Abstract:

The optimal conditions of assaying the activity of acetylcholinesterase (AChE) from the Italian honeybee (Apis mellifera ligustica Spinola) heads were determined by orthogonal matrix method. The concentrations of acetylcholinesterase and substrate,  pH and temperature of the reaction system,  and reaction time were optimized. L₂₅ (5⁶) orthogonal matrix was adopted,  without considering interaction. The specific activity of AChE was determined by colorimetric method of Ellman modified by Gorun (1978),  using acetylthiocholine iodide (ATCI) as substrate and DTNB (5, 5′-dithio-bis-2-nitrobenzoic acid) as chromogen. The optical density was measured by spectrophotometric detection (at 412 nm) of the increase of yellow color produced from thiocholine when reacted with 5,  5′-dithio-bis-2nitrobenzoate ions. Protein concentrations in homogenate supernatants were determined by the method of Bradford (1976). The results revealed that temperature of the reaction system was the most important factor for measuring the activity of AChE from honeybee heads, and the measuring was also remarkably affected by the other four factors. The effects on the activity of acetylcholinesterase were in the order of temperature＞pH＞time＞concentration of AChE＞concentration of substrate. The optimal conditions were: 0.2 heads/mL, 0.8 mmol/L substrate, pH 7.5, 40℃, 5 minutes.

Key words: Apis mellifera ligustica, acetylcholinesterase, orthogonal experiment, variance analysis, optimal conditions