Preparation of Enzymeswith Biological Activity from Physiological Samples

The determination of an absolute enzyme concentration in a physiological sample is principally straightforward; the main problem is the need for a puriRed sample to use as a standard. Enzymes are proteins found in nature in complex mixtures, usually in cells which perhaps contain several hundreds of different enzymes. In order to understand and interpret enzyme data from complex biological systems in, for instance, a subcellular organelle (such as a mitochondrion), a cell or whole organism, we must try to understand its properties in as simple a system as possible. From studies of an isolated enzyme we can learn about its speciRcity for certain substrates, the kinetic parameters for the reaction and the possible means of regulation. All these parameters are useful for understanding the role of the enzyme in more complex systems. The ready availability of isolated enzymes has been of considerable value in a number of medical and industrial applications. To study a given enzyme properly in physiological samples it must be puriRed. Maintenance of biological activity is the goal throughout the whole puriRcation scheme. Extracellular enzymes usually withstand the variety of stresses they are exposed to during the puriRcation. In contrast, when released from their natural protective environment, intracellular enzymes are very sensitive to various steps in the puriRcation scheme. Integral membrane enzymes are especially vulnerable during solubilization. SpeciRc examples, together with trends in enzyme determination in physiological samples, will be discussed in this article.