BRITTON CHANCE 



PHYSICAL METHOD 



Under classical conditions for catalase assay (33) the value 

 of ki declines rapidly above 0.02 M peroxide concentrations, but 

 activity measured in a minute or less gives constant values of 

 Ajj up to 0.05 M peroxide (1). In studies expressly for the pur- 

 pose of investigation of catalase activity at high peroxide con- 

 centrations, George has measured the over-all reaction with 

 rapid manometric methods (35), but an examination of George's 

 data indicates that the values of k^ under these conditions were 

 about one-fifth of the correct value (48) . Thus the manometric 

 data are not suitable for evaluating the constancy of k^ and are, 

 in fact, of questionable value in relation to the reactions of intact 

 catalase. 



We have therefore devised a "quenching" type of rapid- 

 flow apparatus especially designed for studies of catalase at high 

 hydrogen peroxide concentrations (19). With this apparatus 

 catalase is rapidly mixed with \-5 M peroxide, and the reaction 

 is stopped by a second rapid mixing with sulfuric acid a very 

 short time later in order that inactivation of the enzyme be 

 minimized. Figure 1 shows the important features of this 

 apparatus. The plungers are manually driven at a constant 

 rate, and the time required to discharge a given volume is 

 measured with a stop watch. The buffered catalase solution 

 (/-^10~^ M) is rapidly mixed with the strong peroxide solution, 

 the reaction mixture flows down the observation tube a distance 

 of 2 cm, in a time of 0.1 to 0.4 sec, and then it is mixed with 

 about 3 N sulfuric acid to stop the reaction. The remaining 

 peroxide is titrated with permanganate. 



THE LIFETIME OF INTERMEDIATES 

 IN THE CATALASE REACTION 



Dr. Y. Ogura of this laboratory has completed a series of 

 catalase studies with this apparatus (55). As Figure 2 shows, 

 the rate of the decomposition of peroxide by catalase reaches a 

 plateau of 4.6 M/sec. above 5 M peroxide. This corresponds to 



312 



