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.
In the first group are samples that are rather complex not only because they consist of many different cell types but also because they have an extracellular compartment. Such samples include organs, tissues, biological fluids, and microbial cells, together with any other unicellular organisms grown in a culture medium or fermentation broth. For these samples, the initial step is the separation of the cellular from the noncellular compartment. Next, the different cell types within the cellular compartment must be separated, and this set of homogeneous populations of each type of cell becomes the starting point for samples in the second group. With such cells, activities at the cell surface can be directly assayed or the cells can be lysed, providing accessibility to the activities in intracellular organelles and on cytoplasmic fragments.
The samples in the third group are the subcellular fragments liberated by lysis. These include organelles such as mitochondria, as well as those operationally defined as a "membrane fraction" or a fraction containing "soluble components." The initial steps involving samples in this group include the separation of organelles from each other, the separation of insoluble from soluble fractions, and the solubilization of membrane samples or the fractionation and separation of one molecular species from another. Since the strategy developed for purification depends on the choice of starting material, this chapter outlines some of the problems associated with obtaining activities from samples in each group. In addition, we offer solutions to these problems.
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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