Amino acid quantitative tests

Colour Reactions of Proteins - The colour reactions of proteins are of importance in the qualitative detection and quantitative estimation of proteins and their constituent amino acids. Biuret test is extensively used as a test to detect proteins in biological materials. 

This is a very important and well tested method for the quantitative determination of loading of Fmoc protected compounds particularly that of Fmoc (fluorenylmethoxycarbonyl) amino acids on solid support. Fmoc groups can... 

The presence of aromatic amino acid residues results in proteins showing an absorption maximum at 280 nm and comparison of the absorbance value of a test solution with that of a standard solution provides a sensitive method of quantitation. This cannot be considered to be an absolute method of quantitation... 

Post-analytically, schemes are beginning to emerge specifically to compare practice and performance between laboratories pertaining to the interpretation of test results. For instance, in the UK, NEQAS in conjunction with the National Biochemical Genetic network, MetBio.Net, are offering a scheme that provides the opportunity, when given relevant clinical details, to interpret quantitative amino acid results. This proficiency scheme can compare interpretive skills without the need to circulate scarce clinical samples. 

Simple quantitative tests can be used such as measurement of the liver weight/body weight ratio. Overt damage to the liver can be detected by light and electron microscopy of liver sections. However, damage can be detected by other noninvasive means such as the urinary excretion of conjugated bilirubin or the amino acid taurine. 

The interaction of malonaldehyde with amino acids and the presence of other nonlipid TBA-reactive compounds in biological samples may interfere with the quantitation of oxidation products in the TBA test. Due to uncertainty concerning the exact identity of compounds that can react with TBA, the ambiguous term TBARS is now commonly used in lieu of TBA number or value (Ke et al., 1984 Gray and Pearson, 1987 Guillen-Sans and Guzman-Chozas, 1998). Because TBA is not specific for malonaldehyde alone, certain limitations exist when performing the test for evaluation of the... 

Commeyras and co-workers proposed for the first time a prebiotically relevant synthetic pathway [143,144]. They established that the nitrosation of N-carbamoylamino acids 25 (otherwise believed as unreactive in prebiotic environments) by nitrogen oxides (typically obtained by mixing O2 and NO) quantitatively releases NCA. Although first tested in organic solvent [ 143], the reaction proved to work in the solid state [144], and also in aqueous solution [145], where the NCA can be observed for ca. 1 h prior to its conversion into either free amino acids or peptides. 

Based on experience, the following procedure was suggested for the preparation of TFA-amyl esters of amino acids. A sample of amino acids (0.5—2 mg of each acid) was placed into a test-tube and dissolved in 0.2 ml of trifluoroacetic acid. Amyl alcohol (2 ml) was added and dry hydrogen chloride was bubbled through the reaction mixture continuously at 108°C for 25 min. Excess of the reagent was removed under vacuum, dissolved in a small amount of methanol, transferred into a small test-tube and the methanol was evaporated by standing freely at 70°C. TFA anhydride (1 ml) was added and the stoppered test-tube was allowed to stand at room temperature for 1 h. As some of the amino acids (e.g., Arg) were not acylated quantitatively by this procedure, it was recommended that the sealed test-tube be heated at 140°C for 5 min [229]. Excess of anhydride was removed under vacuum and the residue was dissolved in a known volume of dry methyl ethyl ketone. 

One of the first applications of HPLC in the clinical field was the quantitation of theophylline in asthmatic infants. This highly accurate measurement was an important test because of the very low amount of sample required and the accuracy of the determination (see Fig. 1-10). More recent clinically related HPLC separations include drugs and drug metabolites, neurochemicals and their metabolites, histamines, thyroid hormones, and enkephalins. The earliest bioresearch applications of HPLC included the determination of peptides, proteins, and amino acids. Application of HPLC to the analysis of these compounds remains important, as indicated by the rapid growth in references (Fig. 1-11). Bioresearch remains one of the most rapidly expanding growth areas of LC. 

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