Peptides terminal residue analysis

Sequencing the Peptide Terminal Residue Analysis The amino acid analyzer determines the amino acids present in a peptide, but it does not reveal their sequence the order in which they are linked together. The peptide sequence is destroyed in the hydrolysis step. To determine the amino acid sequence, we must cleave just one amino acid from the chain and leave the rest of the chain intact. The cleaved amino acid can be separated and identified, and the process can be repeated on the rest of the chain. The amino acid may be cleaved from either end of the peptide (either the N terminus or the C terminus), and we will consider one method used for each end. This general method for peptide sequencing is called terminal residue analysis. 

Determination of structure of peptides. Terminal residue analysis. 

Several chemical methods have been devised for identifying the N terminal ammo acid They all take advantage of the fact that the N terminal ammo group is free and can act as a nucleophile The a ammo groups of all the other ammo acids are part of amide linkages are not free and are much less nucleophilic Sanger s method for N terminal residue analysis involves treating a peptide with 1 fluoro 2 4 dimtrobenzene which is very reactive toward nucleophilic aromatic substitution (Chapter 23)... 

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 degrada tion IS based on the chemistry shown m Figure 27 12 A peptide reacts with phenyl iso thiocyanate to give a phenylthwcarbamoyl (PTC) denvative as shown m the first step This PTC derivative is then treated with an acid m an anhydrous medium (Edman used mtromethane saturated with hydrogen chloride) to cleave the amide bond between the N terminal ammo acid and the remainder of the peptide No other peptide bonds are cleaved m this step as amide bond hydrolysis requires water When the PTC derivative IS treated with acid m an anhydrous medium the sulfur atom of the C=S unit acts as... 

This reaction forms the basis of one method of terminal residue analysis A peptide is treated with excess hydrazine in order to cleave all the peptide linkages One of the terminal amino acids is cleaved as the free amino acid and identified all the other ammo acid residues are converted to acyl hydrazides Which amino acid is identified by hydrazmolysis the N terminus or the C terminus ... 

Q Use information from terminal residue analysis and partial hydrolysis to determine the structure of an unknown peptide. 

Complete hydrolysis of an unknown basic decapeptide gives Gly, Ala, Leu, He, Phe, Tyr, Glu, Arg, Lys, and Ser. Terminal residue analysis shows that the N terminus is Ala and the C terminus is lie. Incubation of the decapeptide with chy-motrypsin gives two tripeptides, A and B, and a tetrapeptide, C. Amino acid analysis shows that peptide A contains Gly, Glu, Tyr, and NH3 peptide B contains Ala, Phe, and Lys and peptide C contains Leu, He, Ser, and Arg. Terminal residue analysis gives the following results. 

There remains the most difficult job of all to determine the sequence in which these amino acid residues are arranged along the peptide chain, that is, the structural formula of the peptide. This is accomplished by a com >ination of terminal residue analysis and partial hydrolysis. 

Terminal residue analysis is the identifying of the amino acid residues at the ends of the peptide chain. The procedures used depend upon the fact that the residues at the two ends are different from all the other residues and from each other one, the N-terminalresidue, contains a free alpha amino group and the other, the C-terminal residue, contains a free carboxyl group alpha io a peptide linkage. 

In practice it is not feasible to determine the sequence of all the residues in a long peptide chain by the stepwise removal of terminal residues. Instead, the chain is subjected to partial hydrolysis (acidic or enzymatic), and the fragments formed— dipeptides, tripeptides, and so on—are identified, with the aid of terminal residue analysis. When enough of these smaller fragments have been identified, it is possible to work out the sequence of residues in the entire chain. 

Terminal residue analysis (Section 24.5) Methods used to determine the sequence of amino acids in a peptide by reactions involving the N- and C-terminal residues. 

The sequence of amino acid residues in a polypeptide or protein is called its primary structure. A simple peptide composed of three amino acids (a tripeptide) can have 6 different amino acid sequences a tetrapeptide can have as many as 24 different sequences. For a protein composed of 20 different amino acids in a single chain of 100 residues, there are 2 = 1.27 X 10 possible peptide sequences, a number much greater than the number of atoms estimated to be in the universe (9 X 10 ) Clearly, one of the most important things to determine about a protein is the sequence of its amino acids. Fortunately, there are a variety of methods available to determine the sequence of amino acids in a polypeptide. We shall begin with terminal residue analysis techniques used to identify the N- and C-terminal amino acids. 

Tantomerization (Section 18.2) An isomerization by which tautomers are rapidly interconverted, as in keto-enol tantomerization. Tautomers (Section 18.2) Constitutional isomers that are easily interconverted. Keto and enol tautomers, for example, are rapidly interconverted in the presence of acids and bases. Terminal residue analysis (Section 24.5) Methods used to determine the sequence of amino acids in a peptide by reactions involving the N- and C-terminal residues. 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

Figure 10.10 Stability studies analysis by LC-MS. Long-term stability studies (3 months, 30°C) are evaluated by LC-MS analysis of a C-terminal peptide fragment. Various degradation mechanisms are visualized, removal of C-terminal residues due to proteolytic activities, isomerization and deamidation of specific asparagine residues. Future development efforts will allow the use of this methodology to assess progress toward a stable formulation.

---