The thermal stability of phthalic anhydride-modified horseradish peroxidase (HRP) in organic solvents was investigated. The modification increased the tolerance of some organic solvents. In order to investigate the molecular mechanism of the stabilization, the changes between native and modified enzyme were also studied using kinetics and spectroscopic methodology. The modified HRP showed greater affinity and catalytic efficiency in some organic solvents for different substrates than native HRP. The substrate affinity and the catalytic efficiency of native and modified HRP increased with the increases of electron-donating efficiency of substituents at 4-position. The improvements of catalytic properties are related to the changes of the conformation of HRP. The modification changed the environment of both heme and tryptophan, increased alpha-helix content of HRP and decreased the tertiary structure around the aromatic acid residues in HRP. (C) 2004 Elsevier Inc. All rights reserved.