Instrumental Improvements for Rapid, Detailed Kinetic Studies of Oxygenase Activity

The characteristics of an electronic differentiator that facilitates continuous monitoring of changes in the oxygen concentration measured by a polarographic oxygen electrode are described. In addition, some properties of the electrode membranes that increase the sensitivity of the oxygen-sensing electrode are also described. These modifications greatly improve the accuracy and sensitivity of the biological oxygen monitor for use in kinetic studies of complex enzyme systems such as soybean lipoxygenase or prostaglandin cyclo-oxygenase. The electronic differentiator also may be used in conjunction with other rate-monitoring instruments such as spectrophotometers and pH meters for accurate quantita-tion of rapidly changing rates. Since the measurement of oxygen ten-sions in blood and tissues using a polaro-graphic method was first described by Clark (1), numerous modifications to the oxygen-monitoring electrode and various applica-tion of this technique have been reported (e.g., Refs. 2-8). One very useful experi-mental application of the oxygen electrode has been to the measurement of 0, con-sumption or production during enzyme-catalyzed reactions; changes in rates and the total extent of reactions can be determined from continuous monitoring of the electrode signal. For such applications the oxygen electrode is sensitive in a broad range of solutions where photometric procedures for oxygen determination may be complex or inadequate. As an improvement on our approach to studies of the kinetics and mechanisms of fatty acid oxygenation by prostaglandin cyclooxygenase (9- 12) and soybean lipoxy-genase (13,14), we have modified a com