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

Gel filtration of proteins and peptides
Prefractionation of enzymatic hydrolysates on a Sephadex G-50 column

Specific Enzymatic Hydrolysis or chemical Cleavage of any medium-molecular-weight protein yields a rather complex mixture of Peptides. Isolating and purifying all the peptides that made up the polypeptide chain and are present in the unfractionated hydrolysate is a highly challenging task. Therefore, it is recommended to subject the protein hydrolysate to preliminary Separation first. Among modern Fractionation Methods, Gel filtration is the most suitable for this purpose.

1. Sephadex G-50 Treatment. Sephadex is suspended in a 10-fold volume of 0.01 M NH₄OH, stirred for 30 min at room Temperature, and allowed to settle. After that, the supernatant containing fine gel particles is decanted, and the suspension and settling steps are repeated. Then, the Sephadex gel is suspended in a 3-fold volume of 0.01 M NH₄OH and packed into a Column, through which 5 volumes of 0.01 M NH₄OH are passed.

2. Column size Selection. Preliminary fractionation of 100–200 mg of hydrolysate is carried out on a 1.5 x 150 cm column.

3. Sample preparation for fractionation. The sample to be fractionated should be applied to the column in a minimal volume. Usually, 100–200 mg of hydrolysate is dissolved in 3–5 mL of 0.01 M NH₄OH.

4. Sample application. Open the column outlet to drain the excess buffer solution until its level is only 1 mm above the Sephadex gel bed. Then, touching the column wall with the bent tip of a pipette, layer the sample solution onto the Sephadex using gentle circular motions. After this, reopen the column outlet valve and allow the applied sample to slowly enter the gel. Wash down the remaining sample with three 1 mL portions of 0.01 M NH₄OH. Fill the upper part of the column with 0.01 M NH₄OH and connect the column to a Mariotte bottle or a pump.

5. Elution. Elution is performed with 0.01 M NH₄OH at a flow rate of 300 mL/h, collecting 3–5 mL fractions. The fractions are pooled According to the optical density profile of the eluate at 280 nm, obtained using a "Uvicord" or spectrophotometer measurements. The pooled fractions are then lyophilized.

After elution, the column is washed with at least two volumes of 0.01 M NH₄OH solution and can be reused. After 4–5 fractionation runs, the Sephadex gel should be removed from the column and washed with NH₄OH solution. Due to the risk of bacterial growth, storing the column at room temperature is not recommended.

NOTES

1. In accordance with the gel filtration mechanism, the choice of Sephadex grade (G-index) should be guided by the expected Molecular Weight of the peptides. If the hydrolysate contains large peptides, it is advisable to use Sephadex G-50 or G-75. If the average molecular weight of the peptides is small, preliminary fractionation is carried out on Sephadex G-25. The higher the Specificity of cleavage (for example, by Trypsin action on acetyl- and trifluoroacetyl Proteins, or by Cyanogen bromide cleavage), the greater the likelihood of obtaining large peptides. In these cases, Sephadex with a higher G-index is used. Non-specific hydrolysis (hydrolysis by Chymotrypsin, subtilisin, Papain, Pepsin, acid, etc.) typically yields small peptides, which should be pre-fractionated on Sephadexes with a lower G-index.

2. Due to the adsorption properties of Sephadex, the yield of material from protein hydrolysate fractionation is typically 55–60%.

3. Measuring the optical density of solutions at 280 nm can only detect peptides containing Tyrosine and Tryptophan. To detect peptides lacking these Amino Acids, other peptide Determination Methods must be used. These include, for example, direct spectrophotometry at 220 nm, as absorption at this wavelength is characteristic of compounds with peptide bonds.

The ninhydrin reaction can serve as a laborious but highly sensitive method for analyzing individual fractions. Equal aliquots of 0.2–0.3 mL are taken from each fraction, mixed with 1 mL of 2.5 N NaOH, and incubated for 3 h at 90°C. Then, 1 mL of 30% acetic acid, 0.25 mL of a 0.2% SnCl₂ solution in 0.2 M nitrate buffer, and 0.25 mL of a 4% ninhydrin solution in propanol are added to each sample. The samples are placed in a boiling Water bath at 100°C for 10 min, after which 3 mL of 50% propanol is added to each, and the optical density is determined at 570 nm.

4. Preliminary fractionation on a Sephadex column is highly convenient for isolating and purifying components of a mixture selectively labeled with radioactive isotopes. In this case, In addition to determining peptide concentration by spectrophotometry or the ninhydrin reaction, the radioactivity of the collected fractions is also measured using an appropriate instrument (such as a scintillation or other counter).

5. Preliminary fractionation using polyacrylamide gel yields similar results to gel filtration on Sephadex. Unlike Sephadex, which is a natural dextran gel, polyacrylamide gel is a synthetic polymer gel with extremely low adsorption properties. Therefore, separation on polyacrylamide gel can be performed with virtually no loss of the fractionated material. The molecular weight of the fractionated substances is also highly significant for polyacrylamide gel separation. The lower the molecular weight of the components in the mixture, the lower the Bio-Gel index that should be used for fractionation.