The First Goal Selection of the Biological Starting Point

Since enzymes are associated with living systems, the development of a strategy for preparation of an activity begins with the selection of a specific biological starting point. This selection may be difficult, since one can start with an organism like an elephant, an organ such as a liver, biological fluids such as blood or saliva, cells that occur naturally such as bacteria or protozoa, or, finally, cultured cells. Preparing an activity from an elephant will present problems quite different from those experienced when preparing that same enzyme from bacteria.

To deal with the array of choices just outlined, samples have been subdivided into three groups. In group I (see Fig. 5.1) are samples that contain both a cellular and an extracellular compartment. The extracellular compartment can contain low molecular weight compounds such as the nutrients found in a fermentation broth, as well as macromolecular materials such as collagen or proteoglycans found in tissues. Also included in this extracellular compartment are fluids, such as tears, saliva, and urine.

Tissues (organs)

Body fluids

Blood (plasma) Urine Saliva Exudates

Disruption/disintegration ■

Blenders 1 Enzymes

Body fluids

Cellular and noncellular components

Bacteria Yeast

Mammalian cells

Cellular and noncellular components

Bacteria Yeast

Mammalian cells

Separations -

Separations -

{Centrifugation Affinity

Cells

Culture fluid fermentation broth plasma

Differentialseparations or lysis

Cell-free lysate

Centrifugation

Particulate

Soluble

Solubilization

Particulate

Soluble

Purification

Precipitation (salts) Ultrafiltration Chromatography Ion exchange Affinity Gel filtration Hydrophobic

Purified enzymes

Figure 5.1 Anatomy of a purification scheme: the starting and ending points for three types of purification. (/) The starting samples are composed of cells and exracellular materials, the goal of the purification is their separation. (//) The source of the enzyme is a heterogeneous population of cells, and the goal is to produce separate homogeneous populations (1-5). (Ill) The source of the enzyme is a cell, and the goal is to isolate a subcellular organelle, fraction, or purified protein.

While samples in group I contain cells, these usually are not of the same phenotype. For example, tissue samples such as skin can contain cells of several types. Therefore, multicellular samples in group I must contain, as part of their purification scheme, a step for the separation of the different cell types.

Samples in group II are composed of homogeneous populations of cells, and samples in group III are formed by the lysis of these cells and can be anything from organelles to soluble purified proteins. It is often possible to start a "purification" scheme with a sample from group III. In this case the strategy for the purification would include steps such as salt precipitation and chromatography.

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