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

Protein Chromatography
Protein chromatography on a cation-exchange cellulose column
CM-cellulose

A. Preparation of CM-Cellulose for Chromatography. Before chromatography, CM-cellulose should be treated as follows:

1) The required amount of cellulose is stirred for 15 min at room Temperature with a 5-fold volume of 0.5 M NaOH. After the ion exchanger particles settle, the supernatant is decanted.

2) The alkali-treated CM-cellulose is washed with distilled Water until a neutral pH is reached. Granular or fibrous cellulose is recommended to be washed by centrifugation or by filtration on a Büchner funnel. Spherical CM-cellulose settles very rapidly upon standing of the suspension, so it can be washed by decantation in a beaker.

3) To convert the CM-cellulose washed to neutrality into the H⁺ form, it is suspended in 0.5 N HCl and stirred for 15 min at room temperature. The suspension is allowed to settle, the supernatant is decanted, and the acid Treatment is repeated once more. After this, the CM-cellulose is washed with distilled water to neutrality, as described in the previous section.

4) CM-cellulose in the H⁺ form can be stored as an aqueous suspension at 4°C.

B. Column Size Selection. For the fractionation of 100 mg of protein or polypeptide using CM-cellulose with a capacity of 0.7–0.8 meq/g, a 1.5 x 35 cm column is required. Preparative Separation of 1.0 g of protein can be carried out on a 3.5 x 45 cm column.

C. Column Flow Rate. The flow rate is determined by The properties of the CM-cellulose. In a column of fibrous or granular cellulose measuring 1.5 x 35 cm, it reaches 60 mL/h. To obtain good separation during chromatography on spherical CM-cellulose, the flow rate should be slightly reduced.

D. Selection of Chromatographic Conditions. Both stepwise and gradient elution can be used. Stepwise elution is convenient only when the Components of the mixture being fractionated elute at significantly different pH values and eluent molarities.

Gradient elution can be performed in three ways: 1) by increasing the eluent molarity at a constant pH, 2) by changing the eluent pH at a constant molarity, 3) by eluting with a gradient of both pH and eluent molarity.

The selection of optimal fractionation conditions in each case is based on preliminary chromatographic runs. However, as a rule, for the fractionation of natural polymers with cationic properties, such as Polypeptides and Proteins, gradient elution with increasing eluent molarity at a constant pH is preferable.

Chromatography on CM-cellulose can be initiated with the cation exchanger in either the H⁺ or NH⁺₄ form. The conversion of CM-cellulose to the H⁺ form is described above. The Conversion of the cation exchanger from the H⁺ form to the NH₄⁺ form is performed as follows.

CM-cellulose in the H⁺ form is suspended in a 5-fold volume of 0.1 M ammonium acetate solution, pH 4.6, and the suspension is stirred in a beaker for 30 min at room temperature. After the cellulose particles settle, the supernatant is decanted, and the described Procedure is repeated three more times. Then, the column is packed with the cellulose suspension in the same solution, and after appropriate settling, it is washed with 0.01 M buffer solution until the pH and conductivity of the eluate are equal to those of the eluent.

E. Column Chromatography on CM-Cellulose in the H⁺ Form Using Gradient Elution. A 1.5 x 35 cm column is packed with CM-cellulose in the H⁺ form and washed with distilled water,

100 mg of the material to be fractionated is dissolved in 3–5 mL of 0.01 M ammonium acetate buffer solution, pH 4.6. After removing the layer of distilled water above the CM-cellulose in the column, the test solution is applied to the ion exchanger using a bent-tip pipette in a circular motion. Once the applied solution has entered the CM-cellulose bed, any remaining traces are carefully washed down with three 1 mL portions of the buffer solution.

1.5 bed volumes (relative to the ion exchanger volume) of 0.01 M ammonium acetate buffer solution are passed through the column, and gradient elution is started at a constant pH of 6.7, increasing the molarity from 0.01 to 0.5 M, and then, if necessary, from 0.5 to 1.0 M.

To establish the gradient, 100 mL of ammonium acetate buffer, pH 6.7, with molarities of 0.01 and 0.5 M, are poured into the mixer (connected to the column) and the reservoir, respectively. A flow rate of 1 mL/min is established, and after 200 mL of eluent has passed through the column, if necessary, elution with a second molarity gradient is started, increasing it from 0.5 to 1.0 M.

The obtained eluate fractions are pooled according to their absorbance at 280 nm (or the continuous absorbance monitoring profile).

NOTES

1. The main advantage of the ammonium acetate buffer used is its volatility, which allows desalted fractions to be obtained by lyophilization without dialysis. In addition, this buffer can be used over a wide pH range. However, other Buffer solutions can also be used for CM-cellulose chromatography.

2. Fibrous, granular, and spherical CM-cellulose are used for chromatography. Due to their high adsorption activity, fibrous and granular CM-cellulose cannot be regenerated directly in the column. Upon completion of chromatography, the ion exchanger must be removed from the column and regenerated once sufficient quantities have accumulated. Cellulose derivatives are susceptible to bacterial contamination; therefore, CM-cellulose collected for regeneration should be stored at 4°C.

3. The adsorption capacity of spherical CM-cellulose is relatively low, so it can be regenerated directly in the column after 2–4 fractionation runs.

4. For preparative fractionation, The Use of spherical ion exchangers is recommended. They are easier to pack into the column, settle rapidly during washing by decantation, and ensure a constant flow rate of the solution through the column, unlike fibrous and granular Supports, which cause a decrease in the flow rate during chromatography.