[3] Active enzyme centrifugation

This chapter describes active enzyme centrifugation, which is based on the principle that an enzymatic reaction occurring in the cell can be followed by some physical method during centrifugation. In many cases, enzymatic reactions may be monitored spectrophotometrically, which is convenient as well as highly accurate. This technique can be used in the centrifuge by utilizing a photoelectric scanning system. The method is not necessarily limited to reactions in which optical density changes occur in either substrates or products during catalysis. Enzymatic reactions may also be monitored if the enzymatic reaction can be coupled to a secondary reaction, which involves a change in optical density. As only the displacement of the active form of the enzyme is observed with this method, any inactive forms of the enzyme, which may be present in the preparation, are not observed. For this reason, it is not always necessary to use highly purified enzymes to obtain meaningful results. In fact, successful experiments may be done with relatively crude dialyzed tissue extracts. Active enzyme centrifugation may, thus, be applied to many enzyme systems.