Relating to Enzyme Fractionation

Perhaps the most important consequence of the preceding discussion is that enzymes do not come down within fixed limits of salt concentration characteristic of each enzyme, but that the limits vary with the concentration of the enzyme. Fixed characteristic limits can be obtained, however, if the initial solution is always diluted to the same enzyme activity. Unless this is done, the precipitation limits of the enzyme will vary, not only with the stage of purification, but also with the activity of the starting material. Thus in following a published method for purification of an enzyme from yeast, say, one may find that the limits must be modified because the sample of yeast available may have a different content of the enzyme. 

We have stressed the importance of pH and temperature as variables that have a great effect on the positions and separation of the precipitation peaks. They must therefore be carefully controlled. Furthermore, by varying them in accordance with the principles stated above, it should be possible to increase greatly the versatility and effectiveness of the fractionation. 

Temperature is not always easy to control. It is fairly common practice 

Variation of temperature introduces a difficulty in the use of percentage saturation as a measure of salt concentration, since the solubility of the salt will vary with temperature. Some data for ammonium sulfate are given by Taylor (1953). It would be preferable always to state the actual concentration of salt, for example as molarity. 

A survey of the literature of enzyme purification suggests that there has been uncertainty as to the most satisfactory salt concentration increment to use at each step. The theory that we have discussed provides some guidance on this point. It would appear that a span of from 5 to 10 in the percentage saturation with ammonium sulfate, if properly chosen, should provide the best compromise between good purification and good yield. It also appears that repetition of salt fractionation under the same conditions is unlikely to be very useful, but that repetition under different conditions may contribute a considerable further purification. 

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