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    • #16899
      ladwer
      Participant

      Hello,

      In a developing biochemical tools to examine metabolic reactions lap, we measured the absorbance spectra of potential substrates and products as the following:

      OAA stock is 8 mM
      Malate stock is 8 mM
      NADH stock is 1 mM

      we made dilutions of these with water as shown below:

      Dilution of 1:2

      OAA: 500 μL water + 500 μL OAA
      malate: 500 μL water + 500 μL malate
      NADH: 500 μL water + 500 μL NADH

      —-
      Dilution of 1:4

      OAA: 750 μL water + 250 μL OAA
      malate: 750 μL water + 250 μL malate
      NADH: 750 μL water + 250 μL NADH

      —-

      Dilution of 1:10

      OAA: 900 μL water + 100 μL OAA
      malate: 900 μL water + 100 μL malate
      NADH: 900 μL water + 100 μL NADH

      we set the wavelength of the spectrophotometer to 340 nm.
      used a cuvette with 1 mL water as black, and them measured the absorbance of each of the dilutions of OAA, malate, or NADH, mixing each thoroughly before taking a reading.

      the results were as shown below:

      Dilution of 1:2
      OAA= 0.025
      malate= 0.015
      NADH= 0.807

      Dilution of 1:4
      OAA= 0.008
      malate= 0.007
      NADH= 1.002

      Dilution of 1:10
      OAA= -0.054
      malate= -0.037
      NADH= 0.345

      Now my question is, what is the purpose behind these measurements?
      what were they meant to discover that would apply to the development of an assay for measuring the activity of the Enzyme?
      and why we would want to see a relation between the concentration of the substance and its absorbance as measured by the spectrophotometer?

      please explain these to me and thank you in advance!

    • #112591
      JackBean
      Participant

      Did you see some real peaks with the oxaloacetate and malate or was it rather shifted baseline? (especially in the case of 1:10 dilution)

      As you can see, the absorbance of oxaloacetate and malate in range 4 – 0.8 mM is rather low if any. On the other hand, NADH absorbs pretty well, thus if you were about to use these substances to track enzymatic activity you should go for with the NADH, because in the range you have change of absorbance of 0.5 AU and even with 0.8 mM concentration is the absorption still pretty high.

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