Strategy for the Preparation of Enzymatic Activities from

Strategy for the Preparation of Enzymatic Activities from Tissues, Body Fluids, and Single Cells 

In developing a strategy for the preparation of an enzymatic activity, it is useful to consider two factors. The first is the choice or selection of the biological samples to be used as the starting point for the purification. These samples will clearly differ in terms of their complexity, and this complexity can be used to subdivide the samples into groups. 

There is a second consideration involved in the development of a purification strategy. Derived in part from the first, this consideration relates to the question To what extent should the activity be purified The traditional end point of any purification scheme would be a homogeneous protein, given the original demonstration that an enzymatic activity was associated with a single protein molecule. Thus, the question may appear to have only one answer. Several considerations can be used to justify this as the end point of the purification, the most important of which relates to the difficulties associated with the assay of the activity when the preparation is not homogeneous (e.g., when the enzyme remains in a preparation that contains many proteins and many enzymes). 

The development of the HPLC method to assay enzyme activities has made it considerably easier to assay a single activity in the presence of others. Thus, attempts to obtain a pure protein during the purification procedure may not be necessary. Since the advent of HPLC to assay enzyme activities, it is possible to stop the purification at a much earlier stage and still assay for a single enzymatic activity. In fact, for some studies, it is even advantageous to assay the activity of interest in the presence of other activities. 

Finally, this chapter discusses the use of HPLC itself as an aid in the purification of an enzyme activity. Applications are not restricted to the final stages of a purification. Its use of small sample volumes, its sensitivity, and its speed of separation make HPLC an ideal analytical tool to monitor the efficiency of other steps and procedures that are used during a purification. 

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