An integration strategy to measure enzyme activities for detecting irreversible inhibitors with dimethoate on butyrylcholinesterase as a model

Abstract

An integration strategy was investigated to measure initial rates of horse butyrylcholinesterase (BChE) at 50.0 µmol L⁻¹ butyrylthiocholine (BTCh) for detecting irreversible inhibitors as pollutants in environment and foods with dimethoate as a model. In this integration strategy: (a) if BTCh consumption within 5.0 min was >60%, BChE initial rates were derived from maximal reaction rates, estimated by an improved integrated method, according to Michaelies-Menten kinetics at 47.0 µmol L⁻¹ BTCh and Michaelis-Menten constant at 94.0 µmol L⁻¹; (b) or else initial rates were determined by the classical initial rate method. Thus, the differences in BChE initial rates without and after dimethoate treatment indexed final dimethoate contents in reaction mixtures to treat BChE. Results supported that this integration strategy determined BChE activities with a linear range about two magnitudes and an upper limit about twice that by the classical initial rate method alone at 2.0 mmol L⁻¹ BTCh. The coefficient of variation with this integration strategy was below 5%. The difference in BChE initial rates before and after dimethoate treatment was proportional to final dimethoate contents in reaction mixtures. By enzymatic analyses, the molar contents of dimethoate extracted from polluted cabbages were consistent with the summed molar contents of dimethoate and dimethoxon by gas-chromatography. Therefore, this integration strategy was effective to detect irreversible inhibitors as pollutants in environment and foods.

Keywords:

• integration strategy
• butyrylcholinesterase
• enzymatic analysis
• irreversible inhibitors
• dimethoate

List of abbreviations:

• BChE: butyrylcholinesterase
• BTCh: butyrylthiocholine
• CV: coefficient of variation
• GC: gas-chromatography
• K _m: Michaelis-Menten constant
• V _i: initial rate
• V _m: maximal reaction rate

Acknowledgement

This work was supported by the National Natural Science Foundation of China (Nos. 30200266, 30472139, 30672009). This integration strategy was under evaluation for invention patent in China (Application No: 200710093081.4).