Specord 205 spectrophotometer

The AChE activities of the brain slice homogenates were determined spectrophotometrically by monitoring the hydrolysis of S-acetylthiocholine iodide at 30°C (ε₄₀₆ = 13300 M^-1 cm^-1) according to Ellman’s method (Ellman et al., 1961 (link)). Briefly, acetylthiocholine iodide was used as a synthetic substrate for AChE. The sample (7 μL containing 30–50 μg of protein) was mixed with 1 mL of 0.25 mM dithiobisnitrobenzoate (DTNB) in 50 mM phosphate buffer at pH 7.9 and the resulting mixture was incubated at 30°C in a water-jacketed cuvette holder. The reaction was initiated by adding 30 μL of the substrate (5 μmol). The final volume of the reaction mixture was 1037 μL. The thiocholine released by the substrate hydrolysis reacts with DTNB to afford 5-thio-2-nitrobenzoate, which was quantified at 406 nm using a Specord 205 spectrophotometer (Analytik Jena, Germany). The enzymatic activity was normalized in function of the protein content in the assay.