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

Amino Acids
Amino Acids
Specific Reagents for Amino Acids

If for any reason it is impossible to identify a spot by its position and coloration using general tests, specific Reagents for a single amino acid or a group of Amino Acids can be employed.

Tyrosine

Folin-Denis reaction with a-nitroso-β-naphthol. The chromatogram is immersed in a 0.1% solution of a-nitroso-β-naphthol in acetone and allowed to dry at room Temperature. It is then immersed in acetone containing 10% (v/v) conc. HNO₃, dried, and gradually heated starting from one side so that the appearance of spots on the chromatogram can be monitored. The yellow color of the paper fades in the area that is sufficiently heated. Tyrosine is identified by a red color with a sensitivity of ~ 1 µg. Tryptophan produces a grey to brown color with lower sensitivity. Phenol and collidine should be removed by washing with an acetone-diethyl ether (1:1) mixture. Tyrosine can also be identified using the Pauli test (see below).

Millon's reaction – a reaction with mercurous and mercuric salts and nitrous acid. The sensitivity is approximately 50 µg/mL.

Tryptophan and Related Compounds

Ehrlich's Reagent

1. The chromatogram is immersed in a 1% (w/v) solution of p-dimethylaminobenzaldehyde in an acetone-conc. HCl (9:1) mixture and kept at room temperature for several minutes. Tryptophan yields a purple spot, while pyrroles and related compounds produce red spots.

2. Alternatively, the chromatogram can be sprayed with a 2% solution of p-dimethylaminobenzaldehyde in 5% HCl and then dried at room temperature. Tryptophan produces a purple coloration, whereas aromatic amines and citrulline yield a yellow one.

UV Irradiation

1. The paper is dried at room temperature and observed under UV light. Tryptophan either shows no coloration at all or exhibits a very faint one (due to the presence of degradation products, the corresponding spot usually being positioned slightly ahead of the true one). Certain related compounds are clearly visible As a result of interaction with the paper, for example: kynurenine (blue coloration); kynurenic acid (blue-green); hydroxykynurenine (yellowish-green); aminoacetophenone (sky-blue); 2-amino-3-hydroxyacetophenone (yellow); xanthurenic acid (blue); anthranilic acid (blue-violet); hydroxyanthranilic acid (sky-blue). Aromatic Solvents sometimes interfere.

2. The chromatogram is sprayed with a perchloric acid (70–72%)–Water (6:1) mixture. Within 10 min, tryptophan exhibits a stable and intense green-blue fluorescence. Subsequent spraying with a 1% FeCl₃ solution produces a reddish-yellow color.

Terephthalaldehyde in an acidic medium. The chromatogram is sprayed with a 0.2% solution of terephthalaldehyde in acetone containing 10% acetic acid and heated at 105°C for 3 min. Tryptophan produces a blue spot. This reagent is exceptionally sensitive.

Histidine

Pauli's Reagent. Solution 1 (1% sulfanilic acid in 1 M HCl) and Solution 2 (5% aqueous NaNO₂ solution) are kept in a refrigerator. Equal volumes of these solutions are mixed (also while cold) 5 minutes prior to use. The paper is sprayed carefully to avoid washing away the spots, followed by spraying with a 15% Na₂СО₃ solution. Histidine and other imidazoles immediately yield a red color. Colored spots with lower sensitivity are also produced by histamine (pink-brown); tyrosine (pink); xanthine, diiodotyrosine (orange); thiamine, tryptophan, adenine (pink); guanine (orange-purple); catechin (purple).

To reduce the interfering effect of tyrosine, the Procedure can be modified. The chromatogram is sprayed with a 0.1% solution of p-anisidine in a 1:1 mixture of ethanolic solutions of 0.11 M HCl and 10% amyl nitrite, and air-dried at room temperature. It is then sprayed with 1% KOH in ethanol. Recrystallized p-anisidine may fail to work. Other solvents retained by the paper, such as phenol and collidine, must be washed out with acetone and ether.

Terephthalaldehyde. The chromatogram is sprayed with a 0.2% solution of terephthalaldehyde in acetone and heated at 105°C for 10 min. Histidine produces a yellow-green spot, while tryptophan yields a brown spot under UV light. The reagent is highly sensitive, particularly for histidine.

Phenylalanine

Ninhydrin-NaHCO₃. The chromatogram is stained with ninhydrin (see above). When the heated paper is sprayed with a dilute (0.15–10%) NaHCO₃ solution, only the phenylalanine spot remains stable.

Sulfur-Containing Amino Acids: Platinum Tetraiodide

1. The chromatogram is immersed in a solution of: 0.002 M chloroplatinic acid – 1 M KI – 2 M HCl – acetone (4:0.25:0.4:76). After drying at room temperature, the coloration is intensified by exposure to HCl vapors. Cystine, Cysteine, Methionine, cystathionine, lanthionine, djenkolic acid, methionine sulfoxide, and others appear as colorless spots against a pink Background. If phenol or collidine was used as the solvent, the paper should first be washed with an acetone-ether (1:1) mixture.

2. To achieve higher chromatogram contrast (dark brown background), the 0.002 M H₂PtCl₆ solution can be replaced with a 0.002 M PdCl₂ solution in 0.1 M HCl.

Iodine-Azide. The chromatogram is sprayed with a 0.025 M I₂ solution in 50% ethanol containing 1.5% NaN₃. White spots of cysteine appear instantly against a light brown background, followed by cystine (after 15 min), and then methionine (after 60 min). The spots are best viewed under UV light. This reagent also enables the detection of thiamine.

Nitroprusside. Reagent a: Mix 1.5 g of sodium nitroprusside in 5 mL of 1 M H₂SO₄ + 95 mL of MeOH + 10 mL of 28% NH₃, and filter. The chromatogram is immersed in this solution. SH-compounds produce a red color. While the paper is still damp, it is immersed in Reagent b (2% NaCN solution in 95% MeOH). The Cleavage of —S—S— bonds by this reagent leads to the appearance of a red color within a time interval ranging from a few seconds to 10 min. For cystine, the sensitivity is 1 µg. If the detection of SH-compounds is not required, equal volumes of Reagents a and b at twice the concentration are used. In this case, Arginine produces an orange spot on a pale yellow background, which turns grey-blue against an intense green background.

Glycine

o-Phthalaldehyde. The chromatogram is sprayed with a 0.2% solution of o-phthalaldehyde in acetone and heated at 50°C for 10 min. Glycine produces a green spot, which appears chocolate-brown under UV light at 365 nm; histidine and tryptophan exhibit an intense yellow fluorescence at 365 nm. This reagent must not be used after Chromatography in aromatic solvents.

Arginine

Sakaguchi reaction. The chromatogram is immersed in a 0.1% solution of 8-hydroxyquinoline in acetone and air-dried. It is then thoroughly sprayed with a solution of 0.2 mL Br₂ in 100 mL of 0.5 M NaOH. Arginine and other guanidines yield orange-red spots.

Ornithine

Vanillin. The chromatogram is sprayed with a 0.2% solution of vanillin in acetone, followed by a 0.1% solution of KOH in ethanol. After heating at 110°C for 10 min, ornithine produces a red spot. Proline and hydroxyproline react in a similar manner.

Imino acids (proline and hydroxyproline)

Isatin. The chromatogram is sprayed with a 0.2% solution of isatin in n-butanol containing 4% acetic acid, and heated at 105°C for 15 min. Proline and hydroxyproline yield stable blue spots. Although many Other Amino Acids develop colors in this same range, these transient colors fade at room temperature within a few hours. To selectively obtain blue spots for proline and hydroxyproline, the paper is washed in 1 M HCl after development and then rinsed in water.

To differentiate between proline and hydroxyproline, the chromatogram is first immersed in the solution described above and subsequently treated with Ehrlich's reagent (see above). All colors produced by isatin disappear, except for the hydroxyproline coloration, which shifts from blue to light cherry-red.

Multiple Staining

Ninhydrin spraying can be followed by Treatment with α-nitroso-β-naphthol or Ehrlich's reagent, as well as reagents providing lower analytical sensitivity (such as Pauly's or Sakaguchi reagents). Ninhydrin may precede isatin staining, or isatin can be followed by Ehrlich's reagent or α-nitroso-β-naphthol (though the sensitivity of the latter test is thereby reduced). Ehrlich's reagent treatment may be followed by the Sakaguchi reaction, even if prior steps involved spraying with ninhydrin or isatin. UV irradiation and iodine treatment do not interfere with any subsequent tests.

Reactions Involving R-Group Functional Groups

In certain cases, reactions specific to the Functional groups of amino acid R-groups are employed, such as those targeting the SH group of cysteine, the phenolic hydroxyl of tyrosine, or the guanidine group of arginine. The cysteine R-group exhibits exceptionally high reactivity due to the presence of a thiol or sulfhydryl moiety. In alkaline media, cysteine loses a sulfur atom as a result of complex reactions.

Tetranitromethane reacts with the phenyl group of tyrosine under mild conditions with extremely high Specificity:

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Cystine, formed by the covalent linkage of two cysteine residues, plays a vital role in protein architecture by forming cross-linking Disulfide Bonds.