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) The structure of MOPS (3-(N-morpholino) propane sulfonic acid). The molecule is man-made. It has two pKa's, one at the sulfonic acid group (SO3H, pKa around 1.9) and the other at the nitrogen of the morpholino group (pKa 7.2; the N below is shown in its base form). What is the net charge of MOPS at pH 7.4?