Oxazolones as substrates in active site titrations of hydrolases

Active site titrations provide essential information for studies of enzymes, e.g., functional purity and half-site reactivity. The use of a hitherto overlooked class of hydrolase substrates, the oxazolones, for an easy synthesis of reagents possessing desirable properties, e.g., specificity, solubility, and optical parameters, is described. The operational normality may be determined in aqueous media or organic solvents, e.g., dimethyl sulfoxide, which stabilize the enzymes for extended periods at room temperature. Absorbance or fluorescence measurements may be used for detection. The lower limit permitting analyses in this series was found to be 60 pmol of enzyme.