Amino Acids, Peptides and Proteins - Devenyi T., Gergely J. 1976

Ion-exchange chromatography of peptides
Fractionation of peptides on a Dowex 50x2 column in non-volatile buffer solutions

Principle of the method. Dowex 50 x 2 with a low degree of cross-linking in the Na⁺ or NH⁺₄ form binds Peptides consisting of 2–20 amino acid residues. By gradually increasing the pH and Ionic strength of the solution applied to the Column, the bound peptides can be selectively eluted.

Scope of application. Preparative Separation of peptides, Amino Acid Sequence analysis in Polypeptides and Proteins.

PROCEDURE

1. Preparation of Buffer solutions¹. Ammonium formate buffer solution pH 3.08: 0.75 N formic acid + 0.2 N NH₄OH.

Ammonium acetate buffer solution pH 5.06: 1.47 M acetic acid + 1 N NH4OH.

Sodium citrate buffer solution pH 3.1: 0.1 M citric acid + 0.2 N NaOH + 0.138 N HCl.

Sodium citrate-acetate buffer solution pH 5.1 (1 N Na⁺): 0.25 M citric acid + 0.179 N acetic acid + 0.57 N NaOH + 0.5 N sodium acetate.

Sodium citrate-acetate buffer solution pH 5.1 (2 N Na⁺): 0.5 M citric acid + 0.358 N acetic acid + 1.14 N NaOH + 1 N sodium acetate.

Sodium citrate-acetate buffer solution pH 4.3: 5-fold diluted buffer solution pH 5.1 + 0.2 N buffer solution pH 3.1 (1:1).

¹ The indicated molarity corresponds to the final concentration of the given component of the buffer solution.

2. Resin preparation. a) Preparation of the resin in the Na⁺ form. The resin is suspended in 0.25 N HCl and stirred first at room Temperature and then at 60–70°C in a Water bath. After 60 min of stirring, the suspension is cooled to room temperature, the supernatant is removed, the resin is washed three times with distilled water, and suspended in 0.25 N NaOH. The resulting suspension is heated again to 60–70°C in a water bath with stirring for 1 h, then cooled and washed with distilled water until neutral.

b) Preparation of the resin in the NH⁺₄ form. First, the resin is washed with alkali, then with acid. The chloride-free resin in the H⁺ form is suspended in 0.25 N NH₄OH, stirred at 60–70°C for 1 h, and washed with distilled water until neutral.

3. Column packing. A small amount of pH 3.1 buffer solution is poured into a 1.8 x 150 cm column equipped with a thermostatic jacket. The resin is suspended in the same buffer solution heated to 50°C, and after thorough mixing, the degassed suspension under vacuum is loaded into the column. The column should be packed in such a way that a small amount of buffer solution always remains above the resin to ensure free settling of the resin particles. Packing is complete when the resin level is 10 cm below the upper end of the thermostatic jacket.

4. Sample application. About 200 mg of the peptide mixture to be fractionated is dissolved in 25–30 ml of distilled water, the pH of the solution is adjusted to 2 with formic acid, then 25–30 ml of pH 3.1 buffer solution is added, and the mixture is applied to the column.

5. Chromatography. Peptides are fractionated using gradient elution. The starting buffer solution has a pH of 3.1. The mixer contains 2500 ml of 0.5 N buffer solution pH 5.1. The eluate emerging from the column is collected in 10 ml fractions. The ninhydrin reaction is performed either directly with the resulting eluate or after its alkaline Hydrolysis.

Alkaline hydrolysis. From each fraction, 0.3 ml is sampled, mixed with 1 ml of 2.5 N NaOH solution, and incubated at 90°C in a water bath for 2.5 h. During this time, the concentration of the samples increases. To each sample, 1 ml of 30% acetic acid is added, and the ninhydrin reaction is carried out as follows.

To the tubes containing the concentrated samples, 0.25 ml of 0.2 M citrate buffer solution pH 5 containing 0.2% SnCl₂ is added, the mixture is stirred, then 0.25 ml of a 4% ninhydrin solution in propanol is added, placed in a boiling water bath for 10 min, and cooled. To each tube, 3 ml of 50% propanol is added, mixed thoroughly, and the absorbance is measured colorimetrically at 570 nm.

Samples are taken from the eluate fractions corresponding to the peak tops, and their homogeneity is determined by Electrophoresis or the "fingerprinting" method. If a fraction is inhomogeneous, it is subjected to rechromatography.

It is difficult to give any specific recommendations regarding rechromatography. Its conditions are determined by the specific composition, as well as the chemical and Physical Properties of the analyzed material. However, it seems most appropriate to perform rechromatography on the resin in the NH+4 form in buffer solutions containing ammonium ion, since in these cases the obtained fractions contain volatile salts, and Desalting occurs simultaneously with concentration. If, for example, the fractions are eluted with an ammonium acetate buffer solution, they are first dried under vacuum at 50°C, and then dissolved in a few drops of distilled water and lyophilized.