A student runs an enzyme assay using pig heart mitochondrial malate dehydrogenase (MDH; found inthe Krebs cycle).

The assay contains the following:

400 ?l 20 mM MOPS, pH 7.4

10 ?l 10 mM NADH (in H2O)

10 ?l 25 mM oxaloacetate (in H2O)

30 ?l of their MDH solution, in 20 mM MOPS, pH 7.4

They mix everything gently together in a cuvette and then measure the ?A340/? time using a spectrophotometer.Their measured ?A340/20 sec. was “-0.065”.

a) How many I.U.’s of MDH were in the 30 µl assayed? 0.014 I.U.

b) In theory, how long would the assay have to run before the pH in the cuvette rose to pH 7.5? The pKaof MOPS is 7.2.   32.7 min.

c) Will the assay pH actually reach this pH? Explain.  No. Why?? <——Just need to know why, everything else is answered.