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 × = .0025 mol of Na2HPO4 × = 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 × = .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 × = 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
- Wavelength 235 and 265 were monitored for the absorbance of the reagents adenosine and inosine Their absorbance are listed on the table below.
| Absorbance at 235
| Absorbance at 265
- By manipulating Beer's Law, the molar absorptivity was calculated from the absorbance and concentration of the substances.
- The calculation for the molar absorptivities of adenosine and inosine are shown below.
- At 235 = 18566.78
- At 265 = 13029.32
- At 235 = 1400
- At 265 = 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.