Sequenator, automatic

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

Ingenious protein sequenators have been devised to carry out the Edman degradation automatically.242 244-246 Each released phenylthiohydantoin is then identified by HPLC or other techniques. Commercial sequenators have often required 5-20 nmol of peptide but new microsequenators can be used with amounts as low as 5-10 picomoles or less.247 248... 

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. 

Pure peptides (native or reduced and alkylated) are sequenced by automatic Edman degradation on a pulse liquid automatic sequenator. Reagent and solvents are purchased from the manufacturer (Applied Biosystem Division). 

Leuconostoc mesenteroides (4 3), Pseudomonas aeruginosa (4 4), Streptococcus mutans (435), and Bacillus stearothermophilus (486,437). The protein from the latter species was purified by Kolb and Harris (436) and found to have a free N-terminus in contrast to the liver and yeast alcohol dehydrogenases. The bacterial protein was, therefore, submitted to sequence analysis in an automatic sequenator which revealed the 45 first residues (IS). These are listed in Table XIV. It is clear that the structure of the bacterial enzyme is distantly related to those of the yeast and mammalian enzymes, but few residues are identical in all proteins at equivalent positions (Table XIV). Further aspects of this relationship are discussed in Section II,D. 

Automatic sequencing machines ( Sequenators ), first introduced by Edman some 30 years ago, have subsequently been intensively developed. Peptide fragments of 40 or more amino acid residues can usually be easily sequenced. 

These methods are, however, completely destructive of the rest of the peptide chain—a single piece of information is gained at the cost of significant material use. When the Af-terminus of a peptide is reacted with phenyl isothiocyanate, PhN=C=S, the Edman reagent, a phenylthiohy-dantoin, 22.39, is obtained and the rest of the peptide chain is left intact (Figure 22.36). So the process can be repeated, and this methodology is the basis for automatic sequenators. The phen-ylthiohydantoin contains the R group from the Af-terminal amino acid and can thus be identified. 

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