Amino-acids, estimation reactions

It is difficult to estimate what the rate of the metal ion catalyzed oxidative deamination reaction of amino acids would be in natural waters. Hamilton and Revesz (30) found that the rate of oxidation of alanine in the presence of pyridoxal and manganese ions was inhibited by EDTA. Since metal ions in natural waters can be complexed by a variety of organic and inorganic compounds, their effectiveness in catalyzing the oxidative deamination of amino acids may be reduced. Also, the fraction of dissolved amino acids which would be complexed by metal ions at the pH and metal ion and amino acid concentrations found in natural waters must be considered. At neutral pH, where the amino group of the amino acid is protonated, the fraction of the amino acid that would be in the form of the metal ion complex depends upon the equilibrium constant for the formation of the complex and the pK of the amino proton of the amino acid. The reactions for the formation of the Cu2+-alanine complexes can be written as... 

The significance of industrial acrolein production may be clearer if one considers the two major uses of acrolein—direct oxidation to acryUc acid and reaction to produce methionine via 3-methyhnercaptopropionaldehyde. In acryUc acid production, acrolein is not isolated from the intermediate production stream. The 1990 acryUc acid production demand in the United States alone accounted for more than 450,000 t/yr (28), with worldwide capacity approaching 1,470,000 t/yr (29). Approximately 0.75 kg of acrolein is required to produce one kilogram of acryUc acid. The methionine production process involves the reaction of acrolein with methyl mercaptan. Worldwide methionine production was estimated at about 170,000 t/yr in 1990 (30). (See Acrylic ACID AND DERIVATIVES AmINO ACIDS, SURVEY.)... 

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their properties often have to be improved. One way to achieve this is to optimize reaction conditions such as pH, temperature, solvents, additives, etc. [6-9]. Another way is to modulate the substrates without compromising the synthetic efficiency of the overall reaction [10]. In most cases for commercial manufacturing, however, the protein sequences have to be altered to enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial enzymes worldwide have been engineered for industrial applications [11]. Precise prediction of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution... 

In earlier studies [5,6] superoxide detection in mitochondria was equated to hydrogen peroxide formation. However, while it is quite possible that superoxide is a stoichiometric precursor of mitochondrial hydrogen peroxide, it is understandable that the level of hydrogen peroxide may be decreased due to the reactions with various mitochondrial oxidants. Moreover, superoxide level can be underestimated due to the reaction with mitochondrial MnSOD. Several authors [7,8] assumed that mitochondrial superoxide production may be estimated through cyanide-resistant respiration, which supposedly characterizes univalent dioxygen reduction. This method was applied for the measurement of superoxide production under in vitro normoxic and hyperoxic conditions, in spite of the finding [7] that cyanide-resistant respiration reflects also the oxidation of various substrates (lipids, amino acids, and nucleotides). Earlier,... 

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. 

Liquid/liquid partition chromatography was explored by Willstatter from 1913. The process was extensively developed by Martin and Synge (ca. 1941-1948) who partitioned amino acid derivatives between chloroform and water using precipitated silica as support for the aqueous phase. The preparations of silica were again very variable and it was difficult to prevent adsorption which interfered with the expected behavior of the aminoacids. At first methyl orange was added to the water phase to visualize the amino acids the separation of the acids then caused a red band to move down the columns. The quantitative reaction with ninhydrin was introduced by Moore and Stein in 1948 for both the detection and estimation of the amino acids. Consid-... 

Usually the amino acid analyzer is first standardized by running through it a sample containing known quantities of amino acids to account for any differences in their ninhydrin reaction properties. In this way it is possible to relate directly the amount of amino acid present to the amount of colored product formed, as measured by the area under the peak produced on the strip-chart recorder (see fig. 3.15). Similarly, the amino acid hydrolysate of a protein of unknown composition can be run through the analyzer, and the relative peak areas can be used to estimate the ratios of the different amino acids present. 

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. 

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