N-terminal analysis

Figure 26.4 MECHANISM Mechanism of the Edman degradation for N-terminal analysis of peptides.

Microheterogeneity below specified level Polyacrylamide gel electrophoresis C- and N-terminal analysis. Amino acid composition... 

Figure 3 shows the final chromatogram and activity profile of purified alpha-endopsychosin. The first peak contained most of the PCP displacing activity as measured by its ability to inhibit 3H-PCP. An aliquot of the most active material was hydrolyzed in acid and the amino acid composition was determined using OPA detection. It was determined that the peptide contained approximately 26 amino acids, in close agreement with the molecular weight predicted by Sephadex gel filtration studies. N-terminal analysis revealed that the peptide was blocked at this site. The nature of this blockade is yet to be determined. Studies are under way to determine the amino acid sequence of the peptide. 

You have isolated and purified a new enzyme (E) which converts a single substrate (S) into a single product (P). You have determined Mr by gel filtration as 46,400. However, in SDS gel electrophoresis, a molecular mass of 23 kDa was indicated for the single protein band observed. A solution of the enzyme was analyzed in the following way. The absorbance at 280 nm was found to be 0.512. A 1.00 ml portion of the same solution was subjected to amino acid analysis and was found to contain 71.3 nmol of tryptophan. N-terminal analysis on the same volume of enzyme revealed 23.8 nmol of N-terminal alanine. 

The amino-terminal (N-terminal) residue of a protein can be identified by reacting the protein with a compound that forms a stable covalent link with the free a-amino group, prior to hydrolysis with 6 M HC1. The labeled N-terminal amino acid can then be identified by comparison of its chromatographic properties with standard amino acid derivatives. Commonly used reagents for N-terminal analysis are fluorodinitrobenzene and dansyl chloride. If this technique was applied to the oligopeptide above, the N-terminal residue would be identified as Val, but the remainder of the sequence would still be unknown. Further reaction with dansyl chloride would not reveal the next residue in the sequence since the peptide is totally degraded in the acid hydrolysis step. 

Sanger s reagent, l-fluoro-2,4-dinitrobenzene (FDNB), which was used in the earlier days for the quantitation of primary amino groups by colorimetric determination, can also be used in the identification of amino-terminal residue, but not for sequencing. At the present time, N-terminal analysis is performed on a protein sequencer. 

The most useful method of N-terminal analysis is called the Edman degradation. This method allows the N-terminal amino acid to be removed and its identity to be determined without hydrolyzing the other peptide bonds. The reaction initially produces a thiazolinone, which is rearranged by aqueous acid to a phenylthiohydantoin for identification by high-performance liquid chromatography ... 

Both of these peptides exhibited considerable sequence identity with the locust adipokinetic hormone (Lom-AKH-I) and the crustacean red pigment-concentrating hormone (RPCH), as had been suggested by chromatographic behavior, N-terminal analysis, and amino acid analysis (3,4). Indeed, both peptides were shown to be far more potent as stimulators of hyperglycaemia than as myotropins (3). 

N-terminal analysis of the peptide gave IGRTWGSADK (measured molecular mass = 1,091.8 Da theoretical = l,091.2Da). Therefore, the C-terminus of the protein has been extended with the peptide WGSADK (molecular mass = 663.3 Da). This sequence matches perfectly with the translated cloned DNA sequence, before another in-frame termination codon (UAA) was met (Figure 5). 

Most peptide sequencing is now done by Edman degradation, an efficient method of N-terminal analysis. Automated Edman protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by-products interferes with the... 

N-terminal analysis of (F,I,S) peptide S Cyanogen bromide treatment ... 

Another method for sequence analysis is the Sanger N-terminal analysis, based on the use of 2,4-dinitrofluorobenzene (DNFB). When a polypeptide is treated with DNFB in mildly basic solution, a nucleophilic aromatic substitution reaction (SnAt, Section 21.1 lA) takes place involving the free amino group of the N-terminal residue. Subsequent hydrolysis of the polypeptide gives a mixture of amino acids in which the N-terminal amino acid is labeled with a 2,4-dinitrophenyl group. After separating this amino acid from the mixture, it can be identified by comparison with known standards. 

Dinitrofluorobenzene will react with any free amino group in a polypeptide, including the e-amino group of lysine, and this fact complicates Sanger analyses. Only the N-terminal amino acid residue of a peptide will bear the 2,4-dinitrophenyl group at its a-amino group, however. Nevertheless, the Edman method of N-terminal analysis is much more widely used. ... 

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