| Abstract: | The inhibition of horseradish peroxidase (HRP)-cata-lyzed oxidation of indole-3-acetic acid (IAA) by a phenol, caffeic acid (CA), was studied using both a kinetic approach and computer simulation. The presence of CA resulted in a lag period in IAA oxidation. The lag period increased slowly with increasing [CA] until a critical concentration, [CA]cr, was reached, then it increased much faster when [CA] was greater than [CA]cr. The [CA]cr was proportional to [IAA] and did not depend upon [HRP]. Caffeic acid was oxidized by compound I and compound II of HRP with bimolecular rate constants (6.8 ± 107 and 2.1 ± 107M-1s?l), which were much higher than the corresponding rate constants for IAA oxidation (2.3 ± 103 and 2.0 ± 102M?1s?1). Our experimental data show that CA inhibits IAA oxidation because it is able to compete effectively as a peroxidase substrate. A model based on a detailed mechanism of IAA oxidation was investigated using computer simulation. A rate constant driving nonenzymatic hydroperoxide formation in IAA solution was determined, 3.0 × 10?-7 s?1. The model quantitatively describes the experimental results of this work and also qualitatively explains data published earlier. The critical inhibitor concentration is approximately equal to twice the concentration of hydroperoxide in IAA solution at the time of inhibitor addition. Therefore hydroperoxide Concentration can be calculated from the determination of critical inhibitor concentration. |
