Edman s reagent

FIGURE 5.19 N-Tertninal analysis using Edman s reagent, phenylisothiocyanate. Phenylisothiocyanate combines with the N-terminus of a peptide under mildly alkaline conditions to form a phenylthiocarbamoyl substitution. Upon treatment with TFA (trifluo-roacetic acid), this cyclizes to release the N-terminal amino acid residue as a thiazolinone derivative, but the other peptide bonds are not hydrolyzed. Organic extraction and treatment with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative. 

Sequencing is a stepwise process of identifying the specific amino acids at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman s reagent, is... 

Phenyl isothiocyanate is also used for peptide sequencing in combination with colored Edman s reagent 4-W-,/V -dimethy]aminoazobenzene-4 -isothiocyanate (DABITC). Prieto (48) used this DABITC-PITC procedure to determine the partial terminal N-NH2 sequence of the low-molecular-weight peptide fraction from bread dough and bread. 

The primary structure (i.e., the amino acid sequence) of a protein can be determined by stepwise chemical degradation of the purified protein. By far the most powerful and commonly used technique for doing this is the automated Edman degradation. The amino terminal amino acid residue of the polypeptide is reacted with Edman s reagent (phenylisothiocyanate) to form the phenylthiocar-bamyl derivative, which is removed without hydrolysis of the other peptide bonds by cyclization in anhydrous acid. The amino acid derivative is converted to the more stable phenylthiohydantoin and identified by HPLC. The process can be repeated many times, removing the amino acids from the amino terminus of the polypeptide one residue at a time and identifying them until the entire sequence... 

Edman s reagent is also used to determine the amino acid sequence of a polypeptide chain from the N-terminal by subjecting the polypeptide to repeated cycles of Edman degradation. After every cycle, the newly liberated phenylthiohydantoin (PTH) amino acid was identified. The sequence of peptides containing 30-40 amino acids can be determined using a sequencer by adopting the Edman s degradation method. 

In HPLC, after the hydrolysate is treated with compounds such as Edman s reagent, the products are forced at high pressure through a stainless steel column packed with a stationary phase. Each amino acid derivative is identified according to its retention time on the column. Because the time required for amino acid analysis by HPLC (about 1 hour) is significantly shorter than that of other methods, it is becoming the method of choice. 

Each fragment is sequenced through repeated cycles of a procedure called the Edman degradation. In this method phenylisothiocyanate (PITC), often referred to as Edman s reagent, reacts with the N-terminal residue of each fragment. 

There are several ways to identify the N-terminal amino acid of a peptide or protein. One of the most widely used methods is to treat the protein with phenyl isothiocyanate (PITC), more commonly known as Edman s reagent. This reagent reacts with the N-terminal amino group, and the resulting thiazolinone derivative is cleaved from the protein under mildly acidic conditions. The thiazolinone derivative is extracted into an organic solvent and in the presence of acid, rearranges to a more stable phenylthiohydantoin (PTH). 

A nonapeptide undergoes partial hydrolysis to give peptides whose amino acid compositions are shown. Reaction of the intact nonapeptide with Edman s reagent releases PTH-Leu. What is the sequence of the nonapeptide ... 

Chemical reagents Edman s reagent Cyanogen bromide removes the N-terminal amino acid hydrolyzes on the C-side of Met... 

SOLUTION Acid hydrolysis shows that the polypeptide has 13 amino acids. The N-ter-minal amino acid is Leu (Edman s reagent), and the C-terminal amino acid is Val (carboxypeptidase A). 

The primary structure of a protein is the sequence of its amino acids and the location of all its disulfide bridges. The N-terminal amino acid of a peptide or protein can be determined with Edman s reagent. The C-terminal amino acid can be identified with carboxypeptidase. Partial hydrolysis hydrolyzes only some of the peptide bonds. An exopeptidase catalyzes the hydrolysis of a peptide bond at the end of a peptide chain. An endopeptidase catalyzes the hydrolysis of a peptide bond that is not at the end of a peptide chain. 

The Ai-terminus of a polypeptide can be determined by reaction with dansyl chloride, with Edman s reagent or with an aminopeptidase. 

After hydrolysis, the free amino acids are separated by ion exchange chromatography (section 2.3.2) or RP-HPLC (section 2.3.1). They can be identified according to their elution times and quantified according fo fheir elution volumes. To increase sensifivify, the amino acids are usually derivatised either pre- or post-column. Dansyl Chloride, Edman s reagent (Fig. 7.1) as well as ortho-phthalaldehyde (OPA) and 2-mercaptoethanol can be employed to form highly fluorescent adducts (Fig. 7.9) that can be detected easily. 

The remaining peptide has another amino unit, and it is treated with more phenyl isothiocyanate and then cleaved to examine and identify the next amino acid. If a pentapeptide (ala-val-ser-leu-ile) is subjected to the sequential Edman degradations by treatment with Edman s reagent and loss of the phenylthiohydantoin, each amino acid can be identified, in order. Because the process begins at the N-terminus and progresses toward the C-terminus, both the identity of the amino acids and their exact sequence in the peptide are known. This method can be used to identify from 30 to 60 amino acid residues in a long peptide under the right conditions. 

The major limitation of Stark s method was that the insoluble polymeric Edman s reagent kept the cleaved amino acid bound to it as a thiohydan-toin, while the residual peptide remained in solution. It was therefore necessary to apply a subtractive sequencing method by first carrying out... 

Chemical reagents Edman s reagent Cyanogen bromide Exopeptidases ... 

The primary structure of a protein is the sequence of its amino acids and the location of all its disulfide bridges. The N-terminal amino acid can be determined with Edman s reagent. The C-terminal amino acid can be identified with a carboxypeptidase. 

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