Peptides degradation, stepwise, from

The Edman degradation, a process by which amino acids are removed stepwise from the NH 2-terminus of peptides and proteins, has evolved since its introduction (Edman, 1950) to the point where it is currently the most valuable tool for protein sequence analysis. Several innovations have led to its widespread use. One of the first was the dansyl Edman technique (Gray, 1967), which is useful for sequencing at the nanomole level. Accelerated manual procedures have been developed by Niall and Potts (1970) and Tarr (1975). During the past decade the rate of sequencing has been... 

Edman, Pehr Victor, 1916-1977 (pp. 118,240, Plate 15) born in Stockholm in 1916, matriculation examination in 1935, studied medicine at the Karolinska Institute Medical School in Stockholm from 1935, Bachelor of Medicine in 1938, graduation as a physician in 1946. Concurrently with his studies in medicine he started his training in biochemistry with Erik Jorpes, for a short time also with Hugo Theorell, and soon started a project on angiotensin that led to a MD thesis. Then he widened his experience in protein chemistry during one year at the Rockefeller Institute in Princeton with Northrop and Kunitz (crystallization of proteolytic enzymes). On his return to Sweden, Edman was awarded an associate professorship in Lund in 1947 where he conducted his stepwise peptide degradation work (p. 118) between 1950 and 1956. In 1957 Pehr Edman accepted an offer to be Director of Research at St. Vincent s School of Medical Research in Melbourne, Australia, where he remained for 15 years, during which the work on an automated sequence analyzer was finished in 1967. From 1972 until his death from a brain tumor in 1977 he was Director of the Department of Protein Chemistry of the Max-Planck-Institute for Biochemistry in Martinsried near Munich. 

Several enzymes catalyze stepwise removal of amino acids from one or the other end of a peptide chain. Carboxypeptidases232 remove amino acids from the carboxyl-terminal end, while aminopeptidases attack the opposite end. Using chromatographic methods, the amino acids released by these enzymes may be examined at various times and some idea of the sequence of amino acids at the chain ends may be obtained. A dipeptidyl aminopeptidase from bovine spleen cuts dipeptides one at a time from the amino terminus of a chain. These can be converted to volatile trimethylsilyl derivatives and identified by mass spectrometry.233 If the chain is shortened by one residue using the Edman degradation (Section 3) and the dipeptidyl aminopeptidase is again used, a different set of dipeptides that overlaps the first will be obtained and a sequence can be deduced. Carboxypeptidase Y can be used with MALDI mass spectrometry to deduce the C-terminal amino acid sequence for a peptide. However, He and Leu cannot be distinquished. 

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... 

Ingested protein is digested in a stepwise fashion in the stomach, small intestinal lumen, and small intestinal mucosal cells (Chapter 12). Peptides formed in the intestinal lumen are absorbed into the mucosal cells and degraded to free amino acids. The outflow of amino acids to the portal vein does not reflect the amino acid composition of the ingested protein. Thus, alanine levels increase two-to fourfold, and glutamine, glutamate, and aspartate are absent. These changes arise from amino acid interconversions within the intestinal cell. 

Acid treatment of the substituted thioureas resulting from the treatment of esters, amides, or peptides of a-amino acids (10) with isothiocyanates (usually phenyl isothiocyanate) also affords 2-thiohydantoins (11). These reactions furnish the basis of the Edman s method for the stepwise degradation of peptides.3... 

At about the same time, in 1963, it was reported that simple iV-acetyl-peptides (Heyns and Griitzmacher [29]) or iV-trifluoroacetyl peptide esters (F. Weygand et al. [30]) are cleaved under electron impact at the peptide bonds, giving rise mainly to ions corresponding to N-terminal parts of the chain. If such fission occurs at each peptide bond, a series of ions are formed all having the same N-acyl group. This is equal to a stepwise degradation of the chain from its carboxyl end (Fig. 13). 

In the sequencing of bradykinin one of the proline residues was overlooked. The error was corrected both by a reexamination of the degradation study [9] and through experiments with peptides synthesized for this purpose [10]. From the various syntheses of bradykinin the scheme of Nicolaides and de Wald, stepwise chain-lengthening with active esters [11] is mentioned here. Also, bradykinin was the first biologically active peptide synthesized by the solid phase method [12] (Chap. 5). 

Stepwise degradation from the C-terminal end of peptide chains has also been studied, based on the Schlack and Kumpf reaction [141,142), leading to the cleavage of the last amino residue as the cyclic thiohydantoin (Figure 17). 

Again, solid-state procedures have been worked out [143—145] and the products have been identified by chromatographic methods [146—1501 but this approach has not been mechanized, nor has it received anything like the degree of development of the N-terminal approach. The same is true of stepwise degradation from the C-terminal end of a peptide by the iminohydantoin [151] procedure (Figure 18). 

The proteolytic coefficient is largest for compounds with N-terminal leucine and norleucine, but several other amino acids may be attacked at sufficient rates to make this enz3rme a generally useful reagent. Its activity is not restricted to small peptides, and it has been used in the stepwise degradation of proteins from the amino end, in the same manner that carboxypeptidase has been used in the analysis of the carboxyl ends of protein chains. 

Figure 18.5. MALDI-TOF mass spectrum from protein ladder sequencing of the N-terminal peptide of cofilin (G-S-R-S-G-V-A-...). A stepwise degradation was carried out in the presence of 5% phenylisocyanate/95% phenylisothiocyanate. The peptide was subjected to two cycles of ladder generating chemistry. ...

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