User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/18: Difference between revisions

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==Procedure==
==Procedure==
* For details on making stock solutions, see [[User:Melissa Novy/Notebook/CHEM-571/2012/09/18|Melissa's Notebook]] for the final concentration of stock solution for horseradish peroxidase, sodium phosphate buffer, 4-Aminoantipyrine, and 4-Iodophenol.
* For details on making stock solutions, see [[User:Melissa Novy/Notebook/CHEM-571/2012/09/18|Melissa's Notebook]] for the final concentration of stock solution and final concentration reached for horseradish peroxidase, sodium phosphate buffer, 4-Aminoantipyrine, and 4-Iodophenol.
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 17:29, 7 October 2012 (EDT)''':you need to list the final concentrations reached
* 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.
* 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.  
* 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 [[User:Melissa Novy/Notebook/CHEM-571/2012/09/18|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]
* For results obtained from UV-vis spectrometer, see [[User:Melissa Novy/Notebook/CHEM-571/2012/09/18|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]. In those samples, the initial and final concentration reached were depicted in the table below:
 
{| {{table}}
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 17:29, 7 October 2012 (EDT)''':how did you reach all these concnetrations? what volumes were used? be specific. what concentrations specifically were tested and why? how are they labeled? how do i tell which is which?  results shoudl be in your notebook, not referenced.
| align="center" style="background:#f0f0f0;"|'''Stock Solution'''
| align="center" style="background:#f0f0f0;"|'''Initial Concentration, M<sub>1</sub> [M]'''
| align="center" style="background:#f0f0f0;"|'''Volume Added V<sub>1</sub> [mL]'''
| align="center" style="background:#f0f0f0;"|'''Final Concentration, M<sub>2</sub> [M]'''
|-
| H<sub>2</sub>O<sub>2</sub>||0.000034||0.75||0.000017
|-
| AAP||0.0025||0.7||0.001166667
|-
| Phenol||0.018||0.01||0.00012
|-
|}


==Notes==
==Notes==
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* 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 [[User:Melissa Novy/Notebook/CHEM-571/2012/09/18|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.
* 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 [[User:Melissa Novy/Notebook/CHEM-571/2012/09/18|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.


*'''[[User:Abigail E. Miller|Abigail E. Miller]] 17:29, 7 October 2012 (EDT)''':you ne3ed ot include the graphs. and the rates otherwise there is no evidence to back your conclusions.


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Revision as of 20:19, 26 October 2012

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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 and final concentration reached 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]. In those samples, the initial and final concentration reached were depicted in the table below:
Stock Solution Initial Concentration, M1 [M] Volume Added V1 [mL] Final Concentration, M2 [M]
H2O2 0.000034 0.75 0.000017
AAP 0.0025 0.7 0.001166667
Phenol 0.018 0.01 0.00012

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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