Amino acid detection

Thin-Layer Chromatography (tic). Tic (126) is used widely for quahtative analysis and micro-quantity separation of amino acid mixtures. The amino acids detected are developed by ninhydrin coloring, except for proline and hydroxyproline. Isatia has been recommended for specific coloring of pToline (127). 

Colorimetric and Fluorimetric Analysis. The functional groups of amino acids exhibit Htde absorption of uv light from 210 to 340 nm where uv absorption spectrometry is most conveniently conducted. Thus color or fluorescence formation reactions are employed for amino acid detection (128). 

While most amino acids are not electroactive at analytically usable potentials at carbon electrodes, much work is currently directed at general methods of LCEC amino acid detection by electrode surface modification or derivatization of the amino acid. Kok et al. have directly detected amino acids at a copper electrode. Several derivatization methods for amino acids have also been reported 227.228)... 

Several factors indicate that the amino acids detected in all of these carbonaceous chondrites are indigenous and that they must have originated abiotically. First, the presence of protein and non-protein amino acids, with approximately equal quantities of D and L enantiomers points to a nonbiological origin and precludes terrestrial contamination. In addition, the non-extractable fraction of the Murchison is significantly heavier in 13C than terrestrial samples. Finally, the relative abundances of some compounds detected resemble those of products formed in prebiotic synthesis experiments. The aliphatic hydrocarbons are randomly distributed in chain length, and the C2, C3, and C4 amino acids have the highest concentrations (i.e., the most easily synthesized amino acids with the least number of possible structures are most abundant) [4]. 

Edman Degradation. This technique requires more material than MS-based sequencing and its sensitivity decreases with the number of amino acids detected. The use of Edman degradation sometimes allows determination of those N-terminal amino acids that were not detected during MS sequencing. 

Common name Composition and reaction conditions Amino acids detected Colour produced Comments... 

Amino Acids Detection of the molecular ion peaks of amino acids can be difficult. If we examine the mass spectra of amino acids, as well as of steroids and triglycerides, by a variety of ionization techniques, we can appreciate their relative merits. 

Dabsyl Chloride (4-dimethyl-aminoazobenzene-4-sulfonyl chloride) Aabs = 420 nm. Derivatives are very stable (days) and can be formed from both primary and secondary amino acids. Detection is by absorption only. Detection limits are in the high picomole range. Reaction time is typically around 10 minutes at 70°C. Completeness of reaction can be adversely affected by the presence of high levels of various salts. Because reaction efficiency is highly matrix dependent and variable for different amino acids, standard amino acid solution should be derivatized under similar conditions/matrix for accurate calibration. Commercial systems available uti-... 

Fig.4.45. Single-column separation of some amino acids detected with o-phthalaldehyde.

The fluorescent beads are manually isolated, washed with 8 M guanidine-HCl and water, and then submitted for sequence analysis. We expect that some of the peptides on the positive beads will be cleaved at the proteolytic site. As a result, the PTH-amino acids detected during each cycle of Edman degradation will have been generated from both of these peptides. This complicates the sequence analysis somewhat. However, it also allows us to determine the proteolytic cleavage site of the peptide, in addition to its uncleaved sequence. 

Glutamic acid was replaced by tyrosine in position one of a-conotoxin GI to prevent pyroglutamic acid formation at the N-terminal. This strategy also introduced an amino acid detectable at = 280 nm by UV. 

As discussed above DOM contains between 200 and 500 nM of DCAA (Lee and Bada, 1975, 1977) and HMWDOM contains between 178 and 278 nM DCAA (McCarthy et al., 1998). Based on these values, between 30% and 100% of DCAA are in the >1000 Da fraction of DOM. Coffin (1989) found that 60% of aU amino acids isolated from a Delaware estuary were in the <1 kDa fraction and Sommerville and Preston (2001) showed that >90% of total DCAA (as analyzed by GC/MS) are released from peptides with MWs <3 kDa. Taken together, these studies suggest that peptides and small proteins ( 500 and 3000 Da) contain most of the amino acids detected in DOM so far. Unfortunately, precise MW information is not currently available for proteins in HMWDOM or DOM. 

In the Arctic and Antarctic Ocean amino acids were also found in humic substances isolated from DOM by XAD-2 resins (Hubberten et ai, 1995). The concentration of THAA in humic substances was between 233—246 nM, with aU hydrolysable amino acids in the deep ocean and 60% of amino acids in the surface ocean residing in this fraction. Glycine was by far the most abundant amino acid detected in the humic fraction. These authors concluded that amino acids in the XAD-2 extracts represent a refractory protein background that is present throughout the ocean. The dominance of this refractory protein background in the surface and deep ocean could explain the relatively stable amino acid distribution observed by Yamashita and Tanoue (2003) at their open ocean sites. 

Life would have been impossible without the chirality of its molecules, which is required for the larger spectrum of biomolecules, their 3-dimensional assembly, and for the functions of biopolymers. In this balance, the unnatural, nonprotein amino acids detected in some meteorites may have played an especially significant role. 

Table 1.4 Amino acids detected in hydrolysed aqueous extracts of Murchison meteorite (after Engel Nagy 1982)...

To show that the source of the amino acids in our experiments was not the result of the reaction of the various nitrogen species produced in the reaction with ascorbic acid, we reacted ascorbate individually and in combination with ammonia, hydrazine, nitrite, and nitrate. Very low traces of amino acids were produced in these reactions, indicating that the amino acids detected are in fact produced from the electric discharge reaction. While ascorbic acid is not likely to have been an abundant prebiotic species, oxidation could have been inhibited by other available chemical species such as sulfides and reduced metal ions. 

Figure 1. Typical HPLC chromatograms of the OPA-NAC (II) derivitized amino acids detected from the spark discharge reactions. Chromatograms labeled with roman numerals I.) Amino acid standard, II.) CO2/N2 not sparked, III.) CO2/N2 + CaCOs, sparked, hydrolyzed- ascorbate. IV.) CO2/N2, sparked, hydrolyzed - ascorbate V.) CO2/N2, sparked + CaCOs, hydrolyzed + ascorbate. Amino acids I.) DL aspartic acid 2.) DL glutamic acid 3.) DL serine 4.) glycine 5.) P-alanine 6.) DL alanine 7.) a-amino isobutyric acid 8.) DL norleucine (internal standard). The D and L enantiomers of glutamic acid and serine are not separated under these chromatographic conditions.

A wide range in the free amino acid contents of helminths has been reported (1). Concentrations are usually higher than those of vertebrates but lower than in many other invertebrates. In contrast to vertebrates, helminths have one or two dominant free non-essential amino acids, usually alanine, proline, glutamate, serine and/or glycine. Non-protein amino acids detected include 3-alanine, 2-aminoisobutyrate,... 

C [172]. Derivatization of the hydrolyzate with O-phthalaldehyde (OPA) [173], phenyl isocyanate (PTC chemistry) [174,175], or aminoquinolylhy-droxysuccinimidyl carbamate (AQC) [176,177] allows amino acid detection and quantitation by fluorescence (OPA or AQC) or UV absorbance (PTC chemistry) and HPLC separation. 

Method D-Amino acid detected Remarks Reference ... 

AMINO ACIDS DETECTED IN ROOT EXUDATES BY SEVERAL INVESTIGATORS (from ROVIRA, 1965a)... 

Kokoeva, M.V. and Oesterhelt, D. (2000). BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum. Mol. Microbiol. 35, 647-656. 

In the case of the nickel electrode, detection is performed at - -0.49 V versus the standard calomel electrode. At this potential, Ni is present on the electrode surface. The Ni + is reduced in the presence of the amino acids to Ni +. The current produced is proportional to the concentration of the amino acid. Detection limits for glycine have been reported to be in the low nanogram range. 

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