Wheat Germ Lab

The purpose of this experiment was to analyze the activity and kinetic properties of an enzyme derived from wheat germ. For the first part of the experiment, we had to prepare a standard curve for the reaction product by measuring the velocity of the reaction catalyzed by acid phosphate that is extracted from wheat germ. Different enzyme concentrations were tested to see how fast they reacted. In order to quantify the amount of nitrophenol generated, six standards were prepared containing known concentrations of the product nitrophenol. In each cuvette, 1 mL of each of the standards was placed and labeled S1-S6. A spectrophotometer was then turned on and set to a wavelength of 410 nm. The absorbance of each tube was read and recorded on the Nitrophenol standard curve for acid phosphate assays

The nitrophenol standard curve was then constructed by the plotting of A410 of tubes S1-S6 vs. the concentration of nitrophenol. The preparation of acid phosphate from wheat germ extract was previously performed by the instructor at the beginning of the class.
In the second part of the lab the velocity of commercially available purified acid phosphatase was measured. Firstly, 12 test tubes were spitted in groups of six and labeled A1-A6 and B1-B6. Then 0.5 mL of 1.5% (w/v) of KOH was placed in each of the 12 tubes. Following that 5 ml of Phosphate Substrate solution were added to tube A and tube B. While tube A contained the reaction catalyzed by purified acid phosphatase, tube B contained the reaction catalyzed by the extracted acid phosphatase. From tube A 0.5 ml of the substrate solution was transferred to tube A1, likewise 0.5 mL of substrate solution was transferred from tube B to tube B1, at 0 time values each. With the use of a micropipette 50 µL of