In living organisms, enzymes are components of mixtures that contain numerous organic and inorganic substances. These substances may interact with the enzymes, coenzymes, substances, or products of the enzymatic reaction. Study of the properties of an enzyme requires that it be purified. At present, several hundred enzymes are available in purified form. It should be remembered that the characteristics of purified enzymes may not be the same as those they possess in their natural environment. Such variable factors as the stabilizing effect of mitochondria, diffusion rates, and inhibitory feedback mechanisms may alter the behavior of enzymes in vivo. Competition of other substances (e. g., nonspecific proteins) for substrates and inhibitors may also change enzyme activity in vivo. Nevertheless, the investigation of enzyme activity in vivo and the possibility of increasing or inhibiting this activity for experimental or therapeutic purposes is greatly facilitated by studies in vitro of the properties of purified enzymes.Investigation of the properties of purified enzymes requires the availability of: (a) a test system; (b) extraction methods; and (c) fractionation methods. These concepts are considered in detail in various texts (15,22,49).
Test SystemsThe first requirement for the purification of an enzyme is the development of a quantitative test of its activity. The progress of purification is followed by the appropriate test until maximum activity per weight has been reached. It is recommended by the International Commission on Enzymes that enzyme assays should, whenever possible, be based on the measurement of the initial rates of activity; in this way, complications due to reversibility of reactions or 21
