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.
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 micrograms/100mL?

  1. 👍 1
  2. 👎 0
  3. 👁 200
  1. Two dillutions and assume volumes are additive


    1ml was mixed with 4ml giving a total volume of 5ml.


    1ml of X was mixed with 2ml to give a total of 3ml.


    Absorbance was taken of X2:

    Absorbance (A)=l*M*E

    E=molar absorbtivity=6220
    M=Molarity (moles/L)
    l=length of cuvette (1)

    Solve for M:


    Let M=X2

    You know X2, so solve for X:


    You know X, so solve for C1:



    So convert moles to grams using molecular weight (200g/mole):


    And convert L to ml:



    Answer with the appropriate number of significant figures

    1. 👍 0
    2. 👎 0
  2. Additional information for reply:

    The info given will give g/ml. Convert g to ug and do not convert L to ml. Divide answer by 10 to get units desired.

    1. 👍 0
    2. 👎 0

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