BACKGROUND: Peroxidase-catalyzed polymerization of phenols is accompanied by substantial enzyme precipitation with reaction products. The enzyme fate during the polymerization of monobromophenols by horseradish peroxidase (HRP) was studied. Enzyme fate was simultaneously monitored by protein, total nitrogen mass balance and gel electrophoresis (SDS-PAGE) analysis of both soluble and precipitate fractions. RESULTS: SDS-PAGE analysis revealed that molecular weight bands of protein in the precipitate shifted upwards toward higher molecular weights, compared with protein control. When co-polymerization was practiced higher HRP precipitation occurred compared with polymerization of a single substrate, regardless of substrate combination applied. Addition of polyethylene glycol (PEG) to the reaction mixture decreased the extent of HRP precipitation. At 2 mmol L-1 H2O2, corresponding to the stoichiometric equivalent concentration, 50% precipitation occurred after 1 h (similar to 70% after 24 h) compared with 97-98% (similar to 100% after 24 h) without PEG. Nevertheless, further increase of H2O2 increased HRP precipitation regardless of PEG (85% at 4 mmol L-1 and 95% at 5 mmol L-1). The lowest degree of enzyme inactivation was observed for metabromophenol, which displayed the lowest transformation yield, compared to the other congeners. CONCLUSIONS: Results from SDS-PAGE indicate that an interaction stronger than hydrophobic, resisting the denaturative conditions, may take place between HRP and the reaction products, suggesting the occurrence of a covalent link between them. Oxidation was enhanced by inclusion of PEG, which partially suppressed product-dependent inactivation. The extent of enzyme inactivation depends on the substrate used, while highest inactivation occurred when co-polymerization was practiced. (C) 2009 Society of Chemical Industry