ABSTRACT
For the purification of the enzyme, we must take care of the following points in the purification procedures. (1) Temperature should be kept as low as possible, around 5°C, since most proteins are denatured at high temperature. (2) Buffers used for the purification are better to be around pH 7, since most proteins are stable under neutral conditions. As an example, steps of preparation of extracts from liver are described.It is better to get the fresh liver, such as from a slaughterhouse. The liver should be cooled as soon as possible. Then, prepare the extract in a cold room (4-5°C). Cut the liver into small pieces (smaller than 1 cm length in width) and homogenize these in a
buffer using an apparatus such as blender, Potter-Elvehjem homo-genizer, sonicator, and French press. A blender is familiar in kitchen to prepare juices of vegetables
and fruits. It is useful for relatively large-scale homogenization. The Potter-Elvehjem homogenizer is one of a mortar-pestle type homogenizer and is a widely used apparatus. It has been named
after van R. Potter and C. A. Elvehjem. The mortar is made of a cylindrical glass, and the pestle is made of Teflon. The pestle rotates by an electric motor, while small tissues in buffer go through narrow interface between the mortar and the pestle; thus, the tissues are disrupted. The sonicator uses the energy of the ultrasonic sound to disrupt tissues (cells), and is used for a small-scale to large-scale homogenization. However, it is not recommended to use tough tissues. During sonication, it is important to cool the homogenization mixture, since heat is generated during sonication.French press is a method to lyse cells using a high pressure, named after C. S. French. It is useful for the small-scale homo-genization. The cell suspensions to be disrupted are passed through narrow valve under high pressure, and exposed to atmosphere. This causes a large pressure drop, inducing the disruption of cells. During homogenization, samples should be cooled as described above. In addition, buffers used are better to include chemicals to prevent the action of proteases. Some proteases require metal ion; therefore, EDTA (ethylenediamine tetraacetic acid) is better to be added to chelate metal ions. Moreover, a serine protease inhibitor, phenylmethane sulfonylfluoride (PMSF), is also better to be added. 9.2 Purification of Enzyme
The method is used in the initial stage of purification and to concentrate the proteins. 9.2.1.1 Salting-out
Most proteins are soluble in water under low ionic strength, but insoluble at high ionic strength. This precipitation is called salting out. Its principle is briefly as follows. Proteins in water
are surrounded by water molecules, which interact with amino acid residues of protein. When the salt is added to the protein solution, some of the water molecules are used to dissolve the salts, resulting in the decrease in the water molecules that are interacting with the amino acid residues on the surface of proteins. These lead to the increase of protein-protein interaction to precipitate. Table 9.1 Grams of ammonium sulfate necessary to take 1 liter solution from one “percent saturation” to another
Source: Reproduced with permission from Methods Enzymol., 1, Green, A. A., and Hughes, W. L. Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents, 67-90, Copyright Elsevier (1955). Ammonium sulfate is commonly used for the salting out of proteins, since the solubility is almost independent of temperature (70-80 g/100 g water at 0 to 40°C), indicating high solubility at low temperature. It is better to add solid ammonium sulfate to the protein solution for keeping the increase of the volume of the protein solution as small as possible. The ammonium sulfate solution is acidic, so care must be taken to keep the solution neutral by the addition of ammonia solution. It may be better to
start by the addition of ammonium sulfate from 10% saturation. When precipitates appeared, keep the mixture for 10 min or more, and collect the precipitates by centrifugation.To the supernatants, add ammonium sulfate to 20% saturation according to Table 9.1 [3] and perform as described above. The protein precipitates obtained at various saturation of ammonium sulfate are dissolved in small volume of appropriate buffer, and dialyzed against the same buffer. After overnight dialysis, dialysates are collected, and their enzyme activity and protein concentration are measured. The fractions containing the highest total activity are used for further purifications.
