Purification of Thiol-Containing Substances By Covalent Chromatography

Extracted from Affinity Chromatography Principles and Methods, GE Healthcare, 2007

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 51.

Reaction scheme purification of a thiolated substance (RSH) on Activated Thiol Sepharose 4B or Thiopropyl Sepharose 6B

Fig. 51. Reaction scheme purification of a thiolated substance (RSH) on Activated Thiol Sepharose 4B or Thiopropyl Sepharose 6B. The reducing agent is a low molecular weight thiol such as dithiothreitol.

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 52.

Partial structure of Activated Thiol Sepharose 4B

Fig. 52. Partial structure of Activated Thiol Sepharose 4B.

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 53.

Partial structure of Thiopropyl Sepharose 6B

Fig. 53. Partial structure of Thiopropyl Sepharose 6B.


Purification Options

  Binding capacity/ml medium Coupling conditions   Maximum operating flow Comments
Activated Thiol Sepharose 4B Mercaptalbumin, 2–3 mg pH 4–8, 3–16 hours, +4 °C - room temp. 75 cm/h* Low capacity derivative suitable for coupling of high molecular weight substances. Supplied as dry powder, rehydration required.
Thiopropyl Sepharose 6B Ceruloplasmin, 14 mg pH 4–8, 3–16 hours, +4 °C - room temp. 75 cm/h* High capacity derivative suitable for coupling of low molecular weight substances. Supplied as dry powder, rehydration required.

* 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 A280 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.


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.


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

  Density of thiol groups Composition pH stability* Mean particle size
Thiopropyl Sepharose 6B 25 µmoles/ml Mixed disulphide containing 2-thiopyridyl protecting groups attached to Sepharose 6B through a chemically stable ether linkage. Short term 2–8 Long term 2–8 90 µm
Activated Thiol Sepharose 4B 1 µmole/ml Mixed disulphide formed between 2,2’-dipyridyl disulphide and glutathione coupled to CNBr-activated Sepharose 4B. Short term 2–8 Long term 2–8 90 µm

* 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.


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 (see below).

Loss of free thiol content of reduced Thiopropyl Sepharose 6B on storage at +4 °C

Fig. 54. Loss of free thiol content of reduced Thiopropyl Sepharose 6B on storage at +4 °C. The reduced medium was stored in 0.1 M sodium acetate or phosphate, 0.3 M NaCl, 1 mM EDTA at the indicated pH values.

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.



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