Agaricus Bisporus Lab Report

Extract Preparation Several samples of Agaricus bisporus were dissected into their various anatomical components (see Figure 1.) 9.1248 g of the lower stem (S2) was collected and frozen with liquid nitrogen. The frozen sample was ground up and mixed with 28.9 mL ± 0.1 mL of a sodium phosphate buffer (50 mM, pH 6.5). The mixture was then transferred into a centrifuge tube and placed in a Beckman Coulter Avanti J-26 XPI high speed centrifuge. The sample was spun at 20,000 rcf for 20 minutes at 4 ˚C. Six 1.5 mL samples of the supernatant were then taken from the solution; five samples were then taken to be stored at -20 ˚C for future use.
Kinetics Measurements Absorbance values were measured at 400 using a Thermo Scientific Genesys 20 spectrophotometer. Measurements were taken at 23 ˚C. The kinetic samples were created in 2 mL microcentrifuge tubes using a two-fold serial dilution. The catechol concentration of the samples ranged from 8.00 mM to 0.065 mM. Eight uninhibited samples were prepared by mixing 10 μL of extracted enzyme
CU represent the increase in absorbance (at 400 nm) per minute in the total assay volume where catechol is the substrate. The activity was then converted to the relative activity of catechol (in CU/μL), relative activity represents the CU over the volume of enzyme added. The data obtained was then used to generate an Eadie-Hofstee plot to display the kinetic trend of the data (Figure 3). The Eadie-Hofstee plot produced a linear equation of Vo = -Km (Vo/[S]) + Vmax where the y-intercept was maximum velocity (Vmax), and the slope represented the negative Michaelis constant (Km). the uncertainty of Vmax and Km were determined using a LINEST function. Using the obtained uncertainties it was then possible to calculate the uncertainty of Vmax/Km . This process was used again to determine the Kinetic parameters of the inhibited