User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/11/13: Difference between revisions
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M<sub>2</sub> = .0307 mM | M<sub>2</sub> = .0307 mM | ||
[[Image: | [[Image:Adenoino1113labeled.png]] | ||
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[[Image: | [[Image:ADAlabeled.png]] | ||
==Beer's Law== | ==Beer's Law== | ||
* Wavelength 235 and 265 were monitored for the absorbance of the reagents adenosine and inosine Their absorbance are listed on the table below. | * Wavelength 235 and 265 were monitored for the absorbance of the reagents adenosine and inosine Their absorbance are listed on the table below. | ||
* By manipulating Beer's Law, the molar absorptivity was calculated from the absorbance and concentration of the substances. | * By manipulating Beer's Law, the molar absorptivity was calculated from the absorbance and concentration of the substances. | ||
<math>\frac{\epsilon = A}{bc}</math> | <math>\frac{\epsilon = A}{bc}</math> | ||
* The calculation for the molar absorptivities of adenosine and inosine are shown below. | * The calculation for the molar absorptivities of adenosine and inosine are shown below. | ||
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For adenosine | For adenosine | ||
At 235 <math>\frac{\epsilon = .57}{.0000307 M}</math> = 18566.78 | * At 235 <math>\frac{\epsilon = .57}{.0000307 M}</math> = 18566.78 | ||
* At 265 <math>\frac{\epsilon = .4}{.0000307 M}</math> = 13029.32 | |||
For inosine | |||
* At 235 <math>\frac{\epsilon = .14}{.0001 M}</math> = 1400 | |||
* At 265 <math>\frac{\epsilon = .6}{.0001 M}</math> = 6000 | |||
* Evaluating the molar absorptivities for each wavelength, the results indicate that there is a greater difference of molar absorptivities between adenosine and inosine at wavelength 235. The molar absorptivity for adenosine was observed to be higher than inosine. | |||
* We can hypothesized that before running the UV-Vis scans for the kinetic assay of ADA with adenosine, adenosine will produce a more pronounced absorption and signal. Therefore, adenosine should be monitored at 235. | |||
* Adenosine is the substrate of ADA. As time passes, ADA catalyzes the formation of inosine from adenosine. Thus, the concentration of adenosine should decrease over time. When concentration of adenosine decreases, the absorbance monitored at 235 should also decrease over time. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|''Wavelength'' | |||
| align="center" style="background:#f0f0f0;"|''Adenosine'' | |||
| align="center" style="background:#f0f0f0;"|''Inosine'' | |||
|- | |||
| Absorbance at 235||.57||.14 | |||
|- | |||
| Absorbance at 265||.4||.6 | |||
|- | |||
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Revision as of 11:18, 9 December 2012
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ADA Kinetic Assay Preparations
V1 = 0.08934 mL = 89.3 μL in 5 mL of buffer
UV-visible scans of Reagents
M1V1 = M2V2 (3 mM)(10 μL) = M2 (1000 μL) M2 = .0307 mM
3 μL of ADA × 65 μM = M2 (1000 μL) M2 = .195 μM of ADA
M2 = .0975 μM = 97.5 nM
Second dilution M = 48.8 nM Final Concentration = 24.38 nM Beer's Law
[math]\displaystyle{ \frac{\epsilon = A}{bc} }[/math]
For adenosine
For inosine
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