The alpha-chymotrypsin-catalyzed hydrolysis of the p-nitrophenyl acetate in the solvent mixtures containing from 1.6 up to 10%(v/v)acetonitrile in the presence of aqueous Tris buffer at pH 8.0 was investigated at 298 K by use of an isoperibolic batch calorimeter. A special experimental arrangement of the reaction components for the investigation of the hydrolytically instable substrate was used. Furthermore, the release of p-nitrophenol was recorded with an UV-vis-spectrophotometer under comparable conditions. The calorimetric curves consist of two parts. The first part is strongly rising and finished by a break point in the DeltaT (time) curve. This first step is dominated by the enzyme-catalyzed reaction. After the break point a slow non-enzymatic process determines the course of the calorimetric curve. The molar enthalpy changes of overall reaction (ester hydrolysis and buffer protonation) of -100 +/- 8, -106 +/- 5 and -102 +/- 5 kJ/mol were evaluated by a combination of the results from the spectrophotometric and calorimetric data for 1.6, 4.0 and 10.0% acetonitrile mixtures, respectively. The obtained results indicate that the enzyme-catalyzed hydrolysis is suitable for quantitative determination of the hydrophobic ester p-nitrophenyl acetate in water-acetonitrile mixtures using calorimetric detection. (C) 2004 Elsevier B.V. All rights reserved.