Glutamine chromatography

The content of glutamine in muscle is measured in a similar manner to that of glycogen. A biopsy of muscle is taken, extracted and the glutamine content measured by enzymes or by high pressure liquid chromatography (Appendix 2.1). 

The amino-acid composition usually is obtained by complete acid hydrolysis of the peptide into its component amino acids and analysis of the mixture by ion-exchange chromatography (Section 25-4C). This procedure is complicated by the fact that tryptophan is destroyed under acidic conditions. Also, asparagine and glutamine are converted to aspartic and glutamic acids, respectively. 

The purification of glutamine cyclotransferase from papaya latex has been carried out by Messer and Ottesen (116). A batch procedure was used for the removal of impurities by passage of a papaya latex extract through a thin layer of carboxymethyl-Sephadex the active protein was separated by selective elution. Additional purification was achieved by chromatography on a column of carboxymethyl-Sephadex and by gel filtration on Sephadex G-100. The purified enzyme was homogeneous by the criteria of paper electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-100 columns and chromatography on carboxy-methyl- or DEAE-Sephadex. The electrophoretic behavior of the enzyme indicates that it is a basic protein with an isoelectric point near... 

FF Shih, AD Kalmer. Determination of glutamine and asparagine by isocratic elution reverse phase liquid chromatography with fluorescent detection. J Liq Chromatogr 7 1169-1183, 1984. 

Column chromatography yields quantitative results even for constituents present in only small amounts (with the exception of tryptophan, methionine, and glutamine, in which cases the recovery is not complete see below, (b) Dowex 50-X4 columns). It lends itself also to extraction on a preparative scale for further analysis of a particular constituent it does not require previous desalting of urine. The major inconvenience of ion exchange column chromatography for routine analyses is that the technique is elaborate and time consuming, and requires experienced personnel and expensive equipment. Recent developments, however, have allowed for considerable reduction in time consumption, and the latest completely automatic devices open the way to serial analyses. 

Figure 9.45 Chromatography of enzyme assay media. Peaks 1, aspartate 2, glutamate 3, asparagine 4, glutamine 5, Tris-HCl buffer. Elution profile of the assay medium incubated for (A) zero time and (fl) 30 minutes. (From Unnithan et al., 1984.)...

Pahuja, S. L., and Reid, T. W. (1982). Radioisotope assay for glutamine synthetase using thin-layer chromatography. J. Chromatogr. 235, 249—255. 

To analyze potential interference of amino acids in monosaccharide analysis, each of the 20 amino acids (10 /xg each, each injected separately) was subjected to the chromatography conditions used for separating, detecting, and quantifying monosaccharides. In addition to PAD detection, we monitored UV detection at 215 nm after the electrochemical detector to verify amino acid electrochemical detection. Ten amino acids (R, K, Q, V, N, A, I, L, T and C) eluted between 2 and 25 min and were both PAD and UV active. Of these ten, two amino acids could potentially interfere with monosaccharide analysis. Glutamine was found to elute as a shoulder on mannose. However, acid hydrolysis conditions used to release monosaccharides from glycoproteins likely would oxidize glutamine. 

In hyperammonemia, however, there is no single large preponderant ninhydrin-positive band of amino acid visible after paper chromatography and the pattern of urinary amino acid found on chromatography may appear to be normal or nearly so. However, the glutamine band is usually more than normally prominent. Confirmation is by a quantitative ion exchange chromatography as below, which will also reveal the increased excretion of alanine. 

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