The Second Goal Determining the Extent of the Purification or End Point

One of the most important events in the history of biology was the demonstration of enzymatic activity. Next in importance came the isolation and purification of the activity and the demonstration that catalysis is associated with protein molecules. Thus, advances in enzymology have been intertwined with advances in protein chemistry.

Enzyme isolation and purification as practiced today has progressed from the days when the tools available were few and limited to procedures such as "salting out" with ammonium sulfate, filtration, and centrifugation at rather unimpressive g forces. The procedures of the past involved a significant amount of art, as well as science, with the artistry demonstrated by the last step, which in most schemes usually involved crystallization of the enzyme. To watch a pioneer like Moses Kunitz coax enzyme crystals from an alcohol solution was inspirational. Unfortunately, the technology of videotaping was not around in those days to preserve such events.

Today, the enzymologist has a battery of techniques, including improvements in centrifugation and column chromatography, and enzymes can be purified to homogeneity faster than ever before. But should a pure enzyme be your goal?

Imagine looking under the hood of an automobile and trying to find, somewhere in the engine, a screw 0.016 in. long with a 3/32 in. thread and a round head. One approach to finding such a screw, a minute part of the entire assembly, would be to take the engine apart as quickly as possible to obtain all the screws. These could then be spread out on a table top, and by careful inspection the screw of interest could be located.

Similarly, with an enzyme activity, if your goal is to isolate a special enzyme protein to characterize its size, shape, and amino acid composition, of course it would be wise to disrupt the starting material (organ, cell, or organelle) as quickly and completely as possible to obtain all the proteins, and then spread them out or fractionate them to locate the one of interest.

Returning again to the car engine, suppose we know the screw is present, and now we wish to find its location in the engine. The strategy must be different. For example, it would be wiser to remove each part of the engine carefully and disassemble them separately. In this way, when the screw is found, its location in the engine can be established.

When the question of the localization of an enzyme within the cell had to be answered, purification schemes were developed to take cells apart in a stepwise fashion. For this task a variety of tools were used, including rather commonplace scissors as well as sophisticated centrifugation and chromatographic techniques. And it was only after careful dissection that an understanding emerged of where the enzymes were located.

But how do enzymes function at their respective sites? If the question were asked about the automotive engine screw, one answer might be found by examining that component of the engine to which the screw belonged. For example, if this screw was a part of the carburetor, it might function in regulating the intake of fuel or air; but it might operate in several other ways, as well. In fact, it might not be possible to deduce its function merely by inspection, and to really find out how the screw worked, the intact carburetor would have to be returned to the engine and the engine started.

The same is true for an enzyme. Studies on what might be called its "interen-zymatic" function are best carried out while the enzyme is still a part of the organelle or complex to which it belongs. To date such studies have been difficult to perform because of problems in monitoring in a single assay the variety of enzymatic activities that can occur in such complexes. The introduction of HPLC to assay enzymatic activity allows us to consider trying such interenzymatic functional studies, because with this method several activities can be measured simultaneously. Thus, one of the consequences of this advancement in methodology is that we can measure the activity of an enzyme while it remains in a complex and, through such studies, deduce its function within a multienzyme complex.

This advancement in methodology also can affect the strategy employed for the purification of an enzyme, since the goal need not be to isolate the enzyme protein from all other enzyme proteins. Now the goal might be better stated: Remove from the enzyme preparation only those components or structures that are not necessary for function.

The scheme in Figure 5.1 can be used to illustrate this point. While this scheme shows three separate starting points, it also shows potential end points. For example, if the sample is multicellular, the purification could be ended after the removal of the extracellular matrix or the medium or broth used to culture cells. Enzyme activities can be measured directly in the multicellular structures or in the extracellular compartment.

Alternatively, the purification can be continued, the cells lysed, and activities assayed after lysis. It is from lysates so produced that organelles such as nuclei and mitochondria are obtained. The disruption of organelles in turn produces soluble enzymes. Ultimately, the end point is determined by the individual study. If questions related to molecular weight, amino acid composition, or catalytic mechanism are to be answered, a pure protein will be required. In contrast, if questions relating one activity to another are of interest, the purification should be ended at the lysate or organelle level.

The sections that follow focus on samples from each of the three groups shown in Figure 5.1.1 leave the reader to answer the question: Where do I stop?

5.2 PREPARATION AND ASSAY OF ENZYMATIC ACTIVITIES IN SAMPLES OF TISSUES, ORGANS, AND BIOLOGICAL FLUIDS

5.2.1 Separation of Cellular from Extracellular Compartments

5.2.1.1 Samples Obtained Directly from an Organism Tissues, such as connective tissue, and organs, such as skin or liver, can be thought of as being composed of at least two compartments: the cellular compartment and the extracellular compartment. Since enzymes can be localized in either compartment, one of the first problems is to separate the two compartments.

Techniques should be used that will not damage the cells, since any damage is liable to cause leakage of the contents of the cellular compartment into the extracellular compartment. With tissues or organs, where the noncellular compartment is often a stable fibrillar matrix, a two-step procedure such as that shown schematically in Figure 5.2 is helpful in separating the compartments. Often the matrix is disrupted by cutting or dicing with scissors, shearing

Figure 5.2 Methods used in the disruption of tissues or organs, such as liver and skin. The initial step is usually some physical technique; dicing with scissors is illustrated. The fragments are then treated with an enzyme such as trypsin or collagenase to disrupt the fragments further to obtain single cells.

Figure 5.2 Methods used in the disruption of tissues or organs, such as liver and skin. The initial step is usually some physical technique; dicing with scissors is illustrated. The fragments are then treated with an enzyme such as trypsin or collagenase to disrupt the fragments further to obtain single cells.

SINGLE CELLS

SINGLE CELLS

in a blender, or grinding. With such physical techniques, however, some cellular damage is unavoidable.

The disruption of the matrix can be continued by treating the fragments with purified enzymes, which are often commercially available. These materials are ideal, since they can be chosen for their specificity and also chosen with the composition of the matrix in mind. In samples derived from mammalian tissue, the matrix usually contains collagen, and the enzyme collagenase can be used. Trypsin and other proteolytic activities have also been used with great success. As illustrated in Figure 5.2, the end result of this two-step procedure should be a solution containing intact cells, extracellular fluids, and extracellular components, including some insoluble fragments, some soluble components, and, of course, any enzymes added as reagents.

5.2.1.2 Samples Obtained from Tissue or Organ Culture Animal tissues and organs can also be grown using a primary culture system by placing the sample on a support, such as an agar surface, a filter, or even a wire mesh, which can be positioned with the sample bathed in a solution of growth medium. A typical arrangement of the latter (Fig. 5.3), consists of a culture

End View

End View

Medium Wire Mesh Raft

Tissue ,

Medium Wire Mesh Raft

Tissue ,

Top View

Figure 5.3 Diagram of the apparatus used for the culture of a tissue fragment or an organ. The dish contains a central well (diagonal lines), over which is placed a wire mesh raft to support the tissue. The well is filled with sufficient culture medium to make contact with the tissue. The dish is covered (not shown) and incubated under conditions used to maintain viability of the explant. (From Quintner et al., 1982.)

dish containing a centrally placed well filled with culture fluid. Suspended over the well is a wire screen, which acts as a support for the tissue. The well is filled with culture medium, the tissue is placed on the support, and the dish is covered and incubated.

Samples obtained from such a culture system should be processed by the two-step procedure described above to obtain the individual cells, free from the extracellular compartment. However, note that with cultured samples there is an additional extracellular compartment, which is the culture fluid used to support the growth of the sample. It is not at all uncommon to find enzymatic activities in this fluid. Some of these are normal constituents of the culture medium, while some are a consequence of growth of the sample.

5.2.1.3 Samples Obtained from Biological Fluids As illustrated in Figure 5.1, biological fluids, which also have two compartments, are placed in group I. Such fluids include blood (often classified as a tissue), urine, semen, tears, and saliva. Many of these fluids contain cells as a normal component, while in others the cells represent a contamination. Fluids like saliva that contact the "outside" and, when collected, often contain microbes, are examples of contaminated materials. Sometimes such microbes are the result of the collection procedure, and their numbers can often be controlled by careful technique. At other times, as is the case with urine, their presence can indicate an underlying disease process. In any case, the study of enzymes from such fluids again requires the separation of the two compartments.

Biological fluids, however, do not contain a fibrillar matrix material, and their separation does not require the two-step procedure described above. Often, centrifugation at a slow speed, such as 5000g for 10 minutes as illustrated in Figure 5.4, will suffice. The pellet produced during this centrifugation should contain most, if not all, of the cellular elements, including any microbes. It is advisable and informative to recover these pellets. Both a sample of the pellet and a sample of the supernatant solution should be examined microscopically for their cellular content. The supernatant solution produced by the centrifugation can be assayed directly for enzymatic activities. However, if excess protein

gpv:;v

P A

n

Lc.

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CENTRIFUGATION 5000xq lOmin

— Supernatant Pellet

CELLS

CENTRIFUGATION 5000xq lOmin

Figure 5.4 Harvesting cells from a culture medium. A sample of the culture medium is transferred to a centrifuge tube and subjected to centrifugation at rather low g forces such as 5000g for a relatively short time, such as 10 minutes. The cells contained in the pellet can be recovered after the supernatant solution has been decanted.

is present in the sample, it will have to be removed from the enzymatic assay samples before HPLC analysis.

5.2.1.4 Samples Obtained from Cell Cultures Cells of many types are now grown in liquid culture. These include not only mammalian cells but fungi, protozoa, and bacteria. In Figure 5.1 these are placed together under the group I heading because in each case the noncellular elements in the fluid or fermenatation broth should be separated from the cells before analysis. A low speed centrifugation as shown in Figure 5.4 should suffice. The supernatant fraction is collected and assayed for the activity of interest, and the cells set aside for assay or lysis.

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