A-terminal amino acid

A more useful procedure, in that it allows sequential determination of the A-terminal amino acids in a peptide, is the Edman degradation. This process removes the A-terminal amino acid, but leaves the rest of the chain intact, so allowing further reactions to be applied. The reagent used here is phenyl isothiocyanate. 

Thus, the A-terminal amino acid can be identified by analysis of the thiazolinone, and the process can be repeated on the one-unit-shortened polypeptide chain. Under the acidic conditions, the thiazolinone is actually unstable, and rearranges to a... 

Fig. 16. A-Terminal amino acid residues detected in human. serum albumin after treatment with varying amounts of NBS. Yields of DNP-amino acids were corrected for losses on hydrolysis. From Peters (1959).

Tamura, O, Mita, N, Kusaka, N, Suzuki, H, Sakamoto, M, Intramolecular cycloaddition of a-allyloxycarbonylnitrone bearing a chiral sugar auxiliary a short-step synthesis of the A-terminal amino acid component of nikkomycin Bz, Tetrahedron Lett., 38, 428-432, 1997. 

E15. Eriksson, S., and Sjoquist, J., Quantitative determination of A -terminal amino acids in some serum proteins. Biochim. Biophys. Ada 46t 290-296 (1960). 

From these observations it follows that (1) some of the chemical properties of a phosphoamino acid may change considerably on incorporation into a peptide or protein, (2) that the adjacent molecular configuration may be responsible for the stability of the phosphate group, and (3) that the acidity of the medium determines whether migration of a phosphoric acid residue from the —0— to the —— position occurs. As outlined in a previous section in this article A-phosphorylserine and A-phosphorylthreonine, respectively, w-ould represent possible configurations of the A-terminal amino acid of a peptide chain in a native protein. 

A list of key differences between prokaryotes and eukaryotes with respect to protein synthesis is shown in Table 9-1. These include the existence of multiple eukaryotic initiation factors that facilitate the assembly of the riboso-mal protein synthetic machinery, whereas there are only three for prokaryotes. An initiation site on bacterial mRNA consists of the AUG initiation codon preceded with a gap of approximately 10 bases by the Shine-Dalgamo polypurine hexamer, whereas the 5 Cap (a 7-methylguanylate residue in a 5 —>5 triphosphate linkage) acts as an initiation signal in eukaryotes. In prokaryotes, the first or A-terminal amino acid is a formyl-methionine (fMet), but in eukaryotes it is usually a simple methionine. Additionally, the size and nature of the prokaryotic ribosomes are quite different from the eukaryotic ribosomes. 

USE Reagent for labeling a terminal amino acid group in modified Wohl degradations of aldoses. As hapten, Caution,- Vesicant. For proper handling see J. S, Thompson, O. P. Edmunds, Ann. Occup. Hyg, 23. 27 (1980). 

Halogen bound to an aromatic ring is rendered considerably more reactive by nitro groups in the para- or, especially, the ortho-position to it carboxyl, sulfonyl, and cyano groups have considerably less effect. Among such activated halo aromatic compounds the relative reactivities of the halogens are reversed, and fluorine is much the most reactive. This is used for, e.g.9 determination of A-terminal amino acids in peptides by means of l-fluoro-2,4-dinitrobenzene.547 The ease of reaction is illustrated in the following example ... 

A further interesting example in the context of natural products is shown, in the transformation of the p-lactam 102 into the p-amino ketone 103, Scheme 34, which upon carbonyl reduction provides the amino lactone 104, the cyclized form of the A -terminal amino acid residue found in the antibiotic family of nikkomycins. Fig. 6 [101]. 

Fig. 7.2. The Edman degradation is a powerful process for protein sequencing as it allows for sequentially removing the A -terminal amino acid residue in repeated and controlled steps.

The acid-stable a-amylase of Aspergillus niger and the acid-labile a-amylase of A. oryzae have been studied.It was shown that in addition to the more stable acid-resistant properties, the a-amylase of A. niger possesses increased thermal stability in comparison with the a-amylase of A. oryzae. The molecular weight of the acid-stable a-amylase is 5.8 x 10 The amino-acid composition, as well as the C- and A -terminal amino-acids of both forms of a-amylases were determined. It was shown that the enzymes each contain one thiol group, which, being bound to Ca ", plays an important role in maintaining the catalytically active conformation of the enzyme. 

Extracellular a-amylase was purified to homogeneity from Marburg strain of Bacillus subtilis. The enzyme is a single polypeptide chain of molecular weight approximately 67 000. Its A -terminal amino-acid sequence is (15). 

Edman degradation (Section 24.5A) A method for determining the A -terminal amino acid in a peptide. The peptide is treated with phenylisothiocyanate (CgHs — N = C = S), which reacts with the A terminal residue to form a derivative that is then cleaved from the peptide with acid and identified. Automated sequencers use the Edman degradation method. 

Sat er A-terminal analysis (Section 24.5B) A method for determining the A -terminal amino acid residue of a peptide by its SiqAr (nucleophilic aromatic substitution) reaction with dinitro-fluorobenzene, followed by peptide hydrolysis and comparison of the product with known standards. 

Draw a structural formula for Cys-Arg-Met-Asn. Label the A/-terminal amino acid and the C-terminal amino acid. What is the net charge on this tetrapeptide at pH 6.0 ... 

A method for selectively cleaving and identifying the A/-terminal amino acid of a polypeptide chain. 

Of the various chemical methods developed for determining the amino acid sequence of a polypeptide, the one most widely used today is the Edman degradation, introduced in 1950 by Pehr Edman of the University of Lund, Sweden. In this procedure, a polypeptide is treated with phenyl isothiocyanate, C6H5N = C=S, and then with acid. The effect of Edman degradation is to remove the A terminal amino acid selectively as a substituted phenylthiohydantoin (Eigure 18.8), which is then separated and identified. 

The special value of the Edman degradation is that it cleaves the A -terminal amino acid from a polypeptide without affecting any other bonds in the chain. Furthermore, Edman degradation can be repeated on the shortened polypeptide, causing the next amino acid in the sequence to be cleaved and identified. In practice, it is possible to sequence as many as the first 20 to 30 amino acids in a polypeptide by this method, using as little as a few miUigrams of material. 

Edman degradation. Treatment of a polypeptide with phenyl isothiocyanate followed by acid selectively cleaves the A/-terminal amino acid as a substituted phenylthiohydantoin. 

Edman degradation cleaves Glu from the pentapeptide therefore, glutamic acid must be the A/-terminal amino acid, and we have... 

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