Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Amino Acids
Amino Acids
Group Reagents for Amino Acids

Ninhydrin. A 0.25% (w/v) solution of ninhydrin in acetone is used as a group-locating reagent. As a strong oxidizing agent, ninhydrin causes the Deamination of Amino acids, yielding ammonia, carbon dioxide, the corresponding aldehyde, and the reduced form of ninhydrin:

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The reduced form of ninhydrin subsequently reacts with excess ninhydrin and ammonia to form a blue-purple product with λ_mах = 580 nm. The optical density at this wavelength is virtually a linear function of the number of amino groups. Based on this, a photocolorimetric method for the Quantitative determination of Amino Acids has been developed using the ninhydrin reaction. The blue-purple reaction product has the following Structure:

If the reaction with ninhydrin is carried out at pH 1–5, The amount of free amino acid can be determined from the volume of carbon dioxide evolved. Peptides and primary amines also yield a blue-purple coloration with ninhydrin; however, no CO₂ is produced in these cases. This allows for the analysis of amino acids in the presence of peptides. Proline and hydroxyproline react with ninhydrin to form a bright yellow product (λ_mах = 440 nm), which permits the analysis of amino and imino acid mixtures without prior Separation.

Following the ninhydrin reaction and chromatographic separation in thin layers, spots can be visualized by heating at 100°C for 5 min or, to achieve a lighter Background, by keeping them at room Temperature for several hours. Amino acids yield purple spots; Histidine and Glycine, red-gray; phenylalanine, Tyrosine, and aspartic acid, blue; Tryptophan, brown; asparagine, muddy yellow; and proline, yellow. The sensitivity of the method ranges from 0.2 µg for glycine to 2 µg for histidine.

For identification by coloration, a mixture of 50 mL of a 0.1% ninhydrin solution in ethanol and 2 mL of collidine is used. In some Procedures, it is recommended to add 10 mL of СН₃СООН. The lower the purity of the collidine, the better the coloration. The chromatogram is heated while monitoring The Development of color. The color produced by each amino acid is best determined empirically; however, it should be particularly noted that tyrosine changes its color within a few seconds of its appearance, and the spots of many Other Amino Acids turn purple and blue after a few minutes.

Some researchers prefer a modified version of the above procedures involving copper ions. This is dictated by the need to achieve sufficient color differentiation to identify all amino acids. According to this method, two solutions are prepared: (1) a 0.2% solution of ninhydrin in 50 mL of ethanol + 10 mL of acetic acid + 2 mL of collidine; (2) a 1% solution of Сu(NO₃)₂∙3H₂O. The chromatogram is sprayed with a freshly prepared mixture of 25 mL of solution (1) and 1.5 mL of solution (2), dried, and heated for 1.5–2 min at 105°C. TLC plates are processed in the same manner: dried, sprayed with the reagent, and warmed so that the color can be observed as it develops.

To detect N-Methylamino Acids (which typically yield very weak colors with ninhydrin) in the presence of free amino acids, a specialized reagent is used. Equal volumes of a 0.33% solution of ninhydrin in tert-butanol and a mixture of glacial acetic acid–Water–pyridine (1:5:5) are mixed. The chromatogram is sprayed and heated for 10–15 min at 100–110°C. Primary amines and N-methylamino acids give purple spots of comparable intensity. When sprayed with an aqueous ninhydrin solution prepared by dissolving ninhydrin in n-butanol saturated with water and containing 2% acetic acid, N-methylamino acids yield faint spots.

Ninhydrin-stained paper fades under the action of air and light. For longer preservation of the color, an additional spray containing a Cu(CNO₃)₂ reagent can be applied. To 1 mL of a saturated Cu(NO₃)₂ solution, 0.2 mL of 10% HNO₃ is added, and the volume is made up to 100 mL with 95% ethanol. Preferably, the paper should be coated with a film by immersing it in a saturated solution of polymethyl methacrylate in chloroform.

Fluorescamine. This reagent reacts with the primary amino groups of Amino Acids and peptides. The chromatogram is sprayed with a 0.05% solution of fluorescamine in acetone, and the fluorescent spots are observed. Pre- and post-spraying of the chromatogram with a 10% solution of triethylamine in methylene chloride enhances the sensitivity of the method (down to 1 nmol) as well as the Stability of the resulting fluorescent spots. Spots of proline and hydroxyproline develop slowly upon heating for 3 h at 110°C or after two days at room temperature. In an alternative Procedure, the paper or plate treated with fluorescamine is soaked sequentially in a 0.1 M solution of acetic acid in acetone and a 0.1 M solution of N-chlorosuccinimide in acetone, kept for 5 min, rinsed with acetone, and heated for 5–10 min at 110°C.

Isatin. One of the drawbacks of ninhydrin is that proline can easily be "masked" by the spots of other amino acids. Two Reagents have been described that lack this disadvantage while still providing a wide range of colors for the chromatographic Determination of Amino acids.

1. Dissolve 1 g of isatin and 1.3 g of zinc acetate in 70–80 mL of warm isopropanol. After cooling, add 1 mL of pyridine.

2. Dissolve 1 g of isatin and 1.5 g of zinc acetate in 93 mL of warm isopropanol and 3 mL of water. After cooling, add 1 mL of glacial acetic acid.

These reagents yield sets of spots that differ slightly in color. In both cases, the chromatogram is sprayed, dried, and heated at 80–85°C for 10 min. The background can be washed out without altering the color of The amino acid spots by quickly rinsing the chromatogram with water.

UV Observations. The following procedures for Amino acid analysis using UV irradiation are available.

1. Amino groups can react with free aldehyde groups present in Chromatography paper. The resulting Schiff bases exhibit blue fluorescence under UV light. The chromatogram is heated at 100°C for 30 min. N-Substituted amino acids yield dark spots. The sensitivity is slightly lower than that of ninhydrin, and Some amino acids may undergo degradation.

2. The chromatogram is sprayed with a 0.2% solution of salicylaldehyde in acetone. The resulting Schiff bases may decompose upon water-spraying and exposure to air.

3. The chromatogram is impregnated with a 0.01% solution of 1,2-naphthoquinone-4-sulfonate (Folin's reagent) in methanol and heated at 110–120°C for 10 min.

Iodine. A non-destructive yet non-specific method. The paper or thin-layer plate is exposed to I₂ vapors (from a few crystals) in a Glass chamber. After 10–30 s, the chromatogram is removed, and rapidly fading brown spots are marked with a pencil. Alternatively, the paper can be sprayed with petroleum ether (b.p. 60–80°C) saturated with I₂.

Chlorine. Suitable for the determination of amino acids, N-blocked amino acids, and peptides. The test is specific for compounds containing an N–C bond; therefore, Solvents such as pyridine and collidine must be removed. The paper or thin-layer plate is rinsed with ether and then with an acetone-ethanol (1:1) mixture (amino acids and peptides are insoluble in these solvents). It is treated for 5 min with Cl₂ generated by placing it above a vessel containing equal volumes of a saturated KMnO₄ solution and 10% HCl. The chromatogram is removed, aired to remove excess Cl₂, and immersed in a mixture of equal volumes of a saturated solution of o-toluidine in 2% acetic acid and a 0.05 M KI solution. Blue-black spots appear on a light background. The background can be reduced if the chlorine-treated paper is exposed to ammonia vapors for 10 s before immersion in the o-toluidine solution.

Formation of full and partial amino acid anhydrides.

This reaction is of historical interest. The formation of diketopiperazines during Protein Hydrolysis formed the basis for the erroneous diketopiperazine theory of Cell/13.html">Protein Structure proposed by N. Zelinsky in 1914, which was subsequently developed by his students V. Sadikov and N. Gavrilov.