Experiment 4 - BIOC 221 W2019. The Subject for the should be: BIOCHEM 221 EXP4

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1 EXCEL SHEET INSTRUCTIONS 1. a) On the web page there is a file called YOURNAME_Bioc221Exp4W2019.xlsx. Fill in the information colored RED with your own information. (Please do not readjust where the cells are on the Excel sheet or place values in the blue background area... for marking purposes.) b) Under the ABSORBANCE READINGS AT 340 nm on your file YOURNAME_Bioc221Exp4W2019.xlsx, enter the absorbance readings you obtained in the lab using the spectrophotometer. NADH has an extinction coefficient of 6.22 x 10 3 L mol -1 cm -1. Calculate the concentration in the cuvette in units of mole L -1. c) On the Excel sheet YOURNAME_Bioc221Exp4W2019.xlsx under Graph of Concentration... - plot a graph of Concentration NADH ( M) against time (min). On your Excel sheet, fill in the appropriate titles that are lettered in RED coloring. The graph will generate a best fit straight line. You may need to adjust this so that it only considers the linear data points. Adjust the trendline equation to be in scientific notation with 4 SF. d) this Excel file to: tymchakm@uregina.ca The Subject for the should be: BIOCHEM 221 EXP4 The filename should include your name: JOESTUDENT_Bioc221Exp4.xslx (example) Report to hand in is below.

2 Name: Partner: Lab Section: QUESTIONS TO HAND IN Experiment 4 1. On your graph, a best-fit straight line is displayed (with an equation) that should correspond to the initial velocity of the reaction in the cuvette. At this point the concentration of the substrates are high and products are near zero, so this velocity should be at V max. This is necessary to calculate how many units of enzyme are present. (A unit of enzyme is the amount of enzyme which catalyzes the conversion of 1 mole of substrate to product per minute.) When the Malate dehydrogenase solution was made, 2.5 l was taken from a stock bottle and mixed with 1mL of 0.25 M Potassium phosphate buffer (ph 7.4). A 1 in 10 dilution was made of this enzyme solution. It was this diluted enzyme solution which ended up being used in this experiment. What was the equation of your Best fit straight line? Using your graph s best fit straight line, calculate how many units of Malate dehydrogenase were originally present in this stock bottle (The bottle contained 5mL total volume of enzyme originally). (show your work, calculate to 3 SF)

3 2. Our sample was run using the method peptides.met. (See PDF) This involved a flush with water and buffer to waste, then the injection of our sample and water plug. On the diagram below, indicate where vials should be placed and what should be the contents in these vials.

4 3. The conversion of L-malate and NAD + to become Oxaloacetate and NADH + H + is unfavorable. Explain how the citric acid cycle continues to go in the forward direction... if this reaction prefers to go in the reverse direction. (Show the reaction equations to help explain your answer.).

5 4. On the previous page, the CE electropherogram of our reaction at time 47 minutes is shown. (See PDF electropherogram for more info). Can you explain the lettered points on the electropherogram, as to WHAT is being observed by the spectrophotometer at that point and WHY it is giving these results. It would be good to support your answer using other electropherograms from the PDF.