Amino acids detection systems

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-... 

Patients with classic PKU nsnally appear normal at birth. If the disease is not recognized and treated within the first month of life, the infant gradnally develops varying degrees of irreversible mental retardation (IQ scores freqnently nnder 50), delayed psychomotor matnration, tremors, seiznres, eczema, and hyperactivity. The nenrologic seqnelae may result in part from the competitive interaction of phenylalanine with brain amino acid transport systems and inhibition of neurotransmitter synthesis. These biochemical alterations lead to impaired myelin synthesis and delayed nenronal development, which result in the clinical picture in patients such as Piquet Yuria. Because of the simplicity of the test for PKU (elevated phenylalanine levels in the blood), all newborns in the United States are required to have a PKU test at birth. Early detection of the disease can lead to early treatment, and the nenrologic conseqnences of the disease can be bypassed. 

Although the above studies are not specifically instances where array sensing has been employed for amino acid detection and differentiation, they are clear examples of systems amenable to array sensing. Such circumstances where receptors have differential binding to the target analytes represent cases that would benefit from array sensing techniques. 

Bhushan and Ali (1987) tested amino acid separations on silica gel layers impregnated with various metal salts. Bhushan and Reddy (1989) reported the separation of phenylthiohydantoin (PTH) amino acids on silica gel with new mobile phases. Laskar and Basak (1988) de.scribed a new ninhydrin-based procedure that produced different colors and good sensitivity for amino acid detection. Bhushan and Reddy (1987) reviewed the TLC of PTH amino acids. Gankina et al. (1989) described a unidimensional multistep silica gel HPTLC method for the separation and identification of PTH and dansylamino acids. Bhushan et al. (1987) developed numerous solvent systems for effective separations of 2,4-dini-trophenyl-(DNP) amino acids. Bhushan (1988) reviewed the TLC resolution of enantiomeric amino acids and their derivatives. Kuhn et al. (1989) reported the amino acid enantiomer separation by TLC on cellulose of d- and L-tryptophan and methyltryptophan. Guenther (1988) determined TLC-separated enantiomers by densitometry. 

The order of separation of the amino acids, along with approximate Rf values, will be as follows histidine, 0.22 glycine, 0.33 alanine, 0.49 valine, 0.69 leucine, 0.82. Differences in the colors of amino acids detected by this procedure will be very noticeable and are as follows histidine, blue-gray glycine, purple-red alanine, dark purple-violet valine, purple-red leucine, purple-red. The TLC system used as described is extremely sensitive and will achieve good results with applications of 0.1-0.5 lg per amino acid. The choice between silica gel and cellulose for the TLC of amino acids has been dictated mainly by personal preferences rather than clear-cut experimental advantages for one or the other layer. 

Thin layer chromatography, as was previously mentioned, has been automated, with the main emphasis on drug detection. However, it seems reasonable to expect this system to be able to handle carbohydrate and amino acid detection as well, and this would have a practical impact on mass screening for genetic variants. 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

The advantages of this method are a short reaction time and the nonfluorescence of the OPA reagent. Therefore, excess reagent must not be removed before the chromatography stage. Using this method, it is possible to measure tryptophan, but not secondary amino acids such as proline or hydroxyproline. Cysteine and cystine can be measured, but because of the low fluorescence of their derivatives, they must be detected using an UV system, or alternatively oxidized to cysteic acid before reaction. 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

In aqueous solutions at pH 7, there is little evidence of complex formation between [MesSnflV)] and Gly. Potentiometric determination of the formation constants for L-Cys, DL-Ala, and L-His with the same cation indicates that L-Cys binds more strongly than other two amino acids (pKi ca. 10,6, or 5, respectively). Equilibrium and spectroscopic studies on L-Cys and its derivatives (S-methyl-cystein (S-Me-Cys), N-Ac-Cys) and the [Et2Sn(IV)] system showed that these ligands coordinate the metal ion via carboxylic O and the thiolic 5 donor atoms in acidic media. In the case of S-Me-Cys, the formation of a protonated complex MLH was also detected, due to the stabilizing effect of additional thioether coordination. ... 

PelZ is a hydrophilic protein of 420 amino acids with a short hydrophobic sequence at its N-terminal end which has Ae characteristics of the signal sequences of exported proteins. The signal peptide may be 24 amino acids long, which would corroborate wiA the usual length encountered in prokaryotes. The molecular cloning of the pelZ gene in an expression vector pT7-6 allowed for the specific 35S-cysteine-methionine raAo-labelling of PelZ in E. coli K38. We could detect, in crude extracts, the presence of a precursor and a mature form of PelZ. After cell fractionation, Ae mature form of PelZ could be localized in Ae periplasm of E. coli. So PelZ appears to be a protein exported by Ae Sec-dependent system of translocation. 

A large number of amino acid transporters have been detected by isolating mutations which selectively inactivate one permease without altering enzyme activities involving the corresponding amino acid. Competitive inhibition, kinetics and regulatory behaviour have also been used as criteria to distinguish one transport system from another (see section 4.2). 

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol... 

Multiple electrodes have been used to obtain selectivity in electrochemical detection. An early example involved the separation of catecholamines from human plasma using a Vydac (The Separation Group Hesperia, CA) SCX cation exchange column eluted with phosphate-EDTA.61 A sensor array using metal oxide-modified surfaces was used with flow injection to analyze multicomponent mixtures of amino acids and sugars.62 An example of the selectivity provided by a multi-electrode system is shown in Figure 2.63... 

The polyamines putrescine, cadaverine, spermidine, and spermine, which are seen at elevated levels in some victims of cancer, were separated on a Technicon (The Technicon Company Chauncey, NY) TSM Amino Acid Analyzer packed with an 8% divinylbenzene-co-polystyrene sulfonated resin with post-column ninhydrin detection.111 Amines such as ethanolamine, noradrenaline, hexamethylene diamine, methoxytryptamine, spermine, and spermidine were separated from amino acids on a DC-4A cation exchange resin.112 A similar approach, using a Beckman Model 121M amino acid analyzer equipped with an AA-20 column, was also successful.113 A Polyamin-pak strong cation exchange column (JASCO) was eluted with a citrate buffer for the detection of putrescene, spermine, cadaverine, and 1,5-diaminohex-ane from rat thymus.114 A post-column o-phthaldehyde detection system was used. 

Fung, Y.-S. and Mo, S.-Y., Determination of amino acids and proteins by dualelectrode detection in a flow system, Anal. Chem., 67, 1121, 1995. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

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