidic conditions) and the third derivative is phenylthiohydantoin which increases the stability and allows the amino acid to be identified. The identification itself can be performed using, for example, high performance liquid chromatography.
This process described is only for one amino acid and it must be done as many times as needed in order to identify the whole peptide chain. One cycle, determination of one amino acid, lasts approximately one hour.
There are other methods for determining the amino acid sequence in the peptide, like dabsyl method, but the advantage of Edman degradation is that it does not disrupt the rest of the chain; it only cleaves the terminal residue, plus it can be performed with small amount of peptides (about 10 to 100 pico-moles).
Protein sequencing is the way by which we can determine the correct amino acid sequence in the protein. One of the best methods for this is the Edman degradation technique, developed by Pehr Edman, Swedish biochemist, during the middle of the 20th century. The basic principle behind this technique lies in the cyclical derivatization and cleavage of the labeled terminal amino acid residues. It was first done manually, but it was automated later on - machines were built for this process resulting in the less time needed for the procedure to be performed.
One of the main limitations of the Edman degradation sequencing method is that it works only on short polypeptides (up to 50 amino acids long), because the process cannot be performed fully on the longer ones. This limitation is solved by producing more small peptides form the larger one by cleaving it with trypsin, e.g. (it cleaves only peptide bonds that are made by the carboxyl groups of arginine and lysine).
Once smaller peptides are generated, the process can be initiated with the derivatization of amino acids. The first derivative formed from the terminal amino acid is phenylthiocarbamoyl (under alkaline conditions), the second derivative is thiazolinone, which is then cleaved (under acidic conditions) and the third derivative is phenylthiohydantoin which increases the stability and allows the amino acid to be identified. The identification itself can be performed using, for example, high performance liquid chromatography.
This process described is only for one amino acid and it must be done as many times as needed in order to identify the whole peptide chain. One cycle, determination of one amino acid, lasts approximately one hour.
There are other methods for determining the amino acid sequence in the peptide, like dabsyl method, but the advantage of Edman degradation is that it does not disrupt the rest of the chain; it only cleaves the terminal residue, plus it can be performed with small amount of peptides (about 10 to 100 pico-moles).
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