13 Jan They mix everything gently togeth

Homework #3

Know:
a) the components and their functions in the electron transport chain of mitochondria
b) how ATP synthase works
c) the components and their functions in photosynthesis; the concepts of the Calvin cycle
d) the reactions for biosynthesis of palmitic acid
Assigned problems in Biochemical Calculations (Segel):
Oxidation and reduction:
read pages 172 174
examples 3-12, 3-13, and 3-14 on pages 175 – 179

View the following two animations:
1) ATPase Scene 2 which shows ATP synthase as a molecular motor, synthesizing ATP.
www.evolusie.co.za/anim_ATPase3_flv.htm

2) Powering the Cell: Mitochondria at http://multimedia.mcb.harvard.edu/ which illustrates several
things, including ATP synthase and translocation of ATP and ADP across the inner mitochondrial
membrane.
Note: both animations will require either Apple Quicktime Player and/or Flash Macromedia
Plugin. Both may be downloaded for free from the web.

1.
A student has 35 ml of a crude extract of pig heart. They want to determine how many total I.U.s of
malate dehydrogenase they have in the extract. They take 0.1 ml of the extract and add it to 400 µl of 20 mM
MOPS, pH 7.4 (dilution A). After mixing gently, they take 400 µl of dilution A and add it to 800 µl of 20 mM
MOPS, pH 7.4 (dilution B). They then run the assay that is listed below:
400 μl
10 μl
10 μl
30 μl

20 mM MOPS, pH 7.4
10 mM NADH (in H2O)
25 mM oxaloacetate (in H2O)
of dilution B of their MDH solution, in 20 mM MOPS, pH 7.4

They mix everything gently together and then measure the ΔA340/Δ time. Their measured ΔA340/20 sec.
was -0.065.
a) How many total I.U.s of MDH were in the crude extract?
b) Why did you mix gently?
c) Which of the above enzyme assay components should be kept on ice? Why? Which components
should not be kept on ice? Why?

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