Initial Purification and Assay of Activities in Cell-Free

5 INITIAL PURIFICATION AND ASSAY OF ACTIVITIES IN CELL-FREE LYSATES 

Many enzymes are soluble and will be recovered in the S-100 fraction. In addition, their activity can be measured rather conveniently by adding a sample of the S-100 fraction directly to a reaction mixture. Of course, since the S-100 fraction will contain an excessive amount of extraneous protein, it will be necessary to terminate the reaction and filter the sample prior to injecting it onto the HPLC for analysis. 

Note that while the S-100 fraction can be used in this form, it would be best to have it dialyzed to remove unwanted low molecular weight compounds before using it in an assay. In addition to dialysis, a simple salting out can be performed to remove some of the extraneous protein material. Ammonium sulfate is often added for this purpose to remove unwanted proteins or to precipitate the enzyme in question. Any ammonium sulfate should be removed before the sample is used in an assay, because the salt might affect activity. Again, dialysis can be used, or alternatively the sample can be passed through a gel filtration (G-25) column. 

Reversed-phase chromatography has also been used to separate proteins. However, the required use of alcoholic gradients or paired-ion reagents with the reversed-phase support should be avoided, to cancel the potential for inactivation of the enzymatic activities. 

HPLC is useful as an analytical tool in several applications in addition to its role in the purification of an activity. For example, HPLC can be useful in the establishment of gradients. Instruments have been manufactured for use with HPLC that can control the flow and mixing of solvents and thereby generate gradients with a variety of concentrations and shapes. In addition, since it is possible to carry out separations on the HPLC column in a comparatively short time, a number of these gradients can be applied to an analytical scale column, and the one best suited to the separation established fairly rapidly. Armed with this information, it is a relatively simple matter to carry over these gradient conditions to a non-HPLC ion-exchange column. 

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