Estimation of Amino Acids

Determination of Peptide Structure 1. Identification and Estimation of Amino Acids 

The first stage in the determination of the structure of a peptide is to identify the amino acid residues present in it, and the obvious technique for this is paper chromatography. By no other method is it possible to identify completely the amino acids present in a mixture so simply and so rapidly. Unfortunately no solvent has yet been found on which it is possible to separate all the naturally occurring amino acids and it is necessary to run each hydrolyzate on a two-dimensional chromatogram or else to, run aliquots on two separate one-dimensional chromatograms. Where a large number of peptides is to be analyzed, the latter technique is usually preferable (Consden et al., 1949). 

It is often desirable to carry out an approximate amino acid analysis to determine whether there are one or two residues of a particular amino acid in a peptide. This may be done with sufficient accuracy by carrying out the paper chromatography in a semi-quantitative manner (Poison, 1948 Consden et al., 1949). Several modifications of greater accuracy have been described (Martin and Mittelmann, 1948 Wieland and Fischer, 1948 Woiwod, 1949 Fowden and Penney, 1950 Boissonnas, 1950). The question as to how accurately it is possible to determine an amino 

An elegant method for determining the optical configuration of amino acids on paper chromatograms using the enzyme D-amino acid oxidase has been described by Synge (1949). 

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Quantitative estimation of amino acids on paper chromatograms. Ibid., 108, 608 (1948). 

Estimation of amino acids and amines on paper chromatograms. Anal. Chem., 22, 1327 (1950). 

Quantitative Estimation of Amino Acids on Paper Chromatograms. 

Decarboxylase Preparations in the Estimation of Amino Acids and in Protein Analysis. Biochem. J. 39, 46 (1945). 

Method for the Estimation of Amino Acids. J. biol. Chem. 161,99 (1945). 

ES. Efron, M. L., Quantitative estimation of amino acids in physiological fluids using a Technioon amino acid analyzer. In Automation in Analytical Chemistry (L. T. Skeggs, ed.), pp. 637-642. Mediad, New York, 1966. 

The most widely used technique for the estimation of amino acid isomers is gas-liquid chromatography. Two basic strategies have been used to separate isomers by this technique. Both approaches appeared in the mid-1960 s, and both involve derivatiza-tion of the amino acids to suitably volatile acyl-esters (46,47). One method is similar in concept to the separation of disastereo-mers by ion exchange chromatography discussed above. In one step of the two step derivatization, the amino acids are esterified with an optically active agent. This procedure creates molecules with two centers of asymmetry which can be separated on a non-optically active liquid (stationary) phase (46). This method allows any laboratory equipped with a gas chromatograph to perform Isomer analyses. 

The development of chromatographic methods in recent years has greatly aided in the identification and estimation of amino acids in the acid hydrolysates of soil and in other soil organic matter preparations. Bremner (1965) gives a good summary o.f work of this type. About 20 amino acids are commonly present in acid hydrolysates of soils. In addition, many other compounds, some of which have not been identified, have been found in small amounts by means of paper chromatography. Present information indicates that the amino-acid composition of soils is reasonably constant qualitatively... 

Estimation of amino acid levels by radioreceptor binding techniques is a relatively new approach. The chapter on this topic reviews the underlying principles of ligand binding assays and details the practical considerations fundamental to the successful utilization of these techniques. Although this type of assay is extremely sensitive, it is beset with problems of specificity, and only one amino acid (GABA) can currently be determined accurately by the method Future developments should, however, extend its applicability to some of the other ammo acids, but it is likely to remain a method for determining a parhcular ammo acid, rather than one to be utilized for the measurement of a spectrum of ammo acids. 

For quantitative derivative formation Dns-Cl has to be employed in large excess, so that even micro techniques for the preparation of the derivatives are expensive. Users of labelled Dns-Cl are thus frequently tempted to use too little reagent. More importantly, the specificity of the method is not increased, as compared with fluorimetry, by the use of a labelled reagent. Nevertheless, both I C] and [ H]Dns-Cl have been used for the identification or estimation of amino acids and amines [93, 94]. 

For the future, we need more accurate estimates of amino acid fluxes in different tissues, not only in healthy adult subjects but also in patients with injuries or debilitating diseases, and also for children at various stages of development. Such information would allow better prediction of the needs of various groups of patients for amino acids given parenterally. 

After infancy the data are sparse indeed. Other than the limited data available from children age 10 to 12 years, the published estimates of amino acid needs are based upon extrapolation, assuming that amino acid needs change in proportion to somewhat better defined protein needs. While this seems logical, such data as are available suggest that essential amino acid requirements may fall faster than the total protein needs. 

The above method, contrary to the methods of estimation of amino acids based on copper complexes, is selective. A threefold excess of several common amino acids (including histamine) does not interfere. The method thus enables free histidine to be determined in protein hydrolysates, provided that the histidine concentration is sufficiently high. Tryptophane deforms the polaro-graphic waves when present in an amount corresponding to half that of histidine (above 1 mg/10 ml.). Mercaptoamino acids also interfere, but most of the types of compounds are destroyed during the protein acidic hydrolysis. Glycine affects the end-point if present in amounts larger than 10 mg/10 ml. of the sample. 

Hydrophobic interactions [13] are basically entropically driven, largely due to order/disorder phenomena in the surrounding water. Current estimates of amino acid hydro-phobicity are based on the measured free energies of transferring side chains from water to organic solvents, where the latter presumably simulate the polarity of the protein interior. The following subsection is dedicated to explain in more detail the concept of those hydrophobic interactions and why they are thought to be entropically driven. 

With nitrous acid, the a-amiiio acids react very readily to yield nitrogen gas and a-hydroxy acids which usually cannot be isolated with ease. An excellent volumetric method for the estimation of amino acids is based upon this reaction. ... 

Formaldehyde Condensation.—When an amino acid in neutral solution is mixed with excess of neutral formaldehyde solution, the mixture becomes acid, and can be titrated (Sorensen s reaction), thus affording an important method for the estimation of amino acids and ammonium salts. The mechanism of the change has been studied by Levy (1937). According to the older view, formaldehyde condenses with the amino group to form an unstable methylene-imino derivative, thereby destroying the basicity of the group. 

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