Purification and characterisation of proteins

Each cell contains several thousand different proteins. If we want to find out about their properties, we have to first purify them. How else could we be sure that a particular reaction is really caused by a particular protein?

The basic principles behind protein purification (and only those can be covered here) are easily understood, however, it is difficult if not impossible to predict which methods are most suitable for the purification of a given protein. Very rarely is it possible to purify a protein by a single method, usually the judicious combination of several purification steps is required. Protein purification is therefore much more art than science.

3.1 Homogenisation and fractionisation of cells and tissues

If you want to study, say, the enzymes present in liver, the first you have to do is to obtain fresh liver tissue. This is done at the abattoir, immediately after an animal has been slaughtered. The tissue is transported into the laboratory on ice, to minimise proteolytic damage.

Once in the lab, the tissue needs to be disrupted. This is a critical step: Cells should be broken open, but cell organelles should remain intact. Usually the tissue is minced first by hand, then cut into a fine pulp by rotating knifes (for example in a Warring blender like those used to make milk shakes in the kitchen) and finally homogenised by the application shearing forces in specialised equipment (Potter-Elyehjem- or DoUNCE-homogeniser, French press). All these steps are performed on ice, buffer solutions are used to keep the pH at the required value, they usually also contain protease inhibitors, antioxidants and sucrose or mannitol to keep the osmotic pressure in the solutions at the same level as in the cell (« 300-350 mosm). Ions like Na+,

K+, Ca2+ or Mg2+ are added as required by the enzyme to be isolated, some very sensitive enzymes also require the addition of their substrate to stabilise them.

Then the various cell organelles need to be separated from each other. This is done by fractionated centrifugation [26]. First connective tissue, undamaged cells and other debris are removed by a brief spin at low speed (10min at 500 g1). In the next step nuclei and plasma membranes are spun down (10min at 3000 g). Mitochondria, plastids and heavy microsomes require about 30min at 20 000 g to be spun down, 1h at 100 000 g is required for light microsomes. The remaining supernatant contains the cytosol.

These crude preparations are then subjected to further purification steps.

3.2 Precipitation methods

Protein precipitation is a very crude method for purification and rarely achieves enrichment by more than a factor of 2-3. However, it is quick and cheap. If applied to crude homogenisates, it may remove material that would interfere with later purification steps.

3.2.1 Salts

Proteins interact strongly with water, and only the hydrated form is soluble. If the available water concentration is reduced by the addition of salts or water miscible organic solvents (methanol, ethanol or aceton), proteins precipitate out of solution. It is essential to perform these reactions in the cold, proteins are rapidly denatured by precipitating agents at room temperature.

The precipitating agent is slowly added to the well stirred protein solution, until it reaches a concentration where the desired protein is just soluble. Precipitated material is removed by centrifugation, then more precipitant is added, until all desired material has precipitated. It is separated from the solution by centrifugation.

Proteins can be separated into two groups depending on their behaviour towards salt:

Globulins are more or less globular proteins, their electrical charge is evenly distributed across their surface area. Globulins are soluble in distilled water, and can be precipitated by high salt concentrations.

1 The centrifugal acceleration is usually measured as multiple of the gravitational acceleration on earth (@ = 9.81 m/s2).

Albumins have molecules with an elongated, rod-like shape with unsymmet-rical distribution of electrically charged groups. As a result, albumins are insoluble in distilled water, because the molecules form head-to-tail aggregates held together by electrical forces. Low salt concentrations neutralise these charged groups and allow the albumins to go into solution, high salt concentrations precipitate them again.

From the salts ammonium sulfate is most often used, as it is cheap, non-toxic, highly water soluble and strongly ionised. Purified proteins can be crystallised by slow addition of ammonium sulfate, such crystals, suspended in the mother liquor, tend to be very stable if kept refrigerated. Many enzymes are sold in this form by the suppliers. Salts are removed from proteins by dialysis or gel filtration.

3.2.2 Solvents

Organic solvents (ethanol, methanol or acetone) need to be used even more carefully than salts to prevent irreversible denaturation of proteins, precipitation is usually done at subzero temperatures. Particular attention needs to be given to the fact that mixing of solvents with water generates heat. After precipitation the solvent is usually removed by lyophilisation (freeze-drying).

Polyethylene glycol (PEG) may also be used, it is less denaturing and produces no heat when mixed with water.

3.2.3 Heat

Proteins are irreversibly denatured by heat and precipitate out of solution. However, some proteins are more resistant than others, especially in the presence of their ligands. If crude protein extracts are heated to a carefully chosen temperature for a carefully chosen time, some of the extraneous proteins precipitate, whilst the relevant one stays intact. Precipitated material is removed by centrifugation.

3.3 Chromatography

Chromatography was invented by the Russian botanist Mikhail Tsvett for the separation of leaf pigments (on aluminium oxide columns). The method was extended to proteins by Richard Willstatter. Proteins are bound to a solid support and then specifically eluted. Several types of interaction can be used (see also fig. 3.1):

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