Amino detection

Luminescence has been used in conjunction with flow cells to detect electro-generated intennediates downstream of the electrode. The teclmique lends itself especially to the investigation of photoelectrochemical processes, since it can yield mfonnation about excited states of reactive species and their lifetimes. It has become an attractive detection method for various organic and inorganic compounds, and highly sensitive assays for several clinically important analytes such as oxalate, NADH, amino acids and various aliphatic and cyclic amines have been developed. It has also found use in microelectrode fundamental studies in low-dielectric-constant organic solvents. 

Lassaigne s test is obviously a test also for carbon in the presence of nitrogen. It can be used therefore to detect nitrogen in carbon-free inorganic compounds, e.g., complex nitrites, amino-sulphonic acid derivatives, etc., but such compounds must before fusion with sodium be mixed with some non-volatile nitrogen-free organic compound such as starch... 

Note. When this test is applied to amino-acids, e.g. glycine, anthranilic acid, ulphanilic acid, no odour is detected owing to the non-volatility of the acidic isocyanide in the alkaline solution. 

While electrospray is used for molecules of all molecular masses, it has had an especially marked impact on the measurement of accurate molecular mass for proteins. Traditionally, direct measurement of molecular mass on proteins has been difficult, with the obtained values accurate to only tens or even hundreds of Daltons. The advent of electrospray means that molecular masses of 20,000 Da and more can be measured with unprecedented accuracy (Figure 40.6). This level of accuracy means that it is also possible to identify post-translational modifications of proteins (e.g., glycosylation, acetylation, methylation, hydroxylation, etc.) and to detect mass changes associated with substitution or deletion of a single amino acid. 

Biosynthesis. Two closely related genes encode the three mammalian tachykinins. The preprotachykinin A gene encodes both substance P and substance K, while the preprotachykinin B gene encodes neuromedin K (45—47). The active sequences are flanked by the usual double-basic amino acid residues, and the carboxy-terrninal amino acid is a glycine residue which is decarboxylated to an amide. As with most neuropeptide precursors, intermediates in peptide processing can be detected, but their biological activities are not clear (ca 1994). 

CCK has been detected in two principal forms, ie, the traditional 33-amino acid peptide, and an octapeptide CCK-8. The intestine produces mainly CCK-33 (133) and the brain produces mainly CCK-8 (132). The CCK precursor contains one copy of CCK-33 (133,134) this peptide is flanked on both ends with double basic residues, whereas CCK-8 is formed from CCK-33 by cleavage of a single basic residue. 

Chemiluminescent Immunoassay. Chemiluminescence is the emission of visible light resulting from a chemical reaction. The majority of such reactions are oxidations, using oxygen or peroxides, and among the first chemicals studied for chemiluminescence were luminol (5-amino-2,3-dihydro-l,4-phthalazinedione [521-31-3]) and its derivatives (see Luminescent materials, chemiluminescence). Luminol or isoluminol can be directly linked to antibodies and used in a system with peroxidase to detect specific antigens. One of the first appHcations of this approach was for the detection of biotin (31). 

The automated amino acid analy2er depends on ion-exchange chromatography (117) and is now a routine tool for the analysis of amino acid mixtures (118). This most advanced machine can detect as Htde as 10 pmol in ninhydrin reaction analysis. One-half to two hours are required for each analysis. An analysis chart is shown in Figure 2. 

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). 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Enzymatic Determination and Microbial Assay. In these methods, only the desired amino acid is detected ia spite of the presence of other amino acids. No expensive tools are needed for these determiaations. The required nutrients for microorganisms and practical operations for the microbial assay of amino acids have been reviewed (134,135). 

The amino group is readily dia2oti2ed in aqueous solution, and this reaction forms a basis for the assay of sulfas. Aldehydes also react to form anils, and the yellow product formed with 4-(dimethylamino)hen2a1dehyde can be used for detection in thiu-layer and paper chromatography. Chromatographic retention values have been deterrnined in a number of thiu layer systems, and have been used as an expression of the lipophilic character of sulfonamides (23). These values have corresponded well with Hansch lipophilic parameters determined in an isobutyl alcohol—water system. 

Derivatization is useful for detection of compounds such as amino acids and amines that lack easily detectable groups. For similar reasons, saccharides, as a class of compound, ehcit much interest. Two derivatization schemes have been reported using benzamide (61) and FMOC—hydrazine (62) to produce fluorescent products. 

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

Mechanistic studies (6,26,27,67) have shown that the acyl enzyme species is the ring opened compound (13), which can tautomerize to the transientiy inhibited amino acrylate (14), and both of these species can react further to give irreversibly inactivated enzyme. Three inactivated forms of the enzyme have been detected. Two, according to labeling studies, retain the complete clavulanate skeleton and the other retains only the carbon chain of the P-lactam ring. Stmcture (15) has been suggested as one possible inactivated form. 

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