Experiment 7 EXCEL SHEET INSTRUCTIONS

Transcription

1 EXCEL SHEET INSTRUCTIONS 1. a) On the web page there is a file called YOURNAME_Bioc221Exp7W2018.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 RAW DATA FROM YOUR EXPERIMENT on your file YOURNAME_Bioc221Exp7.xlsx, enter the absorbance readings you obtained in the lab using the spectrophotometer. Note that you may need to adjust your times, as each person likely took their first readings at different times. You will notice I have added a Reaction Mix 6 with numbers in blue. I isolated the enzymes as you did in the lab, except instead of waiting a week, I did the WEEK 2 reaction the next day. The activity of the enzymes should be greater as they were not stored for a week, therefore I only used 20 ul of the Heart particle and added 40 ul of water. In contrast, you used 60 ul of the Heart particle. These numbers will be used for comparison to your data. c) Convert your absorbance. DPIP has an extinction coefficient of 20.7 L mmol -1 cm -1. Calculate the concentration in the cuvette in units of mole L -1. You will notice the time automatically gets converted into minutes. d) Under (Titles for Reaction Mixes & Graph), you will notice a graph has been generated.

2 e) Under (Initial Velocity & Activity of Heart Tissue), enter an Excel formula to determine the slope of the initial reaction rate for each reaction mix. (Cells D85 to D89) You may wonder... how do I do this? In the cell (D85-89) you want to make a formula, enter an = sign. To get the formula, look back to some of our previous experiments where we used the LINEST formula. This formula gives you the slope of the line using linear regression. You may notice in the Figure 1 graph that the data lines begin to curve... therefore, to get just the initial velocity, choose only the data points before there is a curve in the line for the LINEST formula. The slope value will likely be negative. Convert this value to a positive value, and then calculate the Apparent Activity that the 500uL Heart Particle Extract would have had in the presence of the reaction mix solution. (In other words, consider how much activity there was in the amount added to cuvette from the initial slope, and multiply to determine how much activity would be displayed if all 500 ul was used.) f) Under (Titles for Reaction Mixes & Graph II), you will notice a graph has been generated. g) Answer the question. h) this Excel file to: tymchakm@uregina.ca The Subject for the should be: BIOCHEM 221 EXP7 The filename should include your name: JOESTUDENT_Bioc221Exp7W2018.xslx (example) Report to hand in is below.

3 Name: Partner: Lab Section: QUESTIONS TO HAND IN Experiment 7 1. In this lab we did several centrifugations. The rotor we used was a fixed angle rotor. The relative centrifugal force (rcf) is the force in terms of the earth s gravitation force (g). This force depends on the radius of the sedimenting particle from the axis of rotation and the revolutions per minute (rpm). Our rotor had a maximum radius of cm. To calculate the g force the following equation is used: N RCF ( r) 1000 where r = radius in centimeters and N = revolutions per minute 2 a) Which particle, Q or Z in picture above should experience the lowest g force? b) In the first centrifugation the procedure says to centrifuge at 800 x g (max force). What should we set the RPM to on our centrifuge? (Show your work)

4 2. In our protocol, homogenisation of our pig heart occurred in a 0.02 M potassium phosphate buffer containing 1 mm EDTA. What is the role of the potassium phosphate? What does EDTA stand for and what is its role in this procedure? 3. Mark on the diagram below where: a) The DPIP accepts electrons, b) the Malonate Inhibits, c) the Antimycin A inhibits

5 4) Explain the difference in activity between Reaction Mix 2, 3, and Reaction Mix 4. State what your results were, if these results might be expected, and explain why.