Purification of Thiol-Containing Substances By Covalent Chromatography

Thiol-containing substances can be isolated selectively by covalent binding to an activated thiolated matrix via thiol-disulphide exchange to form a mixed disulphide bond. After washing away unbound material, the thiol-containing substance is eluted by reducing the disulphide bond. This technique is also known as covalent chromatography. The reaction scheme is shown in Figure 1.

In Activated Thiol-Sepharose 4B the hydrophilic glutathione residue acts as a spacer group thereby decreasing steric interference with exchange reactions at the terminal thiol group. The partial structure is shown in Figure 2.

In Thiopropyl Sepharose 6B the 2-hydroxypropyl residue acts as a hydrophilic spacer group. The partial structure of Thiopropyl Sepharose 6B is shown in Figure 3.

* See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Both media react spontaneously and reversibly under mild reducing conditions or in the presence of denaturing agents with substances containing thiol groups.

Performing a Separation

Binding buffer: 20 mM Tris-HCl, 0.1–0.5 M NaCl, pH 7.0.

If required, include 8 M urea or 6 M guanidine HCl to ensure that the protein is denatured and all thiol groups are accessible for the reaction.

1 mM EDTA can be added to remove trace amounts of catalytic heavy metals.

Elution buffer alternatives:

For covalently bound proteins: 0.025 M cysteine, 50 mM Tris-HCl, pH 7–8.

To minimize reduction of intramolecular disulphide bridges: 5–20 mM L-cysteine, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0 or 20–50 mM 2-mercaptoethanol, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0.

Note: When using Thiopropyl Sepharose, 2-thiopyridyl groups must be removed after the protein has bound. Wash the column with sodium acetate 0.1 M, 2-mercaptoethanol 5 mM, pH 4.0 before beginning elution.

N.B. Degas all buffers to avoid oxidation of free thiol groups.

If the proteins to be purified contain disulphide bonds, the disulphide bridges must be reduced, for example with 2-mercaptoethanol (5 mM).

Analyze the thiol content of the sample by thiol titration to ensure that the capacity of the medium will not be exceeded.

Use preliminary titration studies with 2,2’-dipyridyl disulphide to provide a guide to optimal coupling conditions. A spectrophotometer can be used to determine the release of 2-thiopyridone (absorbance coefficient = 8.08 x 10³ M^-1 cm^-1 at 343 nm) when the sample (1–5 mg in 1–3 mL binding buffer) reacts with 2, 2’-dipyridyl disulphide. Choose the conditions to suit the specific sample. Under standard conditions at pH 7.5, a few minutes is usually enough for a complete reaction.

1. Use a desalting column to transfer pre-dissolved sample into the binding buffer (Buffer exchange and desalting) and to remove any low molecular weight thiol compounds and reducing agents that might interfere with the coupling reaction.
2. Weigh out the required amount of powder (1 g gives about 3 mL for Activated Thiol Sepharose 4B and 4 mL for Thiopropyl Sepharose 6B).
3. Wash and re-swell on a sintered glass filter (porosity G3), using degassed, distilled water or binding buffer (200 mL/g, 15 min at room temperature) to remove additives.
4. Prepare the slurry with binding buffer in a ratio of 75% settled medium to 25% buffer.
5. Pack the column (see Appendix 3, Column packing and preparation) and equilibrate with binding buffer.
6. Load the sample at a low flow (5–10 cm/h) and leave in contact with the medium for at least one hour to ensure maximum binding.
7. Wash the column with binding buffer until no material appears in the eluent (monitored by UV absorption at A_{280 nm}).
8. Elute the target molecules with elution buffer using a low flow (5–10 cm/h).

The coupling reaction can be monitored and, in some cases, quantified by following the appearance of 2-thiopyridone in the eluent at 343 nm during the purification.

Sodium phosphate or ammonium acetate can be used as an alternative to Tris-HCl.

Resolve different thiol proteins by sequential elution: 5–25 mM L-cysteine < 0.05 M glutathione < 0.02–0.05 M 2-mercaptoethanol < and 0.02–0.05 M dithiothreitol in 50 mM Tris- HCl, 1 mM EDTA, pH 7–8.

Reactivation

Pass one to two column volumes of a saturated solution (approximately 1.5 mM) of 2,2’-dipyridyl disulphide, pH 8.0 through the medium.

Prepare 2,2’-dipyridyl disulphide:

1. Make a stock solution by adding 40 mg 2,2’-dipyridyl disulphide to 50 mL buffer at room temperature and stirring the suspension for several hours.
2. Filter off insoluble material.
3. Adjust the pH. The solution will be approximately 1.5 mM with respect to 2,2’-dipyridyl disulphide.

Cleaning

Wash with non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 minute. Re-equilibrate immediately with a minimum of 5 column volumes of binding buffer.

Media Characteristics

* Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. When a molecule has been coupled to the thiolated matrix, the long term and short term pH stability of the medium will be dependent upon the nature of that molecule.

Chemical Stability

Stable to all commonly used aqueous buffers and additives such as detergents. Avoid azides.

Storage

Store freeze-dried powders below +8 °C.

Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at 4 to 8 °C. Storage under nitrogen is recommended to prevent oxidation of thiol groups by atmospheric oxygen.

Avoid using sodium azide, merthiolate or phenyl mercuric salts as bacteriostatic agents. Azide ions will react with the 2,2’-dipyridyl disulphide groups, although low concentrations (0.04%) have been used.

Do not store the suspension for long periods in the free thiol form. Thiol groups are susceptible to oxidation by atmospheric oxygen, especially at alkaline pH. Figure 54 shows the decrease in free thiol content of Thiopropyl Sepharose 6B on storage for moderate periods at three different pH values. The thiol content of partially oxidized medium is restored by treatment with reducing agent under conditions used for removing protecting groups (Figure 4).

Removal of Protecting Groups

Activated Thiol Sepharose 4B and Thiopropyl Sepharose 6B may easily be converted into the free thiol form (i.e. reduced) by removing the 2-thiopyridyl protecting groups with a reducing agent.

1. Prepare the medium as described earlier. Gently remove excess liquid on a glass filter (porosity G3).
2. Suspend the medium in a solution containing 1% (w/v) dithiothreitol or 0.5 M 2-mercaptoethanol,

1. 0.3 M sodium bicarbonate, 1 mM EDTA, pH 8.4.
2. Use 4 ml of solution per gram of freeze-dried powder.
3. React for 40 minutes at room temperature, mixing gently.
4. Wash the medium thoroughly with 0.5 M NaCl, 1 mM EDTA in 0.1 M acetic acid. Use a total of 400 ml of solution per gram of original freeze-dried powder. Perform the washing in several steps.

Estimate the content of free thiol groups by measuring the absorption increase at 343 nm (see above) due to the 2-thiopyridone liberated in the wash solutions. The amount of thiol groups on the medium can be estimated by reacting an excess of 2,2’-dipyridyl disulphide with the medium and measuring the liberated 2-thiopyridone at 343 nm.