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

Protein Chromatography
Fractionation of serum proteins on a DEAE-Sephadex column

Principle of the method. Proteins bound to a DEAE-Sephadex Column at a given pH are eluted by decreasing the pH and increasing the Ionic strength of the eluent.

Applications. Fractionation on DEAE-Sephadex can be successfully used in all fields of biochemistry, Protein Chemistry, immunology, and clinical chemistry to isolate protein fractions.

PROCEDURE

1. Column preparation. DEAE-Sephadex A-50 is commonly used for anion-exchange Chromatography of proteins. 1.0 g of the ion exchanger is suspended in distilled Water and allowed to stand for 1 h, after which the supernatant, along with slowly settling fine particles, is decanted. The ion exchanger is then washed with 0.5 M NaOH to remove Cl^- ions. Excess alkali is washed out with distilled water, followed by an acid wash. Since Blood Serum proteins are typically fractionated in a phosphate buffer, 0.1 M phosphoric acid is used as the acidic washing solution. The excess acid is in turn washed out with distilled water, and then the ion exchanger is washed with the starting buffer until equilibrium is reached. Washing is most easily performed by filtration on a Büchner funnel, as this is convenient not only for washing but also for analyzing the filtrate.

An alternative method to prepare DEAE-Sephadex for chromatography is as follows: the ion exchanger is allowed to swell for 24 hours in a large excess of the starting buffer, which is changed several times. If equilibrium is not reached after 24 hours, decantation and replacement of the supernatant are continued until its pH equals that of the starting buffer.

2. Packing the column. A small amount of buffer is added to the swollen DEAE-Sephadex, which has been equilibrated with the starting buffer. After closing the column outlet, the resulting thin, thoroughly mixed slurry is poured into the column, which has been pre-filled to one-third of its height with the starting buffer. The outlet is then opened, and the column is gradually packed with the ion exchanger to the desired height. Afterwards, the column is checked for equilibration; if it is not equilibrated, washing with the starting buffer is continued until the pH of the effluent matches that of the starting buffer.

3. Setting up the gradient elution system. A 0.5 L flask containing the starting buffer (0.02 M phosphate buffer, pH 8.0) is used as the mixing chamber. The reservoir, which forms a closed system with the mixer, is a 1 L vessel filled with 0.3 M buffer. A pump is used to ensure a continuous flow of the buffer onto the column. By opening the outlet, the buffer level in the column is lowered to the top of the gel bed. The serum sample to be fractionated, previously dialyzed against the starting buffer for 24 hours, is then carefully applied to the ion exchanger, taking care not to disturb the top of the gel bed. The applied sample is washed in with three 2 mL portions of the starting buffer, and chromatography is initiated. Fractionation of 3 mL of serum requires a 2x50 cm DEAE-Sephadex column:

4. Chromatography. Chromatography is performed in the same manner as described for the DEAE-Cellulose column (see p. 209).

NOTES

1. Upon completion of chromatography, the Sephadex gel is immediately removed from the column and regenerated. First, any proteins that likely remain adsorbed to the ion exchanger must be removed. This is achieved by washing the gel with 1 M Na₂HPО₄, followed by washing with distilled water until a neutral reaction is obtained (matching the pH of distilled water). The Ion Exchange gel is then equilibrated with the starting buffer.

2. The capacity of DEAE-Sephadex is typically 3 meq/g. Accordingly, 3 mL of serum can be applied to a DEAE-Sephadex column prepared from 1.0 g of dry ion exchanger.

3. Ion-exchange Sephadexes swell significantly when the ionic strength of the buffer decreases; therefore, using solutions with an ionic strength below 0.05–0.1 is not recommended. If chromatography is initiated in a buffer that is too dilute, a subsequent substantial increase in the ionic strength of the eluting buffer will cause the Sephadex beads to shrink, which can lead to void formation in the gel bed. The formation of voids disrupts the uniform flow of liquid through the column and adversely affects the results of chromatography.

4. It is recommended to regenerate the DEAE-Sephadex gel after removing it from the column and to repack the column before each experiment; these Procedures ensure that the gel bed remains homogeneous.

5. Chromatography on DEAE-Sephadex is highly suitable for isolating IgG from serum and for separating proteins within this fraction that exhibit different electrophoretic mobilities. The starting buffer initially elutes IgG with low electrophoretic mobility from the column. Fast-migrating Proteins of the same Class are eluted only during gradient elution. DEAE-Sephadex chromatography is also a standard method for purifying myeloma IgG.

6. CM-Sephadex G-50 can be used for cation-exchange chromatography. Its pretreatment should be carried out in the same way as for DEAE-Sephadex, with the sole difference that CM-Sephadex is first treated with 0.5 N HCl and then washed with alkali. The chromatographic procedure on CM-Sephadex G-50 is identical to that on CM-cellulose.

7. Based on pore size, ion-exchange Sephadexes are classified into two types, designated as 25 and 50. The ion exchanger designated 25 has smaller pores than the Sephadex designated 50. As in Gel filtration, the pore size determines the size of the molecules that can diffuse into the Sephadex beads. Therefore, Sephadex 25 is used to separate substances with a molecular weight below 10,000, while Sephadex 50 is used for substances with higher molecular weights. Very large molecules cannot penetrate inside the Sephadex beads and are adsorbed on their surface. For the fractionation of high-molecular-weight compounds, ion-exchange Sephadexes of any pore size can be used.

8. Ion-exchange Sephadexes are manufactured as beads with a diameter of 40–120 µm, whose spherical shape ensures a high flow rate through the column. However, it should be kept in mind that swollen Sephadex beads are easily deformed. Therefore, attempting to increase the flow rate by increasing the pressure of the solution applied to the column beyond a certain limit may, conversely, lead to a decrease in flow.

9. The pH and ionic strength of the buffer also affect the Swelling of the Sephadex beads and, consequently, the flow rate through the column. These parameters should be optimized so that maximum swelling or shrinkage of the Sephadex does not disrupt the chromatographic process.

10. For optimal fractionation, it is crucial to correctly calculate The amount of ion exchanger loaded into the column. In each specific case, it is recommended to determine the optimal column dimensions in preliminary experiments. Practice shows that to obtain good results, The ratio of column height to diameter should be approximately 10:1.

11. Swollen ion exchangers can be stored as Suspensions in neutral solutions for several months, even at room Temperature, provided precautions are taken against microbial contamination. Sodium azide (0.02%) is added to suspensions of CM- and sulfoethyl-Sephadex (SE-Sephadex), while 1% butanol is added to DEAE-Sephadex suspensions.