A plasma reaction converts A (Molecular weight = 200) to B and uses NAD as a co-substrate and produces NADH as a by product (1 mole of NADH is produced per every 1 mole of A converted to B). The amount of NADH can be determined by monitoring its’ absorbance at 340nm in a spectrophotometer.

Question

1ml of plasma is taken and diluted with 4 ml of reaction buffer with the enzyme required to convert A to B inhibited. 1ml of this sample is added to a cuvette and the inhibition is then stopped by adding 2 ml of a “inhibition prevention” buffer and the natural conversion of A to B allowed to occur. This conversion is completed in 10 minutes after which time the change in absorbance at 340nm is recorded.

The change in absorbance is 0.82 absorbance units over the 10 minutes. The molar extinction coefficient of NADH at 340nm is 6220.

What is the concentration of compound A in ng/100mL?

Answer 1

A = kc
A = 0.82
k = 6220.
Solve for c in mols/L.
Correct for the two dilutions; i.e., 1 up to 3 and 1 up to 5 and the answer is in mols/L. Convert that to ng/L, then to ng/100 mL.