- to make stock solutions of sodium phosphate, 4-aminoantipyrine (AAP), H2O2, and horseradish peroxidase (HRP)
- to perform an assay on the enzymatic rate of HRP and observing the kinetics of the reaction via real-time UV-vis.
- 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.
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.
- MW of HRP = 40000 g/mol
- 1mg of HRP was dissolved in 1mL H2O, creating 23μM HRP solution.
- The HRP was further diluted to 2.3μM 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.
|Concentration of Reactants
| Phenol||AAP||Hydrogen Peroxide||HRP
| 18 mM||2.5mM||1.7mM||2.3uM
| 18 mM||2.5mM||850uM||2.3uM
| 18 mM||2.5mM||425uM||2.3uM
| 18 mM||2.5mM||212.5uM||2.3uM
| 18 mM||2.5mM||106.25uM||2.3uM
| 18 mM||2.5mM||1.7mM||2.3uM
| 18 mM||1.25mM||1.7mM||2.3uM
| 18 mM||635uM||1.7mM||2.3uM
| 18 mM||312.5uM||1.7mM||2.3uM
| 18 mM||156.25uM||1.7mM||2.3uM
- The concentrations above are the concentrations of each solution before being added to the cuvette.
- Final concentrations of stock solutions in the cuvette for a total volume of 1.5 mL:
||Initial Concentration, M1 [M]
||Volume Added V1 [mL]
||Final Concentration, M2 [M]
- A picture of all the various assay mixtures after the reaction had taken place.
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.
- This graph depicts the absorbance over time of enzymatic activity of HRP in a fix concentration of AAP and varied concentrations of hydrogen peroxide.
- When the AAP was fixed at 2.5mM, the reaction with a concentration of 1.7mM H2O2 took over 300 seconds to be completed. The reaction with a concentration of 850μM H2O2 took about 200 seconds to be completed, and the majority of the reaction took place in the UV-vis, so the change in absorptivity was tracked and measured through out the entire reaction. Reactions with a hydrogen peroxide concentration less that 850μM appeared to occur too quickly such that the change in absorptivity was unable to be effectively tracked by the UV-vis.
- The graph depicts the absorbance over time of enzymatic activity of HRP in a fixed concentration of hydrogen peroxide and varied concentrations of AAP.
- When the H2O2 was fixed at 1.7mM, the reactions within the AAP concentration range of 156.25μM - 1.25mM showed a steady increase in absorptivity. The faster reactions with more absorptivity were the samples containing a more diluted amount of AAP solution.
The protocol that this experiment was based off of was found in the DropBox folder, "CHEM571_Fall2012" → "HRPAssays" → "3760-Protocol HRP_AbsorbanceAssay.pdf"