Poly(ethylene glycol) 4000-alpha -chymotrypsin (with a molar ratio of the polymer/enzyme of 8:1) complex prepared from borate buffer solutions had ca. 6000- and 26-fold higher activity than native alpha -chymotrypsin and a complex prepared using a conventional phosphate buffer, respectively, even at lower concentration (0.0474 mol g(-1) enzyme) than that of inorganic salts which were required for the enzyme activation. These results suggested that relatively hydrophobic buffers are desirable for preparing modified enzymes that are catalytically active in organic media and contributed to develop more effective methodology for the optimal design of enzyme preparations for nonaqueous enzymology.