Amino acids colorimetric methods

Although the free amino acids are present only at very low concentrations in oceanic waters, their importance in most biological systems has led to an inordinate amount of effort toward their determination in seawater. A sensitive, simple, and easily automated method of analysis, the colorimetric nin-hydrin reaction, has been known in biochemical research for many years. In order for the method to be useful in seawater, the amino acids had to be concentrated. This concentration was usually achieved by some form of ion exchange [251]. An automated method not using a concentration step was developed by Coughenower and Curl [252]. While the method was used successfully in Lake Washington, its limit of detection (0.5 imol/l) is just too great for most oceanic samples. 

Introduction of microbiological methods for the determination of amino acids made possible the estimation of the amount of both free and combined amino acids in urine. Dunn et al. (D4), Thompson and Kirby (Tl), Eckhard and Davidson (El), and Woodson et al. (W3) estimated the amount of amino acids liberated in the course of acid or, as in the case of tryptophan determination, alkaline hydrolysis. Microbiological and colorimetric methods used for the determination of certain amino acids present very little opportunity for evaluating the proper quantitative relations between free and combined amino acids, since under the applied condition both combined and free amino acids are equally involved in the reaction. In 1949 Albanese et al. (A3) applied such methods to the quantitative determination of free and combined amino acids in the nondiffusible fraction of urine, and subjected the procedures to broad criticism from just this point of view. 

Ninhydrin (triketohydrindene hydrate) reacts with an amino acid when heated under acidic conditions (pH 3-4) to produce ammonia, carbon dioxide and a blue-purple complex. This reaction forms the basis of many widely used methods (Figure 10.11). One mole of carbon dioxide is liberated from each mole of amino acid, exceptions being the dicarboxylic amino acids, which produce two moles of carbon dioxide, and the a-imino acids, proline and hydroxyproline, which do not produce carbon dioxide. Although this formed the basis of a gasometric technique, colorimetric methods are now the most common. 

The fluorimetric methods often offer improved specificity and sensitivity over colorimetric procedures and the quantitative assays for the aromatic amino acids tyrosine and phenylalanine illustrate this point. 

Some enzymes with improved single amino acid specificity are commercially available. An example is phenylalanine dehydrogenase (EC 1.4.1.1), derived from bacterial sources, which acts on phenylalanine with the simultaneous conversion of NAD to NADH. Quantitation of the phenylalanine is based on determining the amount of NADH produced using standard procedures. In the direct methods, the absorbance at 340 nm is measured, whereas in the colorimetric methods, the reaction is coupled to an electron acceptor... 

Carbon paste and graphite epoxy electrodes modified with RUO2 can be used for detection of amino acids and peptides in F1A systems. Optimal conditions are in strongly alkaline solutions at +0.45 V vs Ag/AgCl electrode, with a fast and linear response. Carbon paste electrodes can be modified also with C03O4371. Colorimetric methods for the determination of amino groups attached to a solid support may give erroneous values... 

There are five categories of protein assay colorimetric assays, direct absorbance methods, fluorescence methods, amino acid analysis, and custom quantitation methods. A brief summary of the principles, advantages, and limitations of these methods follows. 

The quantitative assay for PBG and ALA (Bio Rad, Hercules, CA, USA) that is based on the classical method by Mauzerall and Granick may be used for determination of the porphyrin precursors. PBG is absorbed by the anion-exchange column and ALA by the cation-exchange column interferences are washed out. After elution from the column, ALA is derivatized by acetyl acetone to form a pyrrole. Both ALA and PBG are determined colorimetrically with the modified Ehrlichs reagent. Instead of this broadly used standard method ALA, but not PBG may be detected and quantified using amino acid chromatography. However, our experience has shown that this method is only valid for detecting massively increased concentrations of ALA. 

Many methods are available for measuring TCA-soluble peptides. Possibly the easiest is to measure the absorbance of the solution at 280 nm, as the absorbance at this wavelength is a function of the aromatic amino acid content of the solution. This approach requires a UV spectrophotometer, and the sensitivity of the assay is likely to be lower than that of some of the colorimetric assays. There are also several colorimetric peptide assays that can be applied to this type of peptidase assay, such as the Biuret, Lowry, and Bradford dye-binding methods (for comparison see Piyachomkwan and Penner, 1995). All of these methods measure a relative value rather than an absolute amount of peptide in solution. The results should thus be reported in terms of equivalents, such as BSA equivalents when using a calibration curve prepared using a BSA standard solution. 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

Other methods of estimating total amino acids exist but have not been widely applied to citrus. Various ninhydrin colorimetric methods are published, but different colors produced by individual amino acids caused quantitation problems. Ting and Deszyck (63J attempted to reduce this problem by using two wavelengths, 400 and 570 nm, for proline and the "ninhydrin blue" amino acids. Differences in molar absorption made the results dependent upon the composition of the standard mixture. 

An alternative approach for screening the composition of phytoplankton exudates is to use either 14C-tracer methods combined with chemical fractionation (Hama and Handa, 1987 Siuda and Wcisko, 1990 Sundh, 1991) or colorimetric methods (Obernosterer and Herndl, 1995 Biddanda and Benner, 1997) to characterize the contribution of different classes of organic compounds (carbohydrates and amino acids in polymeric or monomeric forms) to the total pool of exudates. These studies revealed that monomeric and combined carbohydrates were the major components of exudates, typically accounting for 20-90% of the total extracellularly released DOM. 

Recently, in seeking a colorimetric method more sensitive than the Elson-Morgan test (20y), Dische and Borenfreund88 have developed a technique needing only 5y of the amino sugar. The method is based on the deamination of the hexosamine to give the corresponding 2,5-anhydro-hexose with Walden inversion at C2. The anhydro derivatives yield stable characteristic colors when treated with indole in dilute hydrochloric acid, well suited to quantitative colorimetric estimation. 

Column Chromatography. Amino acids were determined in hydrochloric acid hydrolyzates with a 150-cm. column of Amberlite IR-120 (17) using the Technicon AutoAnalyzer. Quantitative measurements were made colorimetrically (17). An alternate method of analysis made use of a 133-cm. column of Technicon chromobead resin and a modification of the sulfur system described by Piez and Morris (14). Although this method gave excellent separation of all classes of amino acids, its value was limited by the failure to separate cystine from glucosamine. Cystine values, therefore, were determined from the IR-120 chromatograms alone. 

As mentioned above, the large majority of recoverable amino acids are in combined form. This result indicates that peptides, and possibly proteins, are important DON constituents. Operational measurements of the amount of total protein in DON can be made by a wide variety of fluorometric and colorimetric assays. A partial hst of those that have been applied to marine samples include coomassie blue (e.g., Mayer et at, 1986 Nunn et ai, 2003 SetcheU, 1981), Lowry s method (Clayton et al., 1988), the fluorescamine assay (Garfield et al., 1979), the bidnchoninic acid assay (Nguyen and Harvey, 1994), and the CBQCA (3 -carboxybenzoyl-quinoline-2-carboxaldehyde) assay (Nunn et al., 2003). A number of the more common methods of protein analysis used in biochemical research, including a discussion of issues affecting their quantitative apphcation, have recently been reviewed by Sapan et al. (1999). 

Tryptophan can be used as an indicator amino acid for the photolysis of protein fibres, such as wool, silk or human hair. Schafer has compared different methods for the quantification of tryptophan and the interference to the colorimetric method (with dimethylaminobenzaldehyde) caused by pigments and dyes. 

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