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

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 × of Na₂HPO₄ = 53.61 g of Na₂HPO₄

x_{60 mL} = = 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.

• 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.

• 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.

• Abigail E. Miller 10:40, 7 October 2012 (EDT):you are missing a bunch or reactants and your data from this date. show what happened and what problem you had to assess for the next day.