Amino acids, analysis peptide linkage

In addition to knowing the sequence of the amino-acid attachment, the total amino-acid content of a peptide or protein aids in the structural composition analysis. Total amino-acid composition gives information against which the sequence information can be referenced. Also the total amino-acid composition is an easier analysis to run routinely to verify protein-peptide composition. To do amino-acid analysis, the first step is to break the linkages between the amino acids in the protein (or the peptide) by hydrolysis of the bonds, often using hydrochloric acid at elevated temperatures. The resulting mixture of amino acids is then separated and quantitated using HPLC. 

A cyclic tetrapeptide, roccanin (17), has been isolated by Bohman-Lindgren from Roccella canariensis (22). Acid hydrolysis of (17) yielded two amino-acids, L-proline (18) and / -P-phenyl- 3-alanine (19), and quantitative amino-acid analysis confirmed that (18) and (19) were formed in equimolar amounts. Mass spectrometry and the observed chemical reactivity confirmed that (17) was a cyclic peptide. Selective reduction of (17) at the more reactive acylproline linkage by treatment with lithium... 

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 ... 

FIGURE 5.2 The peptide bond is shown in its usnal trans conformation of carbonyl O and amide H. The atoms are the oi-carbons of two adjacent amino acids joined in peptide linkage. The dimensions and angles are the average valnes observed by crystallographic analysis of amino acids and small peptides. The peptide bond is the light gray bond between C and N. (Adapted from Ramachandran, G. A., ct ai, 1974. Biochimica Biophysica Acta 359 298-302.)... 

Peptidic photoprobes can be based on the photoreactive amino acid p-benzoyl-L-phenylalanine inserted into a peptide in place of a natural aromatic residue by peptide synthesis [65] or by manipulation of the genetic code [66]. The use of p-benzoyl-L-phenylalanine for this purpose is not new, but the nature of peptide probes naturally offers opportunities for the location of linkage sites by proteomic analysis [67]. 

Several chemical methods have been devised for identifying the N-terminal amino acid. They all take advantage of the fact that the N-terminal amino group is free and can act as a nucleophile. The a-amino groups of all the other amino 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 l-fluoro-2,4-dinitrobenzene, which is very reactive toward nucleophilic aromatic substitution (Chapter 23). 

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. 

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