Comparative kinetic analysis of cholinesterase methods in rat and human erythrocytes and plasma

A kinetic analysis of the substitution of 6,6'-dithiodinicotinic acid (DTNA) for 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) for the determination of rat and human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BuChE; EC 3.1.1.8) is presented. Increasing concentrations of DTNB, but not DTNA, significantly increased Km for the substrate acetylthiocholine but had little or no effect on Vmax for rat or human AChE. The coupling agent DTNA was more efficient than DTNB, as demonstrated by the higher Vmax/Km ratio for the former. DTNB, more so than DTNA, caused linear mixed-type inhibition of rat AChE. Poor precision was observed for the DTNB versus DTNA method. Reagent blanks were a significant component of rat, but not human, AChE activity. The use of DTNA in place of DTNB is recommended for quantitative mechanistic investigations of cholinesterases. The most practical aspect of the DTNA method is that it can be adapted to automated instruments which can monitor the change in absorbance at 340 nm, away from the hemoglobin peak.