Edman degradation is a chemical method developed by a biochemist, Pehr Edman in 1950. This process is used for labeling or purification of proteins by removing the N-terminal residue of peptides without damaging the whole structure of the protein. It sequences the amino acids in short peptides (50-60 residues). For example, alpha and beta melanotropin, lysine, arginine vasopressors, and oxytocin.
After the invention of the amino acid analyzer by Spackman in 1958, it was easy to sequence larger polypeptides with Edman-degradation. Some examples are human hemoglobin (141-146 residues), chicken lysozyme, cytochrome (c), etc.
In 1967, Edman and his co-worker Geoffry Begg introduced the automation of Edman degradation. This helped in increasing the sequencing of amino acids in the polypeptide chains up to sixty steps in minimum time.
Edman Degradation Scheme
The following are the steps of Edman-degradation:
1. Coupling
Phenyl isothiocyanate (PITC) reacts with the peptide (alpha-amino group) in this step. This occurs under basic conditions. After this reaction, phenyl thiocarbamoyl derivative (PTC-peptide) is obtained.
2. Cyclization
The resultant product of the cyclization step is the 2-anilino-thiazo-linon (ATZ amino acid). The rest of the peptide goes for a further degradation cycle.
3. Conversion
In this step, ATZ-amino acid is converted into PTC or PTH amino acids depending on the condition(s). The final product of this step is PTH because it is more stable.
Chemistry of Edman Degradation
During the coupling reaction, reagent PITC reacts with the alpha-amino (α-NH2) terminal group of the peptide. This results in the formation of PTC-peptide. The temperature of the reaction is kept at about 45 – 48 oC and the reaction proceeds for 15 minutes. On the other hand, coupling reactions in older analyzers take a longer time and require higher temperatures.
When a strong acid such as trifluoroacetic acid (TFA) is added to the reaction, it causes cyclization. The first amino acid residue is released from the rest of the peptide chain by cleavage as ATZ amino acid. After the first cycle of degradation, the remaining peptide chains are ready for further degradation.
Edman-degradation allows the conversion of ATZ amino acid into a more stable PTH amino acid by reacting with aqueous acids. In 1975, Edman and Henschen identified PTH amino acids by paper chromatography and staining with reagents, such as iodine and starch.
Identification of PTH in the Edman sequencer is more effective than paper chromatography. During conversion, the formation of side products also takes place. These products interfere with the identification of PTH by chromatographic techniques.
Edman Degradation Sequencer
Several devices sequence the amino acids of polypeptide chains based on Edman degradation called sequencers or Edman sequencers. These sequencers are highly effective, sensitive, and durable. Amino acids are detected with high ultraviolet sensitivity from 254 nm to 268 nm.
Three reactions occur in the sequencer i.e. coupling, cyclization or cleavage conversion, and isomerization. Sequencers perform these reactions automatically and repeat them until they get the sequence. PTH amino acids released in the sequencers are identified by the high-performance liquid chromatography (HPLC) detection system.
Sequencers contain the following parts:
- Reactor
- Converter
- HPLC detection system
- Valve system
- Heaters
- Bottle system
- Inert gas supply
- A computer for data storage
Limitations of Edman Degradation
- Proteins that have blocked N-terminal amino acids cannot be sequenced through Edman-degradation.
- There are unmodified ‘cys’ and glycosylated residues that give a blank cycle.
- Poor results are obtained when impurities such as amine-containing chemicals interfere with Edman’s reaction.
Uses of Edman Degradation
- Edman degradation is used for sequencing amino acids in the proteins.
- There is no need for pre-treatment for the sample protein as required by other techniques.
- It can distinguish the isobaric amino acids, such as leucine and isoleucine.
- Proteins that are not even saved in the database can be identified too.
- Edman sequencers are very accurate and effective in the sequencing of amino acids.
- They do not damage other amino acids in the chain during the reaction.
Concepts Berg
What are the steps of Edman-degradation?
There are 3 steps in Edman degradation: coupling, cyclization, and conversion.
Is Edman-degradation enzymatic?
This degradation process is not enzymatic. There are different reagents used in Edman degradation. These chemical reagents provide an acidic or basic medium for reaction.
Why is Edman-degradation so useful?
It helps sequence the amino acids of polypeptide chains. It is used to purify the proteins by removing residues from the end of the peptides.
What are Edman-based sequencers?
They are devices that work based on Edman chemistry. It gives very effective results.
What reagent is used for Edman’s degradation?
Phenyl isothiocyanate (PTC) is used as an Edman reagent.
References
- Protein structure analysis by R.M. Kamp (Technische Fachhochschule Berlin), T.Choli Papadopoulou (Aristotle University, Thessaloniki, Greece), B. Wittmann Liebold (Max Delbruck Centrum, Germany).
