The Measurement of Enzymatic Activity of Acetylcholinesterase (AChE)
Dan Lappe, Dan Ahmadizadeh, Francesca Iacono
Bio 204 L60
The most important enzyme for muscle and nerve movement is acetylcholinesterase (AChE) (Zapletalova). An enzymes job is to attach to one of the reactants, or the substrate, and lower the biochemical activation energies. AChE uses hydrolysis to break down the neurotransmitter acetylchoine (ACh). This neurotransmitter is found in neuromuscular junction and triggers the contraction of muscles (Sadava). After Ach is broken down it forms choline and acetic acid. The purpose of this experiment is to get a better understanding of how AChE acts as an enzyme. The reason this enzyme and protein is needed for further experimentation is to make the pesticides, used for insects, safe for humans. The pesticides called organophosphates, bind onto the AChE active site and doesn’t allow Ach to bind onto the same active site (Mizyazaki).
The first experiment for this lab is measuring the absorbance of standard cysteine concentrations. To find the free sulfhydryl group in thiocholine, it is needed to use the compound DTNB. Mixing DTNB and a sulfhydryl gives a product that produces a yellow by product, TNB2-. The concentration is determined by the color variation, thus the stronger the yellow allows the concentration of thiocholine to be measured (Mizyazaki). Our hypothesis is that the enzyme, AChE, will increase the reaction rate of thiocholine.
Methods and Materials
To generate a standard curve, the lab section received 6 blank tubes that didn’t have cysteine, which contained a buffer and DTNB. The next 6 tubes all contained a known concentration of 1mM of cysteine with various dilutions of DTNB, 0, 0.025, 0.05, 0.067, 0.08, 0.1, and 0.13 mM. Each lab group received one blank tube and one standard tube (cysteine and DTNB). The absorbance was measured in a Spec20 and calibrated using...