Initial in vitro screening of the enzyme activity against (−)-cocaine was carried out by using a single concentration (1 mM) of (−)-cocaine, assuming that KM << 1 mM such that the enzyme was always saturated by 1 mM (−)-cocaine and the maximum reaction velocity (Vmax) was reached for a given concentration [E] of the enzyme. Vmax = kcat[E]. The relative concentrations of the enzymes were determined by using an enzyme-linked immunosorbent assay (ELISA)26 (link),52 (link) described below. The ELISA buffers used in the present study are the same as those described in literature.26 (link),52 (link) Specifically, the coating buffer was 0.1 M sodium carbonate/bicarbonate buffer (pH 9.5). The washing buffer (PBS-T) was 0.01 M potassium phosphate monobasic/potassium phosphate monohydrate buffer (pH 7.5) containing 0.05% (vol/vol) Tween 20. The diluent buffer (EIA buffer) was potassium phosphate monobasic/potassium phosphate monohydrate buffer (pH 7.5) containing 0.9% sodium chloride and 0.1% bovine serum albumin (BSA).
To measure (−)-cocaine and benzoic acid, the product of the enzymatic (−)-cocaine hydrolysis, we used sensitive radiometric assays based on toluene extraction of [3H](−)-cocaine labeled on its benzene ring.24 (link) In brief, to initiate the enzymatic reaction, 100 nCi of [3H](−)-cocaine was mixed with 100 µl of enzyme solution. For Michaelis-Menten kinetic analysis, the enzymatic reactions proceeded at 37°C and pH 8 with varying concentrations of (−)-cocaine. The reactions were stopped by adding 200 µl of 0.05 M HCl, which neutralized the liberated benzoic acid while ensuring a positive charge on the residual (−)-cocaine. [3H]benzoic acid was extracted by 1 ml of toluene and measured by scintillation counting. Finally, the measured (−)-cocaine concentration-dependent radiometric data were analyzed in terms of the standard Michaelis-Menten kinetics so that the catalytic parameters were determined. The enzyme activity assays with [3H]ACh were similar to the assays with [3H](−)-cocaine. The primary difference was that the enzymatic reaction was stopped by addition of 200 µl of 0.2 M HCl containing 2 M NaCl and that the product was [3H]acetic acid for the ACh hydrolysis.
To measure (−)-cocaine and benzoic acid, the product of the enzymatic (−)-cocaine hydrolysis, we used sensitive radiometric assays based on toluene extraction of [3H](−)-cocaine labeled on its benzene ring.24 (link) In brief, to initiate the enzymatic reaction, 100 nCi of [3H](−)-cocaine was mixed with 100 µl of enzyme solution. For Michaelis-Menten kinetic analysis, the enzymatic reactions proceeded at 37°C and pH 8 with varying concentrations of (−)-cocaine. The reactions were stopped by adding 200 µl of 0.05 M HCl, which neutralized the liberated benzoic acid while ensuring a positive charge on the residual (−)-cocaine. [3H]benzoic acid was extracted by 1 ml of toluene and measured by scintillation counting. Finally, the measured (−)-cocaine concentration-dependent radiometric data were analyzed in terms of the standard Michaelis-Menten kinetics so that the catalytic parameters were determined. The enzyme activity assays with [3H]ACh were similar to the assays with [3H](−)-cocaine. The primary difference was that the enzymatic reaction was stopped by addition of 200 µl of 0.2 M HCl containing 2 M NaCl and that the product was [3H]acetic acid for the ACh hydrolysis.
