# Calculating specific activity of pyruvate kinase

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#1
so, we had to calc specific activity of pyruvate kinase in

μmol pyruvate formed/min
mg enzyme

did the experiment, plotted data, blah blah.

from a graph of absorbance at 340 nm against time I found the ΔA340/min to be
-0.0943

calculated Δconcentration using beer's law rearranged: A/ε = C
-0.0943/6.22x10^3 = -15.1607717 mol/dm^3/min

then because this took place in 3 ml cuvette, I multiplied by 0.003 (3ml = 0.003dm^3) to give Δ in no of moles in the cuvette per min: -0.04548231511 moles/min

then I multiplied by 1000, then 1000 again, to give it in μmol :
- 45482.31511μmol/min
this was the change in NADH concentration, equivalent to
+ 45482.31511 μmol pyruvate formed/min

now for the protein (enzyme):

used a biorad assay to generate a standard curve of absorbance at 595 nm against concentration of protein in mg/ml

Using this and the fact that my neat yeast extract had an absorbance of 1.023, I found the concentration of protein (i.e enzyme) (1.023 divided by gradient of graph, 1.0584) to be 0.96655 mg/ml

we then diluted this 100fold by using 0.01ml of this with 0.99ml of a potassium phosphate buffer
therefore this mixture contained 0.0096655 mg of enzyme (pyruvate kinase)

we then used 0.1 ml of this in the cuvette to test its activity,
therefore there was 0.00096655 mg in each cuvette ----> 9.6655x10^-4 mg

ok, so taking the numbers and plugging in:

μmol pyruvate formed/min = 45482.31511 = 47056350.02
mg enzyme 9.6655x10^-4

so 47056350.02 μmol pyruvate formed/min/mg enzyme

which is the same as

47056350.02 μmol pyruvate formed/mg enzyme/min
which is the final form they wanted it in.

obviously this is completely wrong, and all the other groups I have asked got answers below 1.

sorry to ask such a dumb question on TSR, this is clearly a maths error but I can't see the mistake. I feel like I've calculated properly, and I've gone through it methodically about 6 times now. But my brain ain't working no more

Any kind soul care to help me out?
0
3 years ago
#2
Hi I only have biology knowledge from teaching A level biology and from studying anatomy, physiology, biochemistry, genetics, etc as a med student; I do not have a biology degree.

I note in your 5th paragraph starting calculatiing the change in concentration......."

-0.0943/6.22x10^3 = -15.1607717

you appear to have used the 10^3 as a part of the numerator;if you meant to use it as below, you have introduced an error of millionfold.

This is the only possible error I can see (and I have been named "Hawkeyes" by the editors of my book! [not necessarily a compliment!!]

Good luck and thanks!
Mukesh
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