Amino acids countercurrent chromatography

The great leap forward for chromatography was the seminal work of Martin and Synge (7) who in 1941 replaced countercurrent liquid-liquid extraction by partition chromatography for the analysis of amino acids from wool. Martin also realized that the mobile phase could be a gas rather than a liquid, and with James first developed (8) gas chromatography (GC) in 1951, following the gas-phase adsorption-chromatographic separations of Phillips (9). 

Amino acids have high melting or decomposition points and are best examined for purity by paper or thin layer chromatography. The spots are developed with ninhydrin (see Lederer and Lederer, p.44). Customary methods for the purification of small quantities of amino acids obtained from natural sources (i.e. l-5g) are ion-exchange chromatography (see p. 20) or countercurrent distribution (see p. 28). For general treatment of amino acids see Greenstein and Winitz [The Amino Acids, Vols 1-3, J.Wiley Sons, New York 1961]. 

J. L. Sandlin and Y. Ito, Gram quantity separation of DNP (dinitrophenyl) amino acids with multi-layer coil countercurrent chromatography (CCC), /. Liquid Chromatogr. 7(2) 323-340 (1984). 

The molecular weight of tyrocidine A was found to be about 1,270 . Total hydrolysis and quantitative amino acid analysis revealed the exact composition . Partial hydrolysis, fractionation of the resulting peptide mixture by countercurrent distribution, ion-exchange chromatography and paper-chromatography, followed by sequential analysis, led Paladini and Craig to the structure of tyrocidine A see Table 1.6). 

A most revealing information that can readily be obtained about a peptide is its amino acid composition. Yet, the results of amino acid analysis are really meaningful only if the sample consists of a single peptide. Analysis of mixtures is usually an unrewarding effort. Thus, purification should precede analysis and this generalization is valid for most other methods of structure determination as well. Homogeneity as a prerequisite of analysis can not be overemphasized. Purification is sometimes possible simply by crystallization but in most cases chromatography, electrophoresis or countercurrent distribution or a combination of these techniques is needed. 

The molecule contains four heme groups, which suggests that it is made of four subunits. If all the subunits were identical, the number of amino acid residues obtained by digestion of the molecule would be a multiple of four. But this is not the case for at least some of the amino acids. The ferroprotein contains two identical halves, and each half is subdivided into two subunits called chains. The subunits differ in amino acid composition and sequence. One is called the a-and the other the j8-chain (see Fig. 3-1). The j8-chain ends with valine and leucine, but the a-chain ends with valine. The a-chain can be separated from the j8-chain by chromatography or by countercurrent techniques. 

While the role df lipids in protein s mthesis has not been clarified, there have been reports of a number of different amino acid-lipid complexes. Hendler 93) has investigated in further detail the metabolically active amino acid-lipid compounds which are rapidly formed in intact cells of hen oviduct. By means of countercurrent distribution and chromatography on columns of aliunina-silica and of silicic acid, he has been able to demonstrate a large number of these compounds. These, furthermore, show a distribution pattern which can be altered in a typical fashion by inhibitors, such as dinitrophenol. Lipid-amino acid complexes similar to those in hen oviduct were also found to be present in the membranes and crude ribosome fraction of E. coli. 

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