User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/02

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Preparation of the Reactants of HRP Oxidase Assay

  • The reactants listed in HRP Oxidase Assay were weighed out.
  • The phosphate buffer was made with white granules of sodium phosphate dibasic (FW 268.07, heptahydrate) instead of potassium phosphate. The substitution is not detrimental to the procedure since the ion of interest is the phosphate group. The total volume chosen for the buffer is 60 mL.
  • Calculation for obtaining Na2HPO4:

0.020 mol of Na2HPO4 × [math]\frac{268.07g}{1mol}[/math] of Na2HPO4 = 53.61 g of Na2HPO4

x60 mL = [math]\frac{.060 L * 53. 61 g}{1L}[/math] = 3.22 g of Na2HPO4

  • The required weight for 4-iodophenol is 0.810 g to make 0.0025 M. The actual weighed amount was 0.8109 g. This amount is dissolved in 700 μM dimethyl sulfoxide (DMSO, polar aprotic solvent) due to its immiscibility in water.
  • Dissolved 25 mg of 4-aminoantipyrine (AAP) into 50 mL of water.
  • Weighed out 1 mg of the brown granular solid of horseradish peroxidase (HRP) and dissolved it in 1 mL water.
  • To obtain 0.0017 M of hydrogen peroxide, added 1 mL 30% of the clear and colorless liquid into 100 mL of water. Then, collected 1 mL from the solution to dilute it into 50 mL of the 0.2 M phosphate buffer.
  • Placed the settings of the spectrophotometer on kinetics, at 25°C, 510 nm from the methods function.
  • In reference to the document of Novy, Patel, and Wang (MDK), the group has chosen to prepare the sample solution with the most linearity. The sample solution has the following concentration of reactants listed in the table below. The original concentration of AAP was 2.5 mM amounting to 700 μL.
  • Transferred the reactants to a plastic disposable sample cell using a micropipet. NOTE: The HRP was added last when the sample was already inside the chamber. The addition of HRP initiates the reaction.
  • The solution sample appeared as clear and colorless liquid solution before the reaction. At the end of the reaction, the sample was ruby-red at the top of the sample cell and clear and colorless at the bottom. There is a defined separation between the two color phases.
  • There was difficulty of replicating the sample concentration from MDK. A run using a volume of 35 μL of AAP and 2.5 μL of HRP had a flat signal indicating the possibilities of having no or slow reaction occurring. Increasing the concentration of HRP made the reaction occur at an instant.
  • For the following day, the agenda would be to ascertain the appropriate volume of AAP while keeping the same concentration.
properties 4-iodophenol AAP H2O2 HRP
Molarity 18 mM 156.25 μM 1.7 mM 2.3 μM
Volume 10 μL 750 μL 50 μL

HRP Assay Figures

  • Charts embedded below represent the HRP Assay runs that have varying concentrations of HRP and AAP. The variations in the physical condition of the surrounding environment were done to investigate the appropriate ratio between HRP and AAP.


  • Runs 1, 3, and 4 reflect absorbance trends where the reaction had a low concentration of HRP.
  • Run 2 shows the reaction where there was an error in calculating the proper volume for the HRP and AAP ratio. The group added 50 μL of HRP against a very low concentration of AAP.
  • Run 5 is the replicated absorbance from MDK using the standard condition where the volume of AAP is 700 μL and HRP is 50 μL.


  • The rates of runs 2 and 5 are shown below to indicate that the rate of run 5 (reaction with the proper amount of HRP) has a linear rate compared to run 2.