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

Gel Filtration of Proteins and Peptides
Separation of high-molecular-weight fragments by Sephadex gel filtration

When analyzing Cell/13.html">Protein Structure, one frequently encounters The Study of fragments obtained by Enzymatic Hydrolysis. Preparative Separation of such fragments is a key step in protein research. Gel filtration on a Column of an appropriate grade of Sephadex is a highly suitable method for separating fragments of enzymatically cleaved Proteins. A prime example demonstrating this method is the separation of IgG fractions hydrolyzed by Papain in the presence or absence of reducing agents. If papain hydrolysis occurs in the absence of reducing agents, some IgG molecules remain uncleaved, while others are cleaved to form two large, well-studied fragments (Fab and Fc) and small, low-molecular-weight Peptides, some of which can pass through the membrane during dialysis. The Molecular Weight of native IgG is 150,000, that of the Fab and Fc fragments is about 50,000, and the small peptides have a molecular weight of 5,000 or less. Gel filtration on a Sephadex G-100 column allows for a relatively easy separation of these three fractions of the papain IgG hydrolysate.

1. Treatment of Sephadex G-100. An appropriate amount of dry Sephadex G-100 is suspended in distilled Water, and after settling, the fine particles floating in the supernatant are decanted. This decantation Procedure is repeated several times until the supernatant becomes completely clear. The Sephadex is then left to swell in distilled water for 24 hours. The swollen Sephadex gel is equilibrated with a 0.075 M phosphate buffer solution, pH 7.0, containing 0.075 M NaCl, and packed into the column.

2. Gel filtration. For the fractionation of a papain hydrolysate of approximately 100 mg of IgG, a 2x60 cm column is used. After applying the sample, any remaining residue is washed down with three 4 ml portions of the buffer solution, and gel filtration is initiated at a flow rate of 40–60 ml/h. The elution profile shows two closely consecutive peaks, followed after some time by a third peak. The eluate fraction corresponding to the first peak contains unhydrolyzed IgG (papain-resistant IgG molecules). The fraction corresponding to the second peak contains Fab and Fc fragments. The third fraction, represented by the third peak on the chromatogram, consists of low-molecular-weight peptides. After washing the column with one bed volume of the buffer solution, it can be reused.

NOTES

1. Selection of the chromatographic column. Columns used for Chromatography must have a specific design. Regardless of the column type, the "dead" space at its bottom must be minimized. If the "dead" volume is relatively large, remixing of already separated fractions may occur, reducing fractionation efficiency. Care must be taken to ensure that gel particles do not clog the pores of the supporting porous Glass or metal frit at the bottom of the column. To prevent this, a circle of filter paper of the appropriate size is placed on the frit. Columns manufactured by Pharmacia Ltd. (Sweden) are highly convenient; they have virtually no "dead" space and allow chromatography to be run in both directions.

2. Column length and sample volume. Generally, short columns (20–30 cm) are suitable for Group separation, while longer columns (up to 100 cm) are preferred for fractionation. In analytical Gel chromatography, the sample volume should be small, but not less than 0.02 of the bed volume. In preparative gel chromatography, the goal is to apply the maximum sample volume that still allows for the required resolution.

3. Column diameter. In wide-bore columns (20–30 mm), the "wall" effect interferes less with separation; therefore, wide columns are easier to operate and yield better results than long, narrow ones (10 mm or less in diameter). However, for analytical gel chromatography, it is advisable to use columns with a diameter of 10 mm.

4. Column packing. The simplest way to pack a column is by using an extension tube of the same diameter. This extension tube should extend the column at the top by about one-third of its height. A plastic capillary tube is attached to the bottom outlet of the column, with its free end positioned 4–5 cm below the top of the extension tube. The swollen Sephadex gel suspension is allowed to settle, and the excess supernatant is removed so that the remaining liquid layer is about half the volume of the settled gel. After gently stirring the Sephadex gel, the suspension is poured down a glass rod into the column fitted with the extension tube. It is recommended to pour the entire required volume of the suspension in a single step if possible. The Sephadex is then allowed to settle for 15–20 min before opening the outlet valve to let the buffer flow. The flow rate of the buffer during packing should be lower than the operating flow rate used after Sample application. As soon as the upper bed surface reaches the desired level, the extension tube is removed. Both before and after applying the sample to be fractionated, the gel surface must be carefully protected from disturbance. In long runs, a constant eluent flow rate can be conveniently maintained using a Mariotte bottle.

5. Brief explanation of some terms used in gel chromatography literature.

V_t — total bed volume. (It is recommended to determine the total volume by filling the column with water and measuring its volume rather than by calculation, as wall irregularities in the latter case may lead to inaccurate results.)

V_о — void volume, i.e., the volume of liquid filling the space between the beads of the swollen gel. (It can be determined by chromatographing a substance that cannot penetrate the beads, such as Blue Dextran.)

V_x — volume of the swollen gel in the column (V_t — V_o).

V_t — internal volume, i.e., the volume of solvent inside the pores of the swollen gel.

V_e — elution volume, i.e., the volume of eluting solution required to elute a given substance.

V_s — separation volume, i.e., the volume of eluting solution equal to the difference between the elution volumes of two substances separated by gel chromatography.

V_r — water regain, i.e., the maximum amount of water (in ml) absorbed by 1 g of dry gel.