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 determina tion 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 increa sed K-m for the substrate acetylthiocholine but had little or no effec t on V-max for rat or human AChE. The coupling agent DTNA was more eff icient than DTNB, as demonstrated by the higher V-max/K-m ratio for th e former. DTNB, more so than DTNA, caused linear mixed-type inhibition of rat AChE. Poor precision was observed for the DTNB versus DTNA met hod. Reagent blanks were a significant component of rat, but not human , AChE activity. The use of DTNA in place of DTNB is recommended for q uantitative mechanistic investigations of cholinesterases. The most pr actical aspect of the DTNA method is that it can be adapted to automat ed instruments which can monitor the change in absorbance at 340 nm, a way from the hemoglobin peak.