Edman chemistry amino-acids

The chemistry of this reaction has been extensively studied by Use and Edman. Although the reaction velocity is different for various amino acids, the reaction is complete in 1 N HCl at 80 °C within 10 min, with virtually all amino acids. This treatment, however, would cause partial hydrolysis of the remaining peptide. The method was made amenable to automation when Edman suggested to separate the thiazolinone derivative from the remaining peptide by extraction of the liberated thiazolinone derivative into butyl chloride and to perform the conversion into the PTH-amino acid outside the reaction vessel. 

A major advance was devised by Pehr Edman (University of Lund, Sweden) that has become the standard method for N-terminal residue analysis. The Edman degradation is based on the chemistry shown in Figure 27.12. A peptide reacts with phenyl isothiocyanate to give a phenylthiocarbamoyl (PTC) derivative, as shown in the first step. This PTC derivative is then treated with an acid in an anhydrous medium (Edman used nitromethane saturated with hydrogen chloride) to cleave the amide bond between the N-terminal amino acid and the remainder of the peptide. No other peptide bonds are cleaved in this step as amide bond hydrolysis requires water. When the PTC derivative is treated with acid in an anhydrous medium, the sulfur atom of the C=S unit acts as... 

The Edman degradation method for polypeptide sequence determination. The sequence is determined one amino acid at a time, starting from the amino-terminal end of the polypeptide. First the polypeptide is reacted with phenylisothiocyanate to form a polypeptidyl phenylthiocarbamyl derivative. Gentle hydrolysis releases the amino-terminal amino acid as a phenylthiohydantoin (PTH), which can be separated and detected spectrophoto-metrically. The remaining intact polypeptide, shortened by one amino acid, is then ready for further cycles of this procedure. A more sensitive reagent, dimethylaminoazobenzene isothiocyanate, can be used in place of phenylisothiocyanate. The chemistry is the same. 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

Increase in sensitivity and efficiency of analysis in structural studies of enzymes with a gas phase sequencer have made it possible to determine the primary structure in a shorter period of time with a small amount of enzyme at the picomole or even femtomole level. In addition, thanks to the DNA sequencing technique the number of enzymes (or proteins) whose amino acid sequences are registered in a data base file has expanded explosively. The introduction of mass spectrometry on the primary structure determination of protein has stimulated the search for a new methodology other than Edman chemistry. 

The most popular method is automated Edman chemistry (mn on a sequencer), a method which removes one amino acid at a time from the N-terminus, resulting in the sequential liberation of phenylthiohydantoin (PTH) amino acids, which are identified by on-line HPLC analysis. Edman sequanators are completely automated high-sensitivity instrument systems that can routinely detect and identify as little as 0.2 pmol to 1 pmol of amino acid in a given cycle and carry out more than 20 cycles with 1 to 5 pmol of protein. Most peptides of length 3-30 amino acids can be sequenced completely. Although amino... 

As everybody knows, a sequencing machine digests the amino acid chain starting at the N-terminus and it identifies the amino acid derivatives via a connected HPLC. A requirement is a free N-terminus. The Pierce Catalog, for example, describes the chemistry of the Edman degradation of peptides with phenylisothiocyanate. Baumann (1990) compares the effectiveness of different sequencing techniques. 

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