202 P. M. Ray 



is maintained until exhaustion of substrate, or occurrence of secondary 

 complications such as enzyme inactivation, cause it to fall. These 

 phases are also illustrated in Figure 1 (solid line). The steady rate is, 

 however, considerably lower than can be obtained by adding HoOo 

 to the medium. The rate increases, with increasing concentrations of 

 added HoOo, up to a maximum which we call the HoO-t saturation 

 rate, and which we presume is due to saturation of the peroxidase 

 with HoOo. This occurs at about 2 X lO^^M HoOo with the Omphalia 

 enzyme, and is illustrated by the first part of the broken curve in 

 Figure 1. It is apparent that in the absence of added H^Oo, the 

 enzyme does not become saturated even when the rate has become 

 steady, after induction. A further fact of significance is that when sat- 

 urating amounts of H^Oo are added, it can be demonstrated that 

 HoOo disappears gradually from the system, since after a time the rate 

 falls to the same value as would be reached, after induction, if H2O2 

 had not been added, and at this point the HoOo saturation rate can 

 be restored by adding more HoOo. These effects are shown in the 

 broken curve of Figure 1. They indicate that processes are also oc- 

 curring which lead to loss of oxidation intermediates from the sys- 

 tem, processes which can be called termination reactions by analogy 

 with organic auto-oxidation processes. A probable explanation of 

 the steady rate of oxidation which is reached after induction is that 

 it represents a steady state in which the rates of formation (initia- 

 tion) and disappearance (termination) of oxidation intermediates 

 have become equal — due, for example, to termination increasing 

 more rapidly than initiation as the reaction rate rises (termination 

 is evidently more rapid than initiation at the high rate attained by 

 saturation with HoOo). Examples of possible types of termination re- 

 actions are (a) a catalase-like reaction involving removal of HoOo, 

 and (b) a reaction between free-radical intermediates such as P- -|- 

 POo- -^ POoP. 



It will be evident that the lAA oxidation rate observed at steady 

 state will be influenced not only by effects on enzyme activity and 

 the main catalytic cycle, but also by any factors which influence the 

 initiation or termination processes, and thereby the steady state con- 

 centrations of intermediates. There is thus considerable danger in 

 interpreting rate effects of experimental treatments as if the ob- 

 served effects related only to enzyme activity or to the cyclic lAA 

 oxidation mechanism. To proceed further with interpretation of rate 

 effects on lAA oxidation it will be essential to develop a kinetic 

 analysis of the proposed mechanism which includes assumptions about 

 the nature of initiation and termination reactions. 



In our experience, the rates of lAA oxidation attainable by sat- 



