Purpose
- To make Horseradish Peroxidase Assay in order to test enzymatic activity in UV-vis Spectrometer.
Procedure
- For details on making stock solutions, see Melissa's Notebook for the final concentration of stock solution for horseradish peroxidase, sodium phosphate buffer, 4-Aminoantipyrine, and 4-Iodophenol.
- Samples mixed with altering concentration of H2O2 was made and analyzed under UV-vis spectrometer. The concentration of AAP(4-aminoantipyrine) was set at 2.5mM, 4-Iodophenol was set at 18mM, and horseradish peroxidase at 2.3uM. The concentration of H2O2 was changed from 1.7mM to 106.25uM.
- In the next set of samples, concentration of H2O2 is held constant. The concentration of AAP was altered from 1.25mM to 156.25uM. The concentration of other solutions were kept the same.
- For results obtained from UV-vis spectrometer, see Melissa's Notebook for graph of Absorbance versus Time of enzymatic activity of HRP in Fixed [AAP] and varied [H2O2], and in Fixed [H2O2] and varied [AAP]
Notes
- The above two sets of samples were run to determine the effect on enzymatic function of function of horseradish peroxidase under different concentrations of AAP and H2O2.
- It was discovered that when concentration of AAP is held constant, the enzymatic activity decreases once the concentration of H2O2 increases. This could be concluded by comparing the slope of each sample with different concentrations of H2O2.
- It was discovered that when concentration of H2O2 is held constant, the enzymatic activity increases as the concentration of AAP increases. The results can be shown in Melissa's Notebook in graph Absorbance over time of Enzymatic Activity of HRP in Fixed [H2O2] and Varied [AAP] by comparing the slopes of each sample run.
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Experimental Biological Chemistry I
