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
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 20 mM MOPS, pH 7.4
10 μl 10 mM NADH (in H2O)
10 μl 25 mM oxaloacetate (in H2O)
30 μl 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?