Best Edman degradation assignment help
Edman degradation is an organic chemical reaction that cleaves the C-C bonds in a peptide or protein by beta elimination. This process is also called “sequencing” because it can be used to generate amino acid sequences from polypeptides. It was developed by Nobel Prize winner, Richard Edman who published his findings in 1955. Are you looking for Top Best Edman degradation assignment help? Worry no more! We got you covered!
His discovery has been instrumental not only for biological research but also in drug development. The process of sequencing proteins using this technique is time consuming and requires many steps to produce results.
Edman degradation is a collection of chemical reactions used in biochemistry to cleave the peptide bonds within proteins or peptides. It allows for sequencing of the amino acid sequence within a polypeptide chain and was developed by Nobel Prize winner, Richard Edman who published his findings in 1955.
Uses of Edman Degradation
This method has been instrumental not only for biological research but also in drug development. This is because it can be used to test the protein sequence. It also checks if there are any abnormalities found in the protein structure. It can also be used to determine half-life of proteins and peptide chains, to check protein synthesis and to test the purity of a protein.
Protein sequencing is one of the most important applications of this method. There are many other uses for Edman degradation, such as: determining peptide sequence, confirmation of Nterminal residue in a protein and also analysis for post-translational modifications such as phosphorylation and glycosylation.
Benefits of Edman Degradation
Edman degradation is an easy method and can be used to determine protein sequence. It is more specific than the other protein sequencing methods such as the Sanger method, it only requires pure proteins (unlike liquid chromatography) and there are no hazardous chemicals required in this procedure. Edman degradation is widely used in research, especially in genetics and clinical fields because it can be used to find mutations and diseases that are related to the sequence of proteins.
Edman Degradation Reaction
Edman degradation is a chemical reaction that involves N-terminal sequencing where the C-terminal end of polypeptide is bound with phenylisothiocyanate (PITC) to cleave the peptide bonds. The C-terminal end of the protein is first combined with OH-phenylisocyanate (OPIC) to form an N-acylphenylisothiocyanate (APITC). This APITC then breaks down into phenol and PITC which can be detected through a color change. This is an irreversible method and the product obtained is the amino acid attached to the side chain of OPIC.
After separation, it can be used to determine which amino acid residue is at the C-terminal end of each protein chain where it cleaves off one by one until a stopping point has been reached. The stop points are usually the amino acid residues which do not have a codon, such as phenylalanine and tryptophan.
Edman Degradation Performance
An Edman degradation reaction involves four steps: denaturation, reduction, acylation and finally chromatographic separation. These steps are then repeated until an N-terminal amino acid is detected. Denaturation
Proteins need to be in the denatured state in order for them to detach from their tendrils and also so that they can expose their C-terminal end. This is carried out by heating the protein solution at 100°C for 5 minutes or by adding 1% trifluoroacetic acid (TFA) to the protein solution.
Reduction is carried out by adding 1 mM of dithiothreitol (DTT) to the denatured protein solution for 10 minutes at room temperature under constant stirring. This step reduces all disulfide bridges in the protein.
To carry out an acylation reaction, 1 mM of PITC is added to the reduced protein solution and stood at room temperature for 10 minutes under constant stirring. This step replaces free cysteines with PITC-cys, which can later be removed in step 5.
After the acylation reaction, one has to perform chromatographic separation of all possible N-acetyl-N-protected amino acids in order to get the actual N-terminal sequence of the protein. The column used is a cation exchange column for this purpose and is usually a silica or cellulose column.
Reagents in Edman degradation
The reagents used in an Edman degradation reaction include: phenylisothiocyanate (PITC) and phenylisocyanatminoethylthiol (OPIC). Enzymes used are peptide N-glycosidase F (PNGaseF) and Endopeptidase 24.11 (Endo24.11), which is a protease that break down proteins into smaller polypeptides for sequencing purposes.
Types of Edman Degradation
There are three types of Edman degradation reactions: manual, semi-automated and automated.
In a manual method, 100 mg of protein is dissolved in 0.5 ml of 7% trifluoroacetic acid (TFA) along with 1 mM phenylisocyanatminoethylthiol (PIC). The reaction is left to stand for 10 minutes at 0 °C. This causes the C-terminal amino acid to be lost as ammonia and phenol which can then be detected using a color change.
This method uses different amounts of protein according to the different types of Edman’s degradation machines:
The manual and semi-automated methods use the same reagents as the manual method. The difference between them is that in a semi-automated reaction, an automatic injector injects microliter amounts of protein solution into a peptide sequenator while in a manual reaction, users manually inject the samples into the peptide sequenator.
In an automated Edman degradation, a fixed amount of protein solution is placed in an injection vial which is left to stand for 1 hour at 37°C. The Vials are then vortexed and injected into the analyzer which will run the reactions and use a software to analyze the data. The analyzers for automated Edman degradation include: Vydac C18 Protein Sequencing, Dionex P680 Peptide Sequencer and Beckman P/ACE MDQ.
Edman degradation conditions
The process is affected by extreme conditions. For example, denaturation of protein is extremely sensitive to pH and temperature and if the reaction is carried out at extreme pH or high temperatures, then the N-terminal amino acid may be lost. Also, if the reduction reactions are not done properly or a lot of oxidizing agents are present in the sample, then conversion of cysteine to thiol will occur and this would result in a mixture of glycyl- and N-acetyl-N-protected amino acids which can further lead to misidentification of the N-terminal residue.
Edman degradation cost
The equipment needed for performing an Edman degradation reaction costs $20,000 and each analysis lasts 15 minutes and requires about 10 mg of protein in 0.1 M ammonium bicarbonate buffer (pH 8). This means that one analysis costs about $0.30 and for 30,000 proteins the total cost would be around $9,000.
Protein Sequencing Methods
There are many other protein sequencing methods than Edman degradation such as: Sanger method, MS/MS, liquid chromatography and HPLC. Furthermore, there are some newer methods which include: ion trap MS, matrix-assisted laser desorption/ionization (MALDI) and nano LC.
Detection of products
After the Edman degradation reaction, the peptide is often detected with a UV detector or an evaporative light detector (ELD). There are also other detectors that can be used such as: fluorimeters, spectrophotometers, and charged aerosol detectors. This enzyme assay has also been used to determine the secondary structure of proteins through fluorescence measurements with an antibody probe.
Disadvantages of Edman Degradation
The major problem with Edman degradation is that it can fail. It totally depends on the purity of the protein which might be contaminated by other proteins, nucleic acids or sugars from different organisms.
Another disadvantage is some proteins cannot be analyzed using the Edman degradation because they have modified amino acids or modifications on their side chains which can interfere with the reaction. Also, proteins with charged residues at their C-terminus will not give good results during sequencing.
Future Applications of Edman Degradation
The method is still widely used, but there are new methods which require less labor and time to sequence samples. They are based on mass spectrometry. Other future applications of Edman degradation include sequencing proteins that have longer peptides or sequencing proteins.
Edman degradation is a quick method which can be used to determine the amino acid sequence of an unknown protein. The reagents are cheap and there are no hazardous chemicals required in this procedure. Edman degradation use has many benefits including that it tests the purity of proteins, it gives quick results, has low detection limits and it can be used with mass spectrometry which gives accurate results. However, it fails with hydrophobic or charged amino acid residues at the C-termini of proteins, also some proteins cannot be analyzed using this method because they have modified amino acids.
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