What is the difference between dansyl chloride and Edman degradation? I’ve been
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What is the difference between dansyl chloride and Edman degradation? I’ve been trying to read up on it, but they both sound really similar (they both deal with the N-terminal amino acid, etc.) What would be the beginning and end product of each? What is the difference between dansyl chloride and Edman degradation? I’ve been trying to read up on it, but they both sound really similar (they both deal with the N-terminal amino acid, etc.) What would be the beginning and end product of each? What would be the beginning and end product of each?Explanation / Answer
Dansyl chloride or l-dimethylaminonaphthalene-5-sulfonyl chloride (C12H12ClNO2S) is a reagent that reacts with the free amino groups of peptides and proteins. It was developed by Gray and Hartley for use with peptide but now has also been applied to proteins. The substituted peptide is subjected to total acid hydrolysis (6M HCl) at 1100C to yield a mixture of free amino acid and the dansyl derivative of the N-terminal amino acid.
The SO2Cl group of dansyl chloride forms a bond with the free amino group of the N-terminal amino acid of the protein to form the dansyl derivative of N-terminal amino acid. The bond between the dansyl group and the N-terminal amino acid is resistant to acid hydrolysis. Detection is by UV fluorescence as the dansyl amino acid is fluorescent under UV light. Minute amounts of the N-terminal amino acid can be detected, as the derivative is fluorescent. The derivative can be identified subsequently by thin layer chromatography under polyamide sheets.
It is a sensitive method to identify amino acid and can be used in conjunction with Edman degradation. As all amino acids are released as free amino acid, only one N-terminal amino acid can be identified in each reaction setup. Hence, this method is tedious and complicated. Further, dansyl chloride binds the amide groups, thereby binding to both amino group and side chain of the amino acid. Hence, erroneous results can be obtained.
Edman degradation has an advantage over dansyl chloride method. Edman degradation only identifies the N-terminal amino acid. The bonds between all other amino acids are not affected. In this method, the protein is treated with Edman’s reagent (phenyl isothiocyanate).
Phenyl isothiocyanate reacts with the N-terminal amino acid. It forms a cyclic compound Phenyl thiohydantoin derivative (PTH–amino acid) of N-terminal amino acid under mild acidic condition, which is then released. Amino acid of PTH –amino acid derivative is identified by chromatography. The N-terminal amino acid is detected in the first cycle. However, this method fails if the N-terminal amino acid is blocked in any way. After the first cycle, the next amino acid becomes the N-terminal amino acid and reacts with the Edman reagent. The cycle continues until the end of the protein or until a disulphide bond is encountered in the sequence.
PTH-cysteine derivative remains attached with polypeptide and are not released. Hence, all disulphide bonds need to be reduced before Edman degradation. This method cannot be used for proteins containing more than 50 amino acids as the efficiency decreases after that.
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