User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/18

Objective

1. to make stock solutions of sodium phosphate, 4-aminoantipyrine (AAP), H₂O₂, and horseradish peroxidase (HRP)
2. to perform an assay on the enzymatic rate of HRP and observing the kinetics of the reaction via real-time UV-vis.
   1. Vary the concentration of H2O2, while keeping the concentration of AAP constant, then vary the concentration of APP while keeping the concentration of H2O2 constant.

Description

1. The solutions for the reaction were made.

Making 250mL of 0.2 M Sodium Phosphate Buffer in H₂O

• Measuring and Calculating for Sodium Phosphate Solution
  • MW sodium phosphate: 163.94 g/mol
  • Mass sodium phosphate: 250 mL × (10^-3 L/mL) × 0.2 M × (10^-3 mol/mmol) × 163.94 g/mol = 13.4035 g sodium phosphate
    • Actual mass sodium phosphate: 13.4031 g
  • Actual concentration: 13.4031 g × (1 mol/163.94 g) × (1/0.25 L) = 0.327 M in H₂O
• 13.4031 g sodium phosphate was dissolved in about 200 mL of H₂O.
• The pH was adjusted to 7.00 by adding 20 mL of 1 N HCl dropwise.
• The solution was diluted to a final volume of 250 mL with additional H₂O.

Making AAP Solution

• 0.125g of AAP was added to 5mL H₂O to make 0.0025M solution of AAP.
• Making Phenol solution
  • 0.4054g 4-iodophenol was added to 20mL H₂O to make 0.17M phenol.
    • Because the phenol was not dissolving in the water, 700μM DMSO was added to the mixture.
      • Although the phenol began to dissolve, though not completely, another method was used to create the phenol solution.
  • 0.001 g 4-iodophenol was added to 250μL DMSO and kept separately from the AAP solution, to be mixed in smaller quantities immediately before the reaction.
    • The concentration of this reaction was 18mM phenol.

Making H₂O₂ Solution

• 1mL of 30% hydrogen peroxide was added to 100mL H₂O.
• 1mL of the new hydrogen peroxide solution was added to 49mL of 0.2 M potassium phosphate buffer, creating a 0.0017M hydrogen peroxide solution.

Making HRP

• 1mg of HRP was dissolved in 1mL H₂O, creating 23μM HRP solution.
• The HRP was further diluted to 2.3μMmM by adding 1mL HRP solution to 9mL H₂O.

2. Setting up the spectrophotometer

• The UV-vis was set to detect the absorbance at 510nm at 25°C.

3. Loading the cuvette

• 10μL 18mM phenol
• 700μL AAP solution
• 750μL H₂O₂ solution
• 50μL 2.3mM HRP solution
  • The concentrations for the hydrogen peroxide and AAP varied.
  • A couple of controls were run to verify that the reaction required HRP enzyme to run efficiently.
    • The first control was just buffer [negative control].
    • The second control was phenol, hydrogen peroxide, and AAP (no HRP) [negative control].
    • The table below shows the AAP/H₂O₂ ratio used
      • The concentration of H₂O₂ was varied by serial dilution as follows, while the concentrations of all other solutions were kept constant
• 0.05mL HRP was added to each cuvette at the last moment, right before the cuvette was placed in the UV-vis.

Data

• Final concentrations of stock solutions in total volume of 1.5 mL:

A picture of all the various assay mixtures after the reaction had taken place.

• Please refer to Melissa's entry for graphs depicting absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide and depicting absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.