End-groups, determination

At one end of a peptide chain an amino acid residue is present with a free, that is unacylated amino group. This residue is called the amino terminal or more commonly N-terminal residue. At the opposite end of the chain a residue with a free carboxyl group is the carboxyl terminal or C-terminal residue  

This nomenclature is applied in a broad sense, mainly to indicate the direction of the peptide chain. Thus, in peptides in which an N-terminal glutamine residue is cyclized 

4-dinitrophenyl (DNP) amino acids, under the conditions used for the total hydrolysis of peptides, are not decomposed by boiling hydrochloric acid, Abderhalden (with W. Stix), in 1923, tried to determine amino end groups in a mixture of peptides in a partial hydrolysate of silk fibroin. The experiment, however, gave no encouraging results, because, with the methods available at that time, it was not possible to separate the DNP amino acids formed from the bulk of free amino acids or from each other. 

Decisive progress in the method was achieved in 1945 by Frederick Sanger [6] (Plate 38), who found that 2,4-dinitrofluorobenzene reacts with amino 

DANS-NH-CH-CO2H + amino acids DANSylamino acid 

A second reagent, 5-dimethylamino-naphthalene-l-sulfonic acid chloride (DANSyl-chloride), that allows a very sensitive identification of the N-terminal amino acid, was introduced by Gray and Heartley in 1963 [7]. It reacts with amino groups under mild conditions and yields DANSyl-peptides in which the sulfonamide bond is more resistant to acid hydrolysis than the peptide bonds are. Hence DANSyl-amino acids (Fig. 1) are released intact on hydrolysis and can be visualized, after chromatography on paper or on thin layer plates, even in minute amounts by their fluorescence under UV-light. 

The identification of the carboxyl terminal amino acids of peptides seemed to be of less interest and was experimentally somewhat more difficult. In 1952, S. Akabori (Plate 9) showed that on heating with anhydrous hydrazine to 100 °C for several hours, all peptide bonds of a peptide chain are cleaved by hydrazinolysis. Hereby the amino acid constituents are released as the respective hydrazides, R-CONHNH2, except the C-terminal amino acid which, sometimes with difficulty, is isolated and characterized as such. Shortly before the 

Identification of the C-terminal residue is usually done by reaction with hydrazine (100 °C for 12 h). This converts all amino acids other than the C-terminal residue to the H2N-CHR-CONHNH2 derivatives after solvent extraction to remove these hydrazide derivatives, the intact C-terminal amino acid is treated with 2,4-dinitro-phenol and identified chromatographically as the 2,4-dinitrophenyl amino acid, as above. 

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The reactions of cyanoisopropyl radicals with monomers have been widely studied. Methods used include time resolved EPR spectroscopy,352 radical trappingj53 355 and oligomer00 356 and polymer end group determination. 1 Absolute341 and relative reactivity data obtained using the various methods (Table 3.6) are in broad general agreement. 

Two relatively new techniques, matrix assisted laser desorption ionization-lime of flight mass spectrometry (MALDI-TOF) and electrospray ionization (FS1), offer new possibilities for analysis of polymers with molecular weights in the tens of thousands. PS molecular weights as high as 1.5 million have been determined by MALDI-TOF. Recent reviews on the application of these techniques to synthetic polymers include those by Ilantoif54 and Nielen.555 The methods have been much used to provide evidence for initiation and termination mechanisms in various forms of living and controlled radical polymerization.550 Some examples of the application of MALDI-TOF and ESI in end group determination are provided in Table 3.12. The table is not intended to be a comprehensive survey. 

One system studied by the authors(3) used LG56 triol which had been characterised by molar-mass as well as end-group determinations. The data for the samples used are given in Table III, indicating a true number-average functionality (fn) of between 2.95 and 2.99. The triol was reacted with HDI in bulk (see Figure 1) and at various dilutions in benzene, and values of Nr and ac determined. To estimate functionality from ac, equations... 

Meyer31 has separated maize and potato amyloses into two fractions each, by (1) aqueous leaching, followed by (2) dissolution and precipitation of the remainder. His results of colorimetric, end-group determinations of size were ... 

The third step is to determine the polypeptide chain end groups. If the polypeptide chains are pure, then only one N-terminal and one C-terminal group should be detected. The amino-terminal amino acid can be identified by reaction with fluorodinitrobenzene (FDNB) (fig. 3.18). Subsequent acid hydrolysis releases a colored dinitrophenol (DNP)-labeled amino-terminal amino acid, which can be identified by its characteristic migration rate on thin-layer chromatography or paper electrophoresis. A more sensitive method of end-group determination involves the use of dan-syl chloride (see Methods of Biochemical Analysis 3B). 

Chemical methods for carboxyl end-group determination are considerably less satisfactory. Treatment of the peptide with anhydrous hydrazine at 100°C results in conversion of all the amino acid residues to amino acid hydrazides except for the carboxyl-terminal residue, which remains as the free amino acid and can be isolated and identified chro-matographically. Alternatively, the polypeptide can be subjected to limited breakdown (proteolysis) with the enzyme carboxypeptidase. This results in release of the carboxyl-terminal amino acid as the major free amino acid reaction product. The amino acid type can then be identified chroma-tographically. 

Separation of Methylated Methylglycosides by Adsorption on Alumina. A New Method for End-group Determinations in Methylated Polysaccharides, J. K. N. Jones, J. Chem. Soc., (1944) 333-334. 

Since the signal intensity is proportional to the number of absorbing nuclei, HR NMR can also be used as a quantitative tool, e.g. in polymer end group determinations. 

The inherent viscosity values have been compared with end group determinations of the acetate end groups of completely acetylated poly-n-butyralde-hyde. For this purpose, the intensity of the carbonyl band of the acetate end groups (5.72 p) was determined by infrared and was related to an internal standard giving what we call the end group ratio (15.72 4.75 ) The end... 

This multipart question question illustrates material balance calculations used, for example, in formulating polyurethanes. Refer to Section 1.5.4 for some of the reactions of isocyanate groups. This problem is an extension of the concepts mentioned in Section 3.1.5 on end-group determinations. Some useful definitions follow ... 

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