Effects of phthalic anhydride modification on horseradish peroxidase stability and activity.

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 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees 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.