Amino acid identification

Gas Chromatography (gc). A principal advantage of gas chromatography has been the faciUty with which it can be combined with mass spectrometry for amino acid identification and confirmation of purity. The gc-mass spectrometry combination offers the advantage of obtaining stmctural information rather than the identification by retention time in hplc. 

Sanger s reagent (Section 27.11) Thecompound l-fluoro-2,4-dinitrobenzene, used in N-terminal amino acid identification. 

Humans can synthesize 12 of the 20 common amino acids from the amphiboHc intermediates of glycolysis and of the citric acid cycle (Table 28-1). While nutritionally nonessenrial, these 12 amino acids are not nonessential. AH 20 amino acids are biologically essential. Of the 12 nutritionally nonessential amino acids, nine are formed from amphibolic intermediates and three (cysteine, tyrosine and hydroxylysine) from nutritionally essential amino acids. Identification of the twelve amino acids that humans can synthesize rested primarily on data derived from feeding diets in which purified amino acids replaced protein. This chapter considers only the biosynthesis of the twelve amino acids that are synthesized in human tissues, not the other eight that are synthesized by plants. 

Dansyl chloride (dimethylaminonaphthalene-5-sulphonyl chloride) will react with free amino groups in alkaline solution (pH 9.5-10.5) to form strongly fluorescent derivatives (Figure 10.14). This method can also be used in combination with chromatographic procedures for amino acid identification in a similar manner to the FDNB reagent but shows an approximately 100-fold increase in sensitivity. This makes it applicable to less than 1 nmol of material and more amenable for use with very small amounts of amino acids liberated after hydrolysis of peptides. The dansyl amino acids are also very resistant to hydrolysis and they can be located easily after chromatographic separation by viewing under an ultraviolet lamp see Procedure 10.1. 

Bhown, A.S., J.E. Mole, and J.C. Bennett, improved procedure for high-sensitivity microsequencing use of aminoethyl amino-propyl glass beads in the Beckman sequencer and the ultrasphere ODS column for PTH amino acid identification. Anal Biochem, 1981.110(2) 355-9. 

The dansyl analysis was thereafter directed toward obtaining an NH2-terminal amino acid identification for each of these five major peaks (in Fig. 4.6). In each case, the NH2-terminus was identified by two separate thin-layer chromatographic methods... 

NH2-terminal amino acid identification of the molecular subclasses Composing (dansylated) microbubble glycopeptide surfactant. (Taken from ref. 322.)... 

There are numerous references in the literature to the preparation of quinoxalinones as characterizing derivatives for a-keto acids. Paper chromatographic, " gas-liquid chromatographic, and fluorimetric methods have been used for the microestimation of quinoxalinones. Quinoxalinone formation is also the basis of a method for amino acid identification, as shown in Scheme 18. It has found application in... 

Modem methods of peptide sequencing follow a strategy similar to that used to sequence insulin, but are automated and can be carried out on a small scale. A key feature is repetitive N-terminal amino acid identification using the Edman degradation. 

Edman degradation and subsequent amino-acid identification on HPLC. This sequence shows nearly no homology to the N-terminal sequences of the... 

Brink (1992) described a laboratory experiment for the qualitative analysis of amino acids present in egg lysozyme, involving hydrolysis with HCl, dansyla-tion, and amino acid identification by TLC. The C-terminal amino acid was identified as leucine in a second experiment in which lysozyme was oxidized with acetic acid, hydrolyzed with carboxypeptidase A, and the cleaved acid dansylated and identified by TLC. 

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