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ADA Kinetic Assay Preparations
- A weight of 0.6702 g of sodium phosphate dibasic was dissolved in 50 mL of water to obtain a molarity of 0.05 M. The pH of the solution was adjusted to pH 7.4.
.050 L of water × [math]\displaystyle{ \frac{0.05 mol}{1L} }[/math] = .0025 mol of Na2HPO4 × [math]\displaystyle{ \frac{268.07 g}{1 mol} }[/math] = 0.6702 g
- The pH was was adjusted to 7.4 by the addition of 2 drops of 12 M HCl.
- To obtain 1 mM inosine, 1.5 mg of the solid was dissolved in 1 mL buffer. This was further diluted by collecting 89.3 µL of the 1.5 mg/ml inosine into 5 mL of the sodium phosphate buffer.
.0015 g of inosine × [math]\displaystyle{ \frac{1 mol}{268.2 g} }[/math] = .000005596 mol ÷ .001 L = .005596 M = 5.596 mM
5.596 mM (V1)= 0.1 mM (5 mL)
V1 = 0.08934 mL = 89.3 μL in 5 mL of buffer
- 3 mM adenosine was prepared by dissolving 0.0082 g of the solid into 10 mL sodium phosphate buffer. The stock concentration of adenosine was 3.07 mM.
.0082 g of adenosine × [math]\displaystyle{ \frac{1 mol}{267.24 g} }[/math] = 3.06840 × 10-5 ÷ .010 L = .00307 M = 3.07 mM
UV-visible scans of Reagents
- Mody and Nagle condcuted the runs for the UV-visible scans of ADA, adenosine, and inosine to verify the absorbance peaks for each.
- From the ADA Activity Assay protocol, it was specified to monitor the absorbance of each reagent at wavelengths 235 and 265.
- 1 mL of inosine was transferred to a cuvette. The final concentration of inosine in the cuvette was 1 mM.
- 10 μL of adenosine was diluted to 990 μL of the sodium phosphate buffer in a cuvette. The final concentration of adenosine was .0307 mM.
M1V1 = M2V2
(3 mM)(10 μL) = M2 (1000 μL)
M2 = .0307 mM
- ADA was diluted as follows:
3 μL of ADA × 65 μM = M2 (1000 μL)
M2 = .195 μM of ADA
500 μL × .195 μM = M2 (1000 μL)
M2 = .0975 μM = 97.5 nM
- 2 additional 500 μL dilutions of 97.5 nM to 1000 μL were executed using the dilution equation.
Second dilution M = 48.8 nM
Final Concentration = 24.38 nM
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.
- By manipulating Beer's Law, the molar absorptivity was calculated from the absorbance and concentration of the substances.
[math]\displaystyle{ \frac{\epsilon = A}{bc} }[/math]- The calculation for the molar absorptivities of adenosine and inosine are shown below.
[math]\displaystyle{ \frac{\epsilon_235 = .57}{.0000307 M} }[/math] =
| Wavelength
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Adenosine
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Inosine
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| Absorbance at 235 |
.57 |
.14
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| Absorbance at 265 |
.4 |
.6
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