Investigating The Amount Of Enzyme That Would Produce A Reaction Rate

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To begin the lab, part A was performed to determine the amount of enzyme that would produce a reaction rate that did not proceed too slow or too rapidly. As seen on page 13 of the Lab Handout, varying amounts of tyrosinase and phosphate buffer were added to a cuvette while the amount of the L-DOPA was constant. After all reagents were added to the cuvette, the cuvette was inserted into spectrophotometer and absorbance of product formation at 475 nm was recorded for two minutes at fifteen seconds interval. After absorbance of product formation was measured and recorded for each cuvette, as shown on page 14 of the Lab Handout, graphs of rate of production formation versus time, were made with the data of each cuvettes and the…show more content…
After obtaining all of the absorbance values and unit conversions were calculated, as shown on page 18, a rate of product formation versus time was graphed as well as a Michaelis-Menten plot. Based upon the Michealis-Menten plot, estimated values of Vmax and Km were recorded. Instead of generating a Lineweaver-Burke plot to determine the calculated values of Vmax and Km, our lab instructor provided a video for us to watch, which allowed us to determine the calculated values of Vmax and Km right on the Excel program which were then recorded into our lab notebook. We were then able to calculate out kcat value and our catalytic efficiency using the equations provided on page 20 of the The same experiment was repeated for part C of the lab, however, three different data sets were recorded. The first data set contained the addition of DMSO, a second data set had the addition of the inhibitor quercetin, while the third data set contained fruit extract. The concentration of tyrosinase, quercetin, DMSO, and fruit extract were constant, while concentration of L-DOPA and phosphate buffer differed from each cuvette as recorded on pages 22 and 23 of the Lab Handout. One modification to part C compared to part A and B is that the volumes of each reagent was cut down by a third due to supplies running low, however, this did not affect the experiment in any ways. As seen on pages 22- 25 on the lab manual, after linear plots and Michaelis-Menten graphs
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