Synergistic enhancement of the horseradish peroxidase-catalyzed oxidation of luminol by 4-substituted phenylboronic acids

Combinations of 4-substituted phenylboronic acids [phenyl, iodo, bromo, and trans-4-(3-propenoic acid) substituents] have been discovered to have synergistic effects in the horseradish peroxidase (HRP) catalyzed chemiluminescent oxidation of luminol. Three types of effect have been observed: 1. synergistic reduction in the background light emission of a luminol-peroxide assay reagent to a value lower than the background obtained with either enhancer individually; 2. increase in signal to background ratio (S B ) in the presence of HRP to a value higher than the S B obtained with either enhancer individually (synergy) or to a value higher than the combined S B obtained with each enhancer (synergistic enhancement); and 3. for some combinations of enhancers, an increase in signal in the presence of HRP to a value higher than the signal obtained with either enhancer individually (synergy), or to a value higher than the combined signal obtained with each enhancer (synergistic enhancement). The magnitude of the effect was moderate but the synergistic decreases in background and increases in signal produced increases in S B up to four-fold. Examples of synergistic pairs of enhancers included 4-biphenyl and 4-bromophenylboronic acid; 4-biphenyl and 4-iodophenylboronic acid; and trans-4-(3-propenoic acid) and 4-iodophenylboronic acid. Generally, synergy was obtained at several concentrations of all of the combinations of enhancers tested, and at different time points in the reaction due to the different light emission kinetics of the enhanced reactions. The mechanism of this synergistic effect has not been elucidated but may involve the enhancers acting at different points in the complex chemiluminescent peroxidase catalyzed oxidation reaction.