Most of the enzyme properties are clearly and reliably revealed only with purified enzymes. Prerequisites for the isolation of a pure enzyme are selected protein chemical separation methods carried out at 0-4 degrees C since enzymes are often not stable at higher temperatures.
Tissue Disintegration and Extraction
Disintegration and homogenization of biological tissue requires special precautions: procedures should be designed to rupture the majority of the cells in order to release their contents so that they become accessible for extraction. The tissue is usually homogenized in the presence of an extraction buffer which often contains an ingredient to protect the enzymes from oxidation and traces of heavy metal ions. Particular difficulty is encountered during the isolation of enzymes which are bound tenaciously to membranes which are not readily solubilized. Extraction in the presence of tensides may help to isolate such enzymes. As a rule, large amounts of tissue have to be homogenized because the enzyme content in proportion to the total protein isolated is low and is usually further diminished by the additional purification of the crude enzyme isolate.
Removal of protein impurities, usually by a stepwise process, is essentially the main approach in enzyme purification. Often, as a first step, fractional precipitation, e.g. by ammonium sulfate saturation, is used or the extracted proteins are fractionated by molecular weight e.g. column gel chromatography. The fractions containing the desired enzyme activity are collected and, for example, are purified further by ion-exchange chromatography. Other supplemental options are also available, such as various forms of preparative electrophoresis, e.g. disc gel electrophoresis or isoelectric focusing.
The purification procedure can be substansially, shortened by using affinity column chromatography. In this case, the column is packed with a stationary phase to which is attached the substrate or a specific inhibitor of the enzyme. The enzyme is then selectively and reversibly bound and,thus, in contrast to the other inert proteins, its elution is delayed.
Control of Purity
Previously, the complete removal of protein impurities was confirmed by crystallization of the enzyme. This "proof" of purity can be circumstantial and is open to criticism. Today, electrophoretic methods of high separation efficiency are primarily used.
The behavior of the enzyme during chromatographic separation is an additional proof of purity. A purified enzyme is characterized by a symmetrical elution peak in which the positions of the protein absorbance and enzyme activity coincide and the specific activity (expressed as units per amount of protein) remains unchanged during repeated elutions.
During a purification procedure, the enzyme activities are recorded. Data show the extent of purification achieved after each separation step and show the enzyme yield. Such a compilation of data readily reveals the undesired separation step associated with loss of activity and suggests modifications or adoption of other steps.