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) catalyze d chemiluminescent oxidation of luminol. Three types of effect have be en observed: 1. synergistic reduction in the background light emission of a luminol-peroxide assay reagent to a value lower than the backgro und obtained with either enhancer individually; 2. increase in signal to background ratio (S/B) in the presence of HRP to a value higher tha n the SIE obtained with either enhancer individually (synergy) or to a value higher than the combined S/B obtained with each enhancer (syner gistic enhancement); and 3. for some combinations of enhancers, an inc rease in signal in the presence of HRP to a value higher than the sign al obtained with either enhancer individually (synergy), or to a value higher than the combined signal obtained with each enhancer (synergis tic enhancement). The magnitude of the effect was moderate but the syn ergistic decreases in background and increases in signal produced incr eases in S/B up to four-fold. Examples of synergistic pairs of enhance rs included 4-biphenyl and 4-bromophenylboronic acid; 4-biphenyl and 4 -iodophenylboronic acid; and trans-4-(3-propenoic acid) and 4-iodophen ylboronic acid. Generally, synergy was obtained at several concentrati ons of all of the combinations of enhancers tested, and at different t ime points in the reaction due to the different light emission kinetic s of the enhanced reactions. The mechanism of this synergistic effect has not been elucidated but may involve the enhancers acting at differ ent points in the complex chemiluminescent peroxidase catalyzed oxidat ion reaction. (C) 1997 Elsevier Science B.V.