Protein Edman sequential degradation

A limited amount of information can be obtained by the use of proteolytic enzymes that detach either amino acids or dipeptides sequentially from the C-terminus. They are thus complementary to the aminopeptidases and dipeptidyl aminopeptidases. Two pancreatic enzymes, carboxypeptidases A and B, differ in specificity. The former preferentially liberates C-terminal amino acids with aromatic side chains, somewhat less readily amino acids with alkyl side chains and, more slowly still, other amino acids, but not Pro, Arg, Lys and His. In contrast, carboxypeptidase B releases only C-terminal Arg, Lys and His. Carboxypeptidase Y is much less specific and is capable of removing all amino acids, although Gly and Pro are liberated only slowly. As with aminopeptidases, it is advisable to analyse the hydrolysate at intervals in order to determine the C-terminal sequence of amino acids. An interesting recent development (Carles et al., 1988) uses carboxypeptidase to effect transpeptidation between the protein being sequenced and a tritiated amino acid. The labelled protein is then degraded by various specific methods and then the labelled fragments are isolated by gel electrophoresis and subjected to Edman degradation. 

Taking the purified protein, the N-terminal sequence of 27 amino-acids was determined by Edman automatic sequential degradation ... 

GLC is an important adjunct to protein sequence determination. Automatic "sequenators" based upon the approach developed by Edman are available and have been described in detail by Niall (60). The Edman degradation, summarized in Equation 9.5, makes use of methyl or phenylisothiocyanate which reacts with the N-terminus of a peptide. Exposure of the isothiocyanate derivative of the protein to acid results in cleavage of the terminal amino acid as a thiaxolinones and exposure of the next amine group on the peptide. Thus, the process can be repetitively carried out, each amino acid removed from the peptide, in a sequential manner. Thiazolinones rearrange in acid medium to form thiohydantoin derivatives of amino acids, some of which may be directly gas chromatographed others must be derivatized typically as trimethylsilyl derivatives. 

The N-terminal amino acid of a protein can be determined by reacting the protein with dansyl chloride or fluorodinitrobenzene prior to acid hydrolysis. The amino acid sequence of a protein can be determined by Edman degradation which sequentially removes one residue at a time from the N terminus. This uses phenyl isothiocyanate to label the N-terminal amino acid prior to its release from the protein as a cyclic phenylthiohydantoin amino acid. 

Traditional methods of C-terminal end determination55 were hydrazine degradation, carboxypeptidase digestion, and tritium labeling. Unfortunately, no chemical methods analyzing amino acid sequence sequentially from the C-terminal end is available with reliability similar to that of Edman degradation. Carboxypeptidase digests a protein and... 

Protein sequence determination uses the standard sequential Edman-degradation procedure to cleave of the N-terminal amino acid of the protein followed by the characterization of the released amino acid in each... 

Acid hydrolysis of purified [ Jr-metHuLeptin samples (1-3 nmol) was performed using 6 N HCl, 0.1% 2-mercaptoethanol, and 0.65% phenol at 110 C for 24 hr as described previously (12). The hydrolysates were dried, reconstituted, and injected into a Beckman 6300 amino acid analyzer for compositionad analysis. Sequential Edman degradation was performed on a Hewlett Packard GIOOOA automated protein sequencer using sequencing programs recommended by the manufacturer (Hewlett Packard Inc., Mountain View, CA). 

Determining the structure of a peptide or protein is carried out in several steps. The identity and amount of each amino acid present in a peptide is determined by amino acid analysis. The peptide is hydrolyzed to its constituent a-amino acids, which are then separated and identified. Next, the peptide is sequenced. Edman degradation by treatment with phenyl isothiocyanate (PITC) cleaves one residue from the N terminus of the peptide and forma an easily identifiable phenylthvobydantoin (PTH) derivative of the N-terminal amino acid. A series of sequential Edman degradations allows the sequencing of a peptide chain up to 50 residues in length. 

The sequence of an amino acid in a protein is determined sequentially from the N-terminus. The N-terminus amino acid is identified by the Edman degradation reaction developed at Rockefeller University and later automated in Melbourne, Australia, by Edman and his collaborators (1950, 1967). 

Fig. 7.2. The Edman degradation is a powerful process for protein sequencing as it allows for sequentially removing the A -terminal amino acid residue in repeated and controlled steps.

Separation of phenylthiohydantoin (PTH) amino acid derivatives, which are produced in the sequential analysis of proteins by the Edman degradation, is required. Ashraf-Khorassani etal. showed [21] how SFC on a cyanopropyl-modified silica column allowed the separation of more than twenty PTH-amino acids if an ion-pairing reagent (tetramethylammonium hydroxide) was present at low concentration in the methanol-modified CO2 mobile phase. 

N-terminal sequencing by Edman degradation is frequently used to determine the N-terminal amino acid sequence of a protein. The peptide bonds are sequentially hydrolyzed from the N-terminus, and the released amino acid is... 

Because the Edman degradation does not cleave the peptide bonds in the protein, it can be repeated to sequentially identify the amino acids from the N-terminal amino acid of the molecule. The yield of the Edman degradation approaches 100%, and sequences of 30 residues of a polypeptide can be determined from 5-picomole (5 X 10" mole) samples. This means that the sequence of a peptide with 30 amino acid residues, with a molecular weight of about 3000, can be determined from a 15 nanogram sample ... 

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