Edman procedure

Only the N terminal amide bond is broken m the Edman degradation the rest of the peptide chain remains intact It can be isolated and subjected to a second Edman procedure to determine its new N terminus We can proceed along a peptide chain by beginning with the N terminus and determining each ammo acid m order The sequence is given directly by the structure of the PTH derivative formed m each successive degradation... 

Analogous techniques facilitating sequencing from a polypeptide s C-terminus remain to be satisfactorily developed. The enzyme carboxypeptidase C sequentially removes amino acids from the C-terminus, but often only removes the first few such amino acids. Furthermore, the rate at which it hydrolyses bonds can vary, depending on what amino acids have contributed to bond formation. Chemical approaches based on principles similar to the Edman procedure have been attempted. However, poor yields of derivatized product and the occurrence of side reactions have prevented widespread acceptance of this method. 

Breaking Disulfide Bonds Disulfide bonds interfere with the sequencing procedure. A cystine residue (Fig. 3-7) that has one of its peptide bonds cleaved by the Edman procedure may remain attached to another polypeptide strand via its disulfide bond. Disulfide bonds also interfere with the enzymatic or chemical cleavage of the polypeptide. Two approaches to irreversible breakage of disulfide bonds are outlined in Figure 3-26. 

Sequencing of Peptides Each peptide fragment resulting from the action of trypsin is sequenced separately by the Edman procedure. 

Efficiency in Peptide Sequencing A peptide with the primary structure Lys-Arg-Pro-Leu-Ile-Asp-Gly-Ala is sequenced by the Edman procedure. If each Edman cycle is 96% efficient, what percentage of the amino acids liberated in the fourth cycle will be leucine Do the calculation a second time, but assume a 99% efficiency for each cycle. 

Arens et al. (65) also reported finding only N-terminal Leu in both asparaginases A and B. In addition, using the Edman procedure, they have established the sequence of the first 14 amino acids from the N-terminal end (shown below) still without encountering a difference between the two... 

Because only 40 to 60 amino acid residues can be determined by the Edman procedure, additional methods are needed for larger proteins. Determination of the C-terminal amino acid can be accomplished by treating the protein with carboxypeptidase. This enzyme selectively catalyzes the hydrolysis of the C-terminal amino acid. After the first amino acid has been removed, the enzyme begins to cleave the second amino acid, and so forth. By following the rates at which the amino acids appear, it is possible to determine the first few amino acids at the C-terminal end of the protein by employing this enzyme. However, because the enzyme hydrolyzes different peptide bonds at different rates, it is possible to identify only a few amino acids before the reaction mixture becomes too complex. 

In the sequential degradation according to the Edman procedure methyl or phenyl-isothiocyanate is used as the reagent and the reaction starts from the amine end of the... 

Composition and sequence data 63,86). Simple numbers for peptides refer to Matthews el al. 63), while alpha-numeric designations refer to Brown and Perham 86). — represents a residue placed by the dansyl-Edman or the subtractive-Edman procedures. R/ refers to peptide mobility on electrophoresis at pH 6.5 relative to the mobility of aspartic acid ( — 1.0). 

The cycle of reactions in the Edman procedure can be summarised as follows ... 

Studying chemical modification will be described. Histones are modified in vivo by acetylation (also methylation and phosphorylation, see DeLange and Smith 1971, 1974). In some histones the a-amino groups are acetylated (in addition to lysine residues) and this precludes direct examination of the internal sites of acetylation by the Edman procedure. However, histones IIb2 and III have free a-amino groups and this enabled Candido and Dixon (1972) to examine these histones (from trout testis) which had been acetylated intracellularly with " C-labeled acetate. Each residue that was released by the Edman procedure (using a sequenator) was examined for radioactive material and it was found that lysyl Residues 14 and 23 (major sites) and 9 and 18 (minor sites) were partially acetylated in histone III and lysyl Residues 5,10,13 and 18 were partially acetylated in histone IIb2. This type of approach should be applicable to many other studies of chemical modification. 

It is many years since Schlack and Kumpf showed that a simple A-acyl peptide treated with ammonium thiocyanate and acetic anhydride (Scheme 5.7) underwent cyclisation at the C-terminus to yield l-acyl-2-thiohydantoins (5.29). Mild alkaline hydrolysis then yielded the 2-thiohydantoin (5.30) corresponding to the C-terminal terminal residue and an A-acylpeptide containing one amino acid fewer. This reaction sequence should lead to a cyclic procedure at the C-terminus analogous to the Edman procedure at the A-terminus. Despite several attempts to avoid side reac-... 

In the Edman procedure, PITC reacts under basic conditions with the free a-amino group to form a phenylthiocarbamoyl peptide (Figure 3-9). Treatment with anhydrous acid yields the labeled terminal amino residue plus the remainder of the peptide. In this process, the terminal amino acid is cyclized to the corresponding phenylthiohydantoin derivative (PTH-amino... 

A significant advantage of the Edman procedure is that on removal of the N-terminal residue, the remaining peptide is left intact and its N-terminal remaining peptide group is available for another cycle of the procedure. This procedure can thus be used in a stepwise manner to establish the sequence of amino acids in a peptide starting from the N-terminal. 

Determination of the N-terminal residue by the Edman procedure. After removal of the N-terminal amino acid, the remainder of the peptide remains intact and a new N-terminal amino acid is available for removal by the next reaction cycle. 

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