THE EFFECTS OF MEDIA PROPERTIES ON THE HORSERADISH PEROXIDASE-CATALYZED FLUOROGENIC REACTION

The effects of media properties including buffers, acidity, solvents a nd surfactant on horseradish peroxidase-catalyzed fluorogenic reaction were investigated. The results showed that the so-called non-fluoresc ent hydrogen donors were in fact fluorescent. There existed an acid-ba se equilibrium in the fluorescent dimer product. For p-hydroxyphenylpr opionic acid, a pK(a) value of 8.0 for the product was obtained from i ts titration curve. The product fluorescence increased with higher pH, however, a longer time was needed to reach the reaction equilibrium d ue to the pH mismatch problem. Cationic micelles cetyltrimethylammoniu m bromide and cetyltrimethylammonium chloride could reduce the pH mism atch and offered a way to further increase the determination sensitivi ty. Finally, a micelle-enhanced flow-injection analysis of horseradish peroxidase is suggested.