Inhihition and Retardation of the Oxidation of lAA 



155 



Table 2. Retardation of lndole-3-acetic acid oxidation* by ribollavinphosphate. 

 The effect of enzyme, manganese, dichlorophenol, and indoIe-3-acetic acid concentra- 

 tions. 



* Standard DCP-horseradish peroxidase systems containing 0.6 iiM riboflavin- 

 phosphate. Components were varied individually. 



previously (Table 1) the extent to which systems were retarded by 

 hydroquinone and riboflavinphosphate was almost independent of 

 the nature of the cofactors. 



In order to elucidate the mechanism by which riboflavin or its 

 phosphate exerts a retarding effect on the system in darkness, kinetic 

 studies were undertaken to determine the effect of the concentration 

 of the individual components on the degree of retardation. The data 

 in Table 2 show the effects of varying the concentration of enzyme, 

 substrate, manganese, and dichlorophenol on the retardation brought 

 about by riboflavinphosphate at a concentration of 0.6 fM. Standard 

 DCP-horseradish peroxidase systems were employed. The data indi- 

 cate that under these experimental conditions an increased concen- 

 tration of any of the components was unable to overcome the re- 

 tardation by riboflavin. However, it has been shown previously (6) 

 that it would be more accurate to describe the optimum concen- 

 tration of the phenolic cofactor relative to the substrate concentra- 

 tion since in this delicately balanced system its activity is dependent 

 on the concentration ratio, e.g., lAA/resorcinol = 2.2, and lAA/ 

 DCP =z 4.4. In subsequent kinetic experiments it was shown that 

 when this ratio was maintained in the case of lAA/DCP and the 

 concentration of lAA increased as in the experiments in Table 2, 

 then the retardation by riboflavinphosphate could be partially over- 

 come. In these standard DCP systems with 6.6 /JVf of lAA the rate was 

 retarded 60 per cent by 0.5 jjlM of riboflavinphosphate. When the 

 concentration of lAA was increased to 13.2 /xM and 16.5 fxM and the 

 concentration ratio lAA/DCP maintained at 4.4, the retardation was 

 42 per cent and 24 per cent, respectively. 



