2.3. Cholinesterase Inhibition

Enzyme activities were determined using the Ellman spectrophotometric method [40] at 25 °C in 0.1 M phosphate buffer (pH = 7.4) with 0.3 mM DTNB as the thiol reagent. Substrate stock solutions, ATCh (10 mM), and PTCh (40 mM), and carbamates (50–100 mM), as well as their further dilutions, were prepared in water. Final concentrations of carbamates were in the range of 0.5–200 μM, while substrates were 1.0 mM and 4.0 mM for ATCh and PTCh, respectively. Final dilution of AChE and BChE was 500 and 300 times, respectably.

The extend of inhibition was determined by measuring the time dependence of cholinesterases inhibition by carbamates [16]. An inhibitor was added to the reaction mixture containing DTNB, buffer, and enzyme, and after a given incubation time, a substrate was added followed by measurement of the residual activity of the enzyme. Enzyme activity at “zero” time was measured after adding the enzyme to a reaction mixture containing DTNB, buffer, inhibitor, and substrate immediately before the start of the measurement. With the inhibited probes, the activities of the control probes, which did not contain an inhibitor, were measured.

The increase in absorbance was recorded at 436 nm for AChE or 412 nm for BChE [40,41], on a Cary 300 spectrophotometer (Varian, Inc., Mulgrave, Australia).

Enzyme inhibition proceeds according to the Scheme 1:

Where E, QC, [E] [QC], EC, and Q stands for free enzyme, inhibitor, Michaelis-type complex between enzyme and inhibitor, carbamylated enzyme, and leaving group, respectively. The first-order rate constants (k_obs) were calculated by the linear regression analyses at any given inhibitor concentration ([QC]):

where v₀ and v_i stand for the enzyme activity in the absence and in the presence of inhibitor at time t. When the k_obs was a linear function of [QC], the second-order inhibition rate constant (k_i) was calculated from:

when dependence of k_obs vs. [QC] was not linear, indicating the presence of a reversible enzyme–inhibitor complex, the maximum first-order inhibition rate constant (k_max) and the dissociation constants of enzyme–inhibitor complex (K_a) were determined from:

Then, the k_i constant was the ratio:

All kinetic parameters were calculated using the statistical package GraphPadPrism 8 (Graph Pad Inc., San Diego, CA, USA).