Practical Protein Chemistry - A. Darbre 1989

Traditional strategy for determining protein structure
Fractionation of soluble peptides
Gel filtration

Gel filtration is widely employed as an initial Separation step, typically yielding high recoveries, with large Peptides frequently obtained in a state that requires no further purification. Soluble peptides are loaded onto Sephadex G-50 or G-25 columns in an ammonium bicarbonate buffer and eluted with the same buffer, monitoring the fractionation process spectrophotometrically at 280 nm (absorption maximum for Tryptophan and Tyrosine residues) and 230 nm (absorption maximum for the peptide bond). To fully dissolve the starting mixture in a small volume prior to Column loading, it is sometimes necessary to add a small amount of crystalline urea. Peptides derived from Pepsin Hydrolysis have poor solubility at neutral pH; these are solubilized in a small volume of 100% formic acid, diluted to a 5% concentration, and applied to Sephadex or Bio-Gel columns equilibrated with the same solution.

Small aliquots of each obtained fraction are analyzed by high-voltage paper Electrophoresis; for "conditionally pure" peptides, the N-terminal amino acid is determined, along with partial Amino acid sequences via the dansyl-Edman method (Chapter 11). Aliquots for Amino acid analysis are withdrawn from fractions containing peptides shown to be homogeneous by all criteria, after which the fractions are lyophilized. Large peptides (20–50 residues), even when pure, often appear on electrophoretograms as diffuse bands, in contrast to the smaller fragments generated by their hydrolysis, which form sharp zones upon electrophoresis. This allows minor components—undetectable by other means—to be revealed in "conditionally pure" chromatographic peaks by re-subjecting a small portion of the material to secondary fragmentation and subsequent electrophoresis. Large peptides contaminated with impurities can be successfully purified on DEAE-Cellulose, although recoveries in this Procedure tend to be rather low. Since establishing or monitoring the Amino Acid Sequence of a large peptide generally requires additional hydrolysis, it is advisable to reserve two-thirds of the purified material for this purpose. Even with a low recovery from DEAE-cellulose, The amount of peptide obtained is usually sufficient for amino acid analysis, partial sequencing, and supplementary analytical-scale hydrolysis. The resulting hydrolyzate is analyzed by electrophoresis and Chromatography, followed by preparative hydrolysis of the crude peptide, with fragments belonging to the major component identified by comparison against the analytical map.

Class="center">Table 10.5. Ion-Exchange Columns

General Guidelines

Dowex-50

DEAE-Cellulose

Resin Preparation

Wash in situ with 1 M NaOH, 1 M HCl, and pH 5.0 buffer (2 M pyridine: 645 mL pyridine + 573 mL glacial acetic acid + Water to a total volume of 4 L). After testing the eluate for the absence of Na+ (by flame test), wash with pH 3.1 buffer (0.2 M pyridine: 64.5 mL pyridine + 1114 mL glacial acetic acid + water to a total volume of 4 L) until the column is equilibrated

Process on a Buchner funnel According to the manufacturer's protocol (wash with 0.2 M NaOH, distilled water, 0.2 M HCl, and distilled water again). Leave to stand for several hours in 10% ammonium bicarbonate. Wash with 0.1% ammonium bicarbonate (pH 8.3) and pack the column. Equilibrate the ion exchanger overnight by pumping the same buffer at a flow rate of 100 mL/h

Column Type and Dimensions

Water-jacketed high-pressure column (9.0 x 500 mm)

Glass pipette (25 mL volume) with a glass wool plug at the bottom and a rubber stopper at the top traversed by a syringe needle

Column Temperature

60 °C

Room temperature

Flow Rate

15 mL/h (high-pressure pump, e.g., a Milton Roy chromatographic mini-pump)

5 mL/h

Fraction Volume, mL

2

2

Sample application

Dissolve the sample in 1 mL of water with The addition of a few drops of conc. HCl to pH 2. Centrifuge to remove any precipitate (if turbidity develops), apply to the column, flush with nitrogen, and fill the void volume with pH 3.1 buffer

Dissolve the desalted sample in 2 mL of 0.1% ammonium bicarbonate (pH 8.3). Add crystalline urea if necessary to dissolve any precipitate

Wash 1

Wash with pH 3.1 buffer until acidic peptides are completely eluted (one column volume). Monitoring: high-voltage paper electrophoresis at pH 6.5

Wash with 0.1% ammonium bicarbonate (pH 8.3) until basic peptides are completely eluted. Monitor by measuring optical density at 230 nm

Buffer pH Gradient

300 mL of pH 3.1 buffer + 600 mL of pH 5.0 buffer (i.e., a 1:2 ratio in a gradient mixer) provide a linear gradient of pyridine ion concentration

First gradient: 200 mL of 0.1% ammonium bicarbonate (pH 8.3) + 200 mL of 1% ammonium bicarbonate (pH 8.3); elutes basic and neutral peptides. Second gradient: 200 mL of 1% ammonium bicarbonate (pH 8.3) + 200 mL of 3% ammonium bicarbonate (pH 8.3); elutes predominantly acidic peptides

Wash 2

pH 5.6 buffer (8.5 M pyridine: 684 mL pyridine + 180 mL glacial acetic acid + water to a total volume of 1 L)

50 mL of 5% ammonium bicarbonate, followed by 10 mL of 6 M guanidine hydrochloride. Upon completion of the procedure, the column must be regenerated to its initial conditions

The remaining peptides are pooled and loaded onto a Dowex-50 column or, in several portions, onto DEAE-cellulose.



Last update: 06/08/2026

Editorial and Educational Adaptation: This material has been compiled based on the primary/original source text. The project team performed an editorial review, corrected technical inaccuracies, structured sections, and adapted the content for an educational format.

What was processed:

  • elimination of formatting defects (OCR errors, structural breaks, corrupted characters);
  • editorial organization of content;
  • standardization of terminology in accordance with academic sources;
  • verification of factual statements against the original source text.

All mentions of the author, publication year, and origin of the primary text have been preserved in accordance with the source.