New reagents for spectral titration of active site of chymotrypsin

The reaction of alpha-chymotrypsin (ChT) with 2-phenyl-(E)-[4-(E)-cinnamylidene]oxazolin-5-one (E-PCO) or 2-phenyl-(Z)-[4-(E)-cinnamylidene]oxazolin-5-one (Z-PCO) at pH 4.5 and 25 degrees C led to the quantitative accumulation of the corresponding acyl-ChT intermediate. Very large changes in molar extinction coefficient were observed in the visible region during the conversion of the oxazolinones to the acyl-ChT intermediates. This afforded a reliable method for the spectral titration of ChT in the visible region. Both E-PCO and Z-PCO were stable under these conditions and did not cause any complication in the titration of the active site.