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

Protein chromatography
Protein chromatography on a DEAE-cellulose column
Chromatography on DEAE-cellulose in the presence of urea

In addition to the Chromatography of native Proteins, several Applications require the chromatographic Separation of modified (reduced, oxidized, carboxymethylated, alkylated, etc.) protein derivatives. This is primarily essential when determining the Amino Acid Sequence of proteins composed of multiple polypeptide chains with different structures. In such cases, these chains must be separated before proceeding with The amino acid sequence analysis.

Chromatography on DEAE-Cellulose in the presence of high urea concentrations is one of the best Methods for fractionating denatured or chemically modified proteins.

Preparation of DEAE-cellulose for chromatography in the Cl^- form is performed as described above, but Column equilibration, dissolution of the sample to be fractionated, and elution are carried out using Buffer solutions containing 8 M urea. In this case, a Tris-HCl buffer (pH 8) containing 0.05 M Cl^- and 8 M urea is used as the starting buffer. To prevent aggregation and association of the sample molecules in the presence of urea, 0.1% sodium dodecyl sulfate is added to the buffer solution. The sample to be fractionated is dissolved in the resulting solution containing both detergents (urea and sodium dodecyl sulfate) and then dialyzed against the same solution for 24 h.

The binding conditions for the sample and the Selection of the elution gradient do not differ from those described above; however, all buffer solutions must contain 8 M urea.

Urea removal. Almost all the urea in the collected fractions can be removed by dialysis. After 72 h of dialysis, no more than 1–5% of the initial urea content remains in the solution. If the fraction is Water-soluble, urea can be removed by Gel filtration. Water-insoluble Proteins can be freed from urea by washing the precipitate formed during dialysis several times with distilled water using centrifugation.

Following this operation, the aqueous suspension of the insoluble protein is lyophilized.

NOTES

1. Powdered or fibrous DEAE-cellulose cannot be regenerated in the column. Once chromatography is complete, the ion exchanger is removed from the column and stored in the cold. When a sufficient amount of used DEAE-cellulose has accumulated, it is regenerated as described above.

2. Due to the risk of bacterial growth, a column containing regenerated ion exchanger ready for fractionation should not be left at room Temperature.

3. The preparation of a column packed with spherical DEAE-cellulose (beaded polymer) does not differ from that of a column with granular or fibrous cellulose. The Use of spherical DEAE-cellulose offers several significant advantages, despite a slightly lower resolution:

1) The flow rate of the liquid through the column packed with spherical DEAE-cellulose is constant, and no shrinkage of the ion exchanger occurs during chromatography.

2) Packing the column with spherical DEAE-cellulose is very simple; a homogeneous ion-exchange bed is quite easy to obtain.

3) Unlike Other types of ion exchangers that bind denatured proteins very tightly, spherical DEAE-cellulose has a lower adsorptive capacity, allowing it to be regenerated directly in the column.

4) The ease of Column packing, constant flow rate through the ion exchanger, and straightforward regeneration process make spherical DEAE-cellulose suitable for use in large preparative columns.

Recommended reading

Block H. J., Bolling D., The Amino Acid Composition of proteins and Foods.

Analytical Methods and Results, Springfield, Thomas Publ., 1951.

Smith I., Chromatographic and Electrophoretic Techniques, Vols 1 and 2, London, Heinemann Publ., 1960.