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Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65°C and half-lives at 65°C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60°C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 233–240, 2003.

The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 Å of the EGNHS crosslinker. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 277–284, 2001.

The phenol precipitation and dye bleaching capabilities of phthalic anhydride–modified horseradish peroxidase C (PA–HRP) were compared with those of native HRP C and ethylene glycol- bis-(succinic acid N-hydroxysuccinimide ester)–modified HRP (EG–HRP) reported previously. Removal efficiency (percentage of phenol removed from solution under experimental conditions) was determined for native HRP and both modified forms. Removal efficiencies at 37 °C were very similar, with >95% removal in each case. Removal efficiencies were less at 70 °C overall (range 25–45%) but PA–and EG–HRP removed up to 50% more phenol than native HRP. The three HRP forms showed similar dye bleaching performance at 37 °C in the presence of H2O2 and accelerators (up to 86% colour removal). PA–and EG–HRP showed slightly greater bleaching abilities at 65 °C than native HRP for some of the dye/accelerator combinations tested. Modified HRPs performed better in 40% (v/v) mixtures of dioxane or dimethylformamide. © 2000 Society of Chemical Industry